Diagnostic efficacy of elevated serum angiotensin-converting enzyme and lymphopenia between presumed sarcoid uveitis and presumed tubercular uveitis.

PURPOSE: To evaluate the diagnostic efficacy of elevated serum angiotensin-converting enzyme (sACE) and lymphopenia in presumed sarcoid and tubercular uveitis. METHODS: A single-centre retrospective study was conducted on a cohort of 755 adult patients with uveitis between January 2019 and June 2020. Demographic, clinical and laboratory data were retrieved from our hospital database. Measurements of serum angiotensin-converting enzyme (sACE) and lymphocyte counts were analysed. RESULTS: The mean age of the patients was 41 ± 13 years. Presumed sarcoid uveitis was diagnosed in 50 (7%) patients, presumed tubercular uveitis in 222 (29.4%) and other uveitic entities noted in 483 (64%). Intermediate and posterior uveitis were the most common anatomical diagnosis in presumed sarcoid uveitis (59% and 20%, respectively) and in presumed tubercular uveitis (46% and 38%, respectively). Elevated sACE was noted in 76% of presumed sarcoid uveitis and 46% in presumed tubercular uveitis. The combination of high serum angiotensin-converting enzyme along with lymphopenia was only in 17% in presumed sarcoid uveitis and 9.7% in presumed tubercular uveitis. sACE was found to be a significant risk factor for presumed sarcoid uveitis with an odds ratio of 3.603 (p < 0.002), and in presumed tubercular uveitis odds ratio was not significant with odds ratio of 1.19. Lymphopenia was not found to be a significant factor in both groups. CONCLUSION: Elevated sACE activity was an independent risk factor for presumed sarcoid uveitis over lymphopenia alone or in combination with lymphopenia.

Biochemical Analysis of Aqueous Humor in an Elderly Patient with Multiple Myeloma Presenting First as Bilateral Chronic Uveitis.

PURPOSE: To study the biochemical analysis of aqueous humor in a patient with multiple myeloma presenting first as chronic uveitis. METHODS: Observational case report. RESULTS: A 63-year-old healthy woman presented with blurred vision in both eyes for 9 months. Slit-lamp examination showed bilateral conjunctival congestion, corneal oedema, and anterior uveitis. Fundus exam revealed normal optic disc with fine retinal folds in the macula. Serum protein electrophoretogram showed a monoclonal M protein band in the gamma globulin region. The bone marrow biopsy revealed hypercellular marrow with trilineage haematopoiesis and the bone marrow aspirate showed clonal plasma cells >10%, confirming the diagnosis of multiple myeloma. Aqueous fluid showed a differential band in electrophoretic profile of aqueous humor protein that on mass spectrometry analysis was strongly suggestive of immunoglobulin band. CONCLUSION: The biochemical analysis of aqueous humor is another diagnostic test to monitor M protein in patients with multiple myeloma.

Three-dimensional spheroids of choroid-retinal vascular endothelial cells as an in-vitro model for diabetic retinopathy: Proof-of-concept investigation.

Diabetic retinopathy (DR) is a primary microvascular complication of diabetes mellitus and a vision-threatening condition. Vascular endothelial growth factor (VEGF) induces neovascularization and causes metabolic damage to the retinal and choroidal vasculature in diabetic patients. Existing drug screening models and treatment strategies for DR need to be refined through the establishment of relevant pre-clinical models, which may enable development of effective and safe therapies. The present study discusses the development of an in-vitro three-dimensional (3D) spheroid model, using RF/6A choroid-retinal vascular endothelial cells, to closely mimic the in-vivo disease condition. Compact, reproducibly-sized, viable and proliferating RF/6A spheroids were fabricated, as confirmed by microscopy, live/dead assay, cell proliferation assay and histological staining. In-vitro angiogenesis was studied by evaluating individual effects of VEGF and an anti-VEGF monoclonal antibody, Bevacizumab, and their combination on cellular proliferation and 3D endothelial sprout formation. VEGF stimulated angiogenic sprouting while Bevacizumab demonstrated a dose-dependent anti-angiogenic effect, as determined from the cellular proliferation observed and extent and length of sprouting. These investigations validated the potential of RF/6A spheroids in providing an alternative-to-animal, pathophysiologically-relevant model to facilitate pre-clinical and biomedical research related to DR.

RETRACTED: Young adults with myopia have lower concentrations of neuromodulators-dopamine and melatonin in serum and tear.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors, as questions have arisen in the interim regarding the validity of some of the methods and therefore the results. The authors have provided the following statement: "We have addressed a question raised by the reader on the higher levels of dopamine and melatonin in serum and tear levels reported in this study in Myopia along with controls. Serum and tear levels of dopamine and melatonin in Myopia cases were estimated by HPLC (UV detector) is reported by us in a higher order of magnitude (ng/mL) in both specimens compared to most of the reported data (pg/mL) that are reported based on LC/MS/MS or kit based immunoassay(pg/mL). The sample extraction protocol that we followed before estimation of the analytes was based on removing protein by precipitation methods with no further purification by liquid/liquid extraction/use of SPE cartridges as reported in other published reports. This could have possibly extracted related compounds, such as the closely related indoles in the case of melatonin extraction; the other Dopamine related metabolites respectively. The possibility of confounding by other metabolites in our estimation has not been ruled out by any targeted analysis. This we agree is a limitation in the specificity of the protocol we have followed that may have contributed to the nmol range of detection. We have done the HPLC method of analysis and detection (UV) and not LC-MS/MS or immunoassays by kit method whose sensitivity and specificity are reportedly higher based on the range of testing reported in serum. The lower detection limit of our protocol was not in pg/mL but in ng/mL. Parity in the levels reported was discussed in the manuscript attributing to the analytical method adopted and interpretation based on relative changes. Amongst ocular fluids, Aqueous humor by similar HPLC analysis reportedly shows up to 200 ng/ml in literature (Alkozi, H. et al 2017). However, based on our serum data, we feel that further validation studies and method comparisons are required. The method of estimation of the neurotransmitters HPLC (UV detection) seems to have influenced the absolute levels of dopamine and melatonin in the cases and controls studied and therefore casts doubts on the validity of these data. Though further interpretations can be made on relative changes, we decide to withdraw our paper, to work further on the method comparison. We sincerely apologize for the inconvenience to the readers."

Elevated serum OxLDL is associated with progression of type 2 Diabetes Mellitus to diabetic retinopathy.

Hyperlipidemia is associated with the progression of diabetic retinopathy (DR). Paraoxonase 1 (PON1), an esterase is known to prevent systemic LDL oxidation. This study assessed if serum oxLDL is associated with the progression of Type 2 DM to DR. This study is part of a three-year hospital based prospective study where 87 subjects were recruited. This included T2DM without DR (n = 22); Non-Proliferative (NPDR) (n = 21) and Proliferative DR (PDR) (n = 22) along with age/sex matched controls (n = 22). Serum oxLDL-Ab was estimated by ELISA. Serum PON esterase activity and plasma Malondialdehyde (MDA) level were estimated by spectrophotometry and the serum Advanced Glycation End products (AGE) level by spectroflourimetry. The systemic levels of oxLDL, AGE and MDA were increased with the progression of T2DM without DR to DR as seen by ANOVA (P < 0.05). Serum oxLDL-Ab levels showed a positive correlation to total cholesterol (P = 0.04) as evaluated in the DR group. Statin intake was found to lower PON esterase activity (P < 0.05). Based on this pilot study, it is proposed that elevated serum oxLDL should be validated in larger cohort studies to ensure it could be potential risk factor for the progression of T2DM to DR.

Isolation of acid from eye drop bottles being used by patients presenting with presumed scleritis.

Purpose: The aim of the study was to report the occurrence of contamination/replacement of ophthalmic eye drops with liquids of acidic nature in patients treated for nonresponding scleritis. Methods: This was a retrospective interventional case series study. Results: Of the three patients (4 eyes) referred as necrotizing scleritis, two were found to have acid as the content in the bottle/s being used as eye drops, confirmed using biochemical tests. All four eyes had tarsal ischemia and tarsal conjunctival defect in addition to severe scleral ischemia involving the inferior bulbar area. All four eyes required tenonplasty with amniotic membrane transplant more than once for the ocular surface to heal. Two of the three patients were on systemic immunosuppressives including pulse cyclophosphamide for refractory necrotizing scleritis. Sulfuric and hydrochloric acid was isolated from the bottles of 2nd and 3rd patient using confirmatory biochemical tests. Conclusion: It is important to be aware of the possibility of contaminating or replacing contents of eye drops with harmful agents of acidic nature and should be considered in situations that resemble the clinical picture described herein.

Probing the intermolecular interactions of PPARγ-LBD with polyunsaturated fatty acids and their anti-inflammatory metabolites to infer most potential binding moieties.

BACKGROUND: PPARγ is an isoform of peroxisome proliferator-activated receptor (PPAR) belonging to a super family of nuclear receptors. PPARγ receptor is found to play a crucial role in the modulation of lipid and glucose homeostasis. Its commotion has been reported to play a significant role in a broad spectrum of diseases such as type 2 diabetes mellitus, inflammatory diseases, Alzheimer's disease, and in some cancers. Hence, PPARγ is an important therapeutic target. Polyunsaturated fatty acids (PUFAs) and their metabolites (henceforth referred to as bioactive lipids) are known to function as agonists of PPARγ. However, agonistic binding modes and affinity of these ligands to PPARγ are yet to be deciphered. METHODS: In this study, we performed a comparative molecular docking, binding free energy calculation and molecular dynamics simulation to infer and rank bioactive lipids based on the binding affinities with the ligand binding domain (LBD) of PPARγ. RESULTS: The results inferred affinity in the order of resolvin E1 > neuroprotectin D1 > hydroxy-linoleic acid > docosahexaenoic acid > lipoxin A4 > gamma-linolenic acid, arachidonic acid > alpha-linolenic acid > eicosapentaenoic acid > linoleic acid. Of all the bioactive lipids studied, resolvin E1, neuroprotectin D1 and hydroxy-linoleic acid showed significant affinity comparable to proven PPARγ agonist namely, rosiglitazone, in terms of Glide XP docking score, H-bond formation with the key residues, binding free energy and stable complex formation with LBD favouring co-activator binding, as inferred through Molecular Dynamics trajectory analysis. CONCLUSION: Hence, these three bioactive lipids (resolvin E1, neuroprotectin D1 and hydroxy-linoleic acid) may be favourably considered as ideal drug candidates in therapeutic modulation of clinical conditions such as type 2 DM, Alzheimer's disease and other instances where PPARγ is a key player.

Correlation of Aqueous Humor Lysyl Oxidase Activity with TGF-ß Levels and LOXL1 Genotype in Pseudoexfoliation.

PURPOSE: Pseudoexfoliation (PXF) is a microfibrillopathy involving disordered elastogenesis. Abnormal extracellular matrix (ECM) production underlies the pathophysiology of PXF. The enzyme Lysyl oxidase (LOX) and its isoforms are known to cross-link the elastin and collagen. Though the etiopathogensis of PXF is not well understood, studies report on the genetic risk involving LOXL1 gene. This study aims to screen LOXL1 coding variants rs1048661 and rs3825942 in the South Indian population and the implication of the single nucleotide polymorphism (SNP) with LOX activity. The levels of transforming growth factor ß (TGF-ß) in aqueous humor and its correlation with the LOX activity were also examined. METHODS: Blood, plasma, and aqueous aspirates were prospectively collected from PXF cases with and without glaucoma and cataract cases as controls. DNA was extracted from 48 PXF cases without glaucoma, 12 PXF cases with glaucoma, and 40 age-matched cataract-alone controls without PXF/glaucoma for analyzing LOX SNPs. LOX activity was measured in aqueous humor and plasma of 30 PXF cases without glaucoma, 24 age-matched cataract-alone controls without PXF/glaucoma, and 14 PXF cases with glaucoma. Protein levels of LOX, LOXL1, LOXL2, and total TGF-ß were estimated in plasma and aqueous humor by ELISA. RESULTS: The specific activity of LOX in aqueous humor was found to be significantly lowered in PXF cases compared with cataract-alone controls (p = 0.014). This decrease in LOX activity in PXF cases was associated with high-risk GG haplotype. However, this was not statistically significant and a larger sample size is warranted. TGF-ß1 and TGF-ß2 negatively correlated with LOX activity in aqueous humor (p = 0.028; p = 0.046, respectively). CONCLUSIONS: The LOXL1 SNPs, rs1048661 and rs3825942, are associated with PXF in the South Indian population correlating with lowered LOX activity in the aqueous humor. The increased level of total TGF-ß in the aqueous humor of PXF cases is possibly associated with LOX regulation which needs further investigation.

Development and validation of a LC-MS/MS method for homocysteine thiolactone in plasma and evaluation of its stability in plasma samples.

The present study demonstrates the development and validation of a sensitive method for the quantification of homocysteine thiolactone (HCTL) in human plasma using the technique of LC-MS/MS. The gradient elution of HCTL was achieved within 5min using ZIC HILIC column having acetonitrile with 0.1% formic acid and water with 0.1% formic acid. The method was validated for the linearity, sensitivity, accuracy, precision, recovery, matrix effect and stability. A good linearity was found within a range of 0.5-32.5nmol/ml. Quantification was performed using multiple reaction monitoring (MRM) mode based on the molecular/fragment ion transitions for HCTL (118/56) and homatropine (276.1/142.2) as internal standard. Generally, HCTL levels in plasma were found to be highly unstable. In order to verify the stability of the HCTL levels in plasma for a longer period, the samples were extracted immediately and stored at -86°C. Using the above method it was found to be stable for a period of 1 month. The method was well applied for quantification of HCTL in plasma of healthy human volunteers.

Increased homocysteine, homocysteine-thiolactone, protein homocysteinylation and oxidative stress in the circulation of patients with Eales' disease.

BACKGROUND: Eales' disease (ED) is an idiopathic retinal vascular disorder. It presents with inflammation and neovascularization in the retina. Adult men, aged between 15 and 40 years are more susceptible than women. Homocysteine has been implicated in other ocular diseases including age-related macular degeneration (ARMD), central retinal vein occlusion (CRVO) and optic neuropathy. The present study investigates the role of homocysteine in ED. METHODS: Forty male subjects, 20 with ED and 20 healthy controls, were recruited to the study. Their blood samples were used to measure thiobarbituric acid reactive substances (TBARS), glutathione (GSH), homocysteine, homocysteine-thiolactone, extent of homocysteine conjugation with proteins and plasma copper concentration. RESULTS: In the ED group, plasma homocysteine (18.6 ± 1.77 µmol/L, P < 0.001) and homocysteine-thiolactone (45.3 ± 6.8 nmol/L, P < 0.0001) concentrations were significantly higher compared to homocysteine (11.2 ± 0.64 µmol/L) and homocysteine-thiolactone (7.1 ± 0.94 nmol/L) concentrations in control subjects. TBARS (P < 0.011) and protein homocysteinylation (P < 0.030) were higher in the ED group while GSH (5.9 ± 0.44 µmol/L, P < 0.01) and copper (6.6 ± 0.42 µmol/L, P < 0.001) were lower compared to GSH (8.1 ± 0.41 µmol/L) and copper (15.4 ± 0.73 µmol/L) concentrations in control subjects. CONCLUSIONS: Increased homocysteine, and its metabolite thiolactone, is associated with the functional impairment of protein due to homocysteinylation in ED.

Lacrimal proline rich 4 (LPRR4) protein in the tear fluid is a potential biomarker of dry eye syndrome.

Dry eye syndrome (DES) is a complex, multifactorial, immune-associated disorder of the tear and ocular surface. DES with a high prevalence world over needs identification of potential biomarkers so as to understand not only the disease mechanism but also to identify drug targets. In this study we looked for differentially expressed proteins in tear samples of DES to arrive at characteristic biomarkers. As part of a prospective case-control study, tear specimen were collected using Schirmer strips from 129 dry eye cases and 73 age matched controls. 2D electrophoresis (2DE) and Differential gel electrophoresis (DIGE) was done to identify differentially expressed proteins. One of the differentially expressed protein in DES is lacrimal proline rich 4 protein (LPRR4). LPRR4 protein expression was quantified by enzyme immune sorbent assay (ELISA). LPRR4 was down regulated significantly in all types of dry eye cases, correlating with the disease severity as measured by clinical investigations. Further characterization of the protein is required to assess its therapeutic potential in DES.

Agonistic effect of polyunsaturated fatty acids (PUFAs) and its metabolites on brain-derived neurotrophic factor (BDNF) through molecular docking simulation.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is a potent neurotrophic factor that is implicated in the regulation of food intake and body weight. Polyunsaturated fatty acids (PUFAs) localised in cell membranes have been shown to alter the levels of BDNF in the brain, suggesting that PUFAs and BDNF could have physical interaction with each other. To decipher the molecular mechanism through which PUFAs modulates BDNF's activity, molecular docking was performed for BDNF with PUFAs and its metabolites, with 4-Methyl Catechol as a control. RESULTS: Inferring from molecular docking studies, lipoxin A4 (LXA4), and a known anti-inflammatory bioactive metabolite derived from PUFAs, with a binding energy of -3.98 Kcal/mol and dissociation constant of 1.2 mM showed highest binding affinity for BDNF in comparison to other PUFAs and metabolites considered in the study. Further, the residues Lys 18, Thr 20, Ala 21, Val 22, Phe 46, Glu 48, Lys 50, Lys 58, Thr 75, Gln 77, Arg 97 and Ile 98 form hot point motif, which on interaction enhances BDNF's function. CONCLUSION: These results suggest that PUFAs and their metabolites especially, LXA4, modulate insulin resistance by establishing a physical interaction with BDNF. Similar interaction(s) was noted between BDNF and resolvins and protectins but were of lesser intensity compared to LXA4.

Intra-ocular expression of vascular endothelial growth factor (VEGF) and pigment epithelial-derived factor (PEDF) in a case of Eales' disease by immunohistochemical analysis: a case report.

We report a immunohistochemical study of expression of VEGF and PEDF in Eales' disease. An enucleated eye from a patient with Eales' disease and an age and sex-matched donor eyeball were subjected to immunohistochemical and histopathological analysis to study the expression of VEGF and PEDF in the retinal region. Strong positive anti-VEGF immunostaining was found around the vessel walls in the retina in the eyeball from the Eales' disease patient compared with the donor control, from which it was almost absent. PEDF staining was detected in the normal donor eyeball retinal layers surrounding the blood vessels, and the photoreceptor and ganglion cell layer, whereas in the eyeball from Eales' disease patient PEDF staining was restricted to the surrounding blood vessels. The strong expression of VEGF in eyes with Eales' disease as opposed to weak expression of PEDF indicates the propensity of the disease to show neovascularization and recurrent hemorrhages.

Tear ascorbic acid levels and the total antioxidant status in contact lens wearers: a pilot study.

AIMS: The tear ascorbate owing to its high concentration, functions as an effective antioxidant against the oxidative damage of cornea. Contact lens wearers (CLW) are prone to oxidative stress due to the lens-induced hypoxic conditions. A pilot study was done to compare the tear ascorbic acid level and the total antioxidant capacity give as in normal and CLW. MATERIALS AND METHODS: In this study 21 CLW (Mean age 23 +/- 3 years ; M-2, F-19), who were daily wear users, with duration of wear not more than four years, along with age-matched 28 controls (Mean age 28 +/- 3 ; M-15, F-13) were recruited in the study for collection of reflex tears using Schirmer's strip. Ascorbic acid in tears was determined using high-performance liquid chromatography (HPLC), total antioxidant capacity (TAC) and total protein assay by spectrophotometric analysis. RESULTS: CLW showed no significant change in the tear ascorbic acid levels (0.4 +/- 0.26 mM) compared to the control subjects (0.61 +/- 0.59 mM). The amount of ascorbic acid in tears did not correlate with the TAC or the total protein of the tears. The mean TAC in CLW was 0.69 +/- 0.16 mM, with a total protein of 1.35 +/- 0.46 mg/ml while in controls it was 0.7 +/- 0.18 mM and 1.21 +/- 0.47 mg/ml respectively. CONCLUSIONS: Soft contact lens wear did not show any significant change in tear ascorbic acid, TAC and total protein levels compared to controls.

Hyperhomocysteinemia and low methionine stress are risk factors for central retinal venous occlusion in an Indian population.

PURPOSE: The underlying cause of disturbed homocysteine metabolism is incompletely understood in young persons with central retinal vein occlusion (CRVO) with mild hyperhomocysteinemia (HHcys) and no other systemic disease in India. A 2-year prospective study was undertaken to determine whether HHcys is a risk factor for CRVO in an Indian population. METHOD: The prevalence of fasting HHcys was evaluated in a consecutive series of 29 patients with CRVO (mean age, 30 +/- 6 years) along with 57 age- and sex-matched control subjects (healthy subjects, mean age 27 +/- 5 years). Strict inclusion and exclusion criteria were used. Plasma levels of homocysteine (Hcys), methionine, cysteine, glutathione, B(12), and folate were measured. Multivariate logistic regression analysis was performed to determine the risk factors for CRVO. RESULT: Fifteen of 29 patients with CRVO (51.72%) exhibited HHcys (>15 muM). The mean Hcys level was significantly elevated in the patients with CRVO (19.1 +/- 13.1 muM) compared with that in the healthy control subjects (14.7 +/- 6.2 muM) with P = 0.04. The increased Hcys levels in CRVO cases was associated with decreased methionine (P = 0.052) and decreased B(12) (P = 0.001). A multivariate logistic regression analysis revealed an odds ratio of 1.9 (95% CI = 0.50-7.16) for Hcys and 15.9 for methionine (95%CI = 1.50-169.62; P = 0.022). CONCLUSION: Elevated Hcys and low methionine were risk factors for CRVO in an Indian population.

Glycation and glycoxidation studies in vitro on isolated human vitreous collagen.

BACKGROUND: Structural and functional impairment in vitreous collagen plays an important role in the pathogenesis of diabetic retinopathy. Collagen being a long-lived protein is prone to both glycation and glycoxidation, resulting in accumulation of advanced glycation end products (AGE). The objective of our study was to explore the extent of glycation by glucose, and iron- and copper-mediated glycoxidation of human vitreous collagen, and also to study the beneficial effects of lysine, inositol and aminoguanidine as antiglycating and anti-cross linking agents. MATERIAL/METHODS: Vitreous from human donor eyeballs was pooled and collagen was extracted using 0.9 M NaCl. Collagen was estimated by measuring the hydroxyproline content. The extracted collagen was used for glycation and glycoxidation studies. Glycation studies were conducted using U14C glucose, along with anti-glycating agents, such as lysine and aminoguanidine. Metal-mediated glycoxidation studies were done by measuring collagen content in cyanogen bromide insoluble fraction, in the presence and absence of an anti-cross linking agent, inositol. RESULTS: Human vitreous collagen extractable with 0.9 M NaCl was glycated by glucose at 5 and 10 mM concentrations under physiological conditions of temperature and pH. An anti-glycating effect was exhibited by lysine, inositol and aminoguanidine, of which lysine was the best (76% antiglycating activity) followed by inositol. Inositol was also found to be useful in inhibiting glycoxidation. CONCLUSIONS: Vitreous collagen undergoes glycation, as well as iron- and copper-mediated glycoxidation, leading to possible structural and functional impairment. Glycation and glycoxidation are inhibited, significantly by lysine and inositol respectively.