Diabetes, Diabetic Retinopathy, and Inflammatory Disorders.

This review summarizes the impact of systemic and ocular inflammatory disorders on diabetes mellitus (DM) and diabetic retinopathy (DR). Local inflammation is a key pathology in diabetic retinopathy (DR) and is also an evolving target for clinical therapy. The legacy effects of local inflammation at the intracellular level make DR a persistent self-driven vicious process. Ocular inflammation is accompanied as well as incited by systemic inflammation due to diabetes mellitus (DM) itself. Over the years, a multitude of studies have evaluated the impact of systemic inflammatory disorders (SIDs, like rheumatoid arthritis, lupus, psoriasis, etc.) and anti-inflammatory drugs prescribed for managing them on manifestations of DM. Recent studies have indicated increased insulin resistance to be a result of chronic inflammation, and the anti-inflammatory drugs to have a protective effect towards DM. Very few studies have evaluated the impact of SIDs on DR. Furthermore, the evidence from these studies is conflicting, and while local anti-inflammatory therapy has shown a lot of clinical potential for use in DR, the results of systemic anti-inflammatory therapies have been inconsistent. The impact of local ocular inflammation due to uveitis on DR is a crucial aspect that has not been evaluated well at present. Initial pre-clinical studies and small-sized clinical reports have shown a strong and positive relationship between the presence of uveitis and the severity of DR as well as its progression, while larger cross-sectional patient surveys have refuted the same. The long term impact of ocular inflammation due to uveitis on DR needs to be studied while adjusting for confounders.

Optical Coherence Tomography Features of Ocular Cysticercosis: A Review of Literature With Observer Variation.

Ocular cysticercosis is a sparsely reported condition, requiring urgent management. The gold standard for diagnosis is an in toto extraction of the cyst with subsequent histopathology. The procedure can be demanding in contrast to the frequently adopted practice of in vivo cyst lysis. The latter, however, obviates a conventional biopsy. We reviewed published optical coherence tomography (OCT) images of ocular cysticercosis for their suitability to surrogate a conventional biopsy and identified commonly reported features. We also used triple masking and ascertained the observer agreement on identification of these features. We found that the features of the parasite are much more clearly discernible as compared with features of the involved ocular tissue itself. The hyperreflective cyst wall and scolex and the hyporeflective cyst cavity had the highest frequency and observer agreement among all the analyzed features, suggesting their use for diagnosis. We could match many of the OCT features with the previously reported histopathological findings, supporting the role of OCT as a diagnostic adjunct and a substitute for conventional biopsy. Conversely, features of the ocular tissue could be judged poorly with low observer agreement, suggesting poor prognostic ability of OCT. [Ophthalmic Surg Lasers Imaging Retina 2022;53:446-454.].

Evidence Suggesting the Role of Gut Dysbiosis in Diabetic Retinopathy.

Purpose: Gut dysbiosis has been identified and tested in human trials for its role in diabetes mellitus (DM). The gut-retina axis could be a potential target for retardation of diabetic retinopathy (DR), a known complication of DM. This study reviews the evidence suggesting gut dysbiosis in DR. Methods: The published literature in the past 5 years was reviewed using predetermined keywords and articles. The review intended to determine changes in gut microbiome in DR, the hypothesized mechanisms linking to the gut-retina axis, its predictive potential for progression of DR, and the possible therapeutic targets. Results: The gut microbiota of people with DM differ from those without it, and the gut microbiota of people with DR differ from those without it. The difference is more significant in the former (DM versus no DM) and less significant in the latter (DM without DR versus DM with DR). Early research has suggested mechanisms of the gut-retina axis, but these are not different from known changes in the gut microbiome of people with DM. The current evidence on the predictive value of the gut microbiome in the occurrence and progression of DR is low. Therapeutic avenues targeting the gut-retina axis include lifestyle changes, pharmacologic inhibitors, probiotics, and fecal microbiota transplantation. Conclusions: Investigating the therapeutic utility of the gut ecosystem for DM and its complications like DR is an emerging area of research. The gut-retina axis could be a target for retardation of DR but needs longitudinal regional studies adjusting for dietary habits.

Retinitis pigmentosa in Laurence-Moon-Bardet-Biedl syndrome in India: Electronic medical records driven big data analytics: Report II.

Purpose: To describe the clinical presentation and demographic distribution of retinitis pigmentosa (RP) in Laurence-Moon-Bardet-Biedl (LMBB) syndrome patients. Methods: This is a cross-sectional observational hospital-based study wherein 244 patients with RP in LMBB syndrome presenting to our hospital network between March 2012 and October 2020 were included. An electronic medical record database was used for data retrieval. Results: There were 244 patients in total, with a hospital-based prevalence rate of 0.010% or 1000/100,000 population. The mean and median age of patients was 15.22 ± 7.56 and 14 (IQR: 10-18.5) years, respectively, with the majority being in the age group of 11-20 years (133/244 patients; 54.50%). Males were more commonly affected (164 patients; 67.21%), and the majority (182 patients; 74.59%) were students. All 244 patients (100%) complained of defective central vision at presentation. More than one-fourth of the patients had severe visual impairment to blindness at presentation. Prominent retinal feature at presentation was diffuse or widespread retinal pigment epithelial degeneration in all patients. Conclusion: Patients with RP in LMBB syndrome present mainly in the first to second decade of life with severe visual acuity impairment to blindness early in life. It is important to rule out LMBB syndrome in early-onset RP with central visual acuity impairment. On the contrary, all patients diagnosed or suspected with LMBB syndrome systemic features at physician clinic should also be referred for ophthalmic evaluation, low vision assessment, rehabilitation, and vice versa.

Scleral Fixation of Intraocular Lens in Aphakic Eyes without Capsular Support: Description of a New Technique.

PURPOSE: To study the efficacy and safety of a modified trans-scleral intraocular lens (IOL) fixation technique in aphakic eyes when performed by ophthalmologists in training. METHODS: The study was conducted in an institutional setting that included 43 surgeries performed by surgeons training in small incision cataract surgeries. The data were analyzed for stability and position of IOL, refractive changes, best-corrected vision, and associated complications. RESULTS: Mean age of the subjects was 53.8 ± 18.5yrs (range 6-81yrs). Surgical aphakia (58.14%) was the most common cause. The corrected distance visual acuity improved significantly at six weeks (p = 0.0003). The mean residual spectacle correction was +0.74 ± 1.2D spherical equivalent (cylinder -1.6±1.5D at 84 ± 50°) at the 6th-month follow-up (24.35 ± 6.71wks). Lens tilt on ultrasound biomicroscopy (kappa 0.762; p < 0.001) and the IOL centration (kappa 0.411; p = 0.001), assessed by two independent masked observers, were satisfactory at the 6th-month visit. Transient postoperative vitreous hemorrhage was the most common complication (46.5%). Cellular deposits on the IOL surface (18.6%), cystoid macular edema (11.6%), subconjunctival haptic exposure (4.66%), and haptic slippage (2.33%) were the other complications. CONCLUSION: This method of trans-scleral IOL fixation is an effective rescue procedure for eyes with deficient capsular support when ophthalmologists perform in training.

Management of a misidentified and misnumbered intraocular glass foreign body with an endoscope in a case of endophthalmitis.

An endoscope is a useful adjunct for the retinal surgeon to overcome haze of a compromised anterior segment. It allows early surgery in trauma and infections which translates to better results. Intraocular glass foreign body is a challenging condition, demanding highly skilled surgical expertise. We present endoscopic removal of an intraocular foreign glass body in a badly traumatised and infected eye. The surgical challenge was accentuated by an imaging misdiagnosis of 'twin metallic foreign bodies'.

Phytochemicals perturb membranes and promiscuously alter protein function.

A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding.

Bidirectional coupling between ryanodine receptors and Ca2+ release-activated Ca2+ (CRAC) channel machinery sustains store-operated Ca2+ entry in human T lymphocytes.

The expression and functional significance of ryanodine receptors (RyR) were investigated in resting and activated primary human T cells. RyR1, RyR2, and RyR3 transcripts were detected in human T cells. RyR1/2 transcript levels increased, whereas those of RyR3 decreased after T cell activation. RyR1/2 protein immunoreactivity was detected in activated but not in resting T cells. The RyR agonist caffeine evoked Ca(2+) release from the intracellular store in activated T cells but not in resting T cells, indicating that RyR are functionally up-regulated in activated T cells compared with resting T cells. In the presence of store-operated Ca(2+) entry (SOCE) via plasmalemmal Ca(2+) release-activated Ca(2+) (CRAC) channels, RyR blockers reduced the Ca(2+) leak from the endoplasmic reticulum (ER) and the magnitude of SOCE, suggesting that a positive feedback relationship exists between RyR and CRAC channels. Overexpression of fluorescently tagged RyR2 and stromal interaction molecule 1 (STIM1), an ER Ca(2+) sensor gating CRAC channels, in HEK293 cells revealed that RyR are co-localized with STIM1 in the puncta formed after store depletion. These data indicate that in primary human T cells, the RyR are coupled to CRAC channel machinery such that SOCE activates RyR via a Ca(2+)-induced Ca(2+) release mechanism, which in turn reduces the Ca(2+) concentration within the ER lumen in the vicinity of STIM1, thus facilitating SOCE by reducing store-dependent CRAC channel inactivation. Treatment with RyR blockers suppressed activated T cell expansion, demonstrating the functional importance of RyR in T cells.

Density of functional Ca2+ release-activated Ca2+ (CRAC) channels declines after T-cell activation.

CRAC channel-mediated Ca(2+) entry plays a crucial role in T lymphocyte activation. Activated T cells display enhanced Ca(2+) signaling compared with resting T cells; this is partially attributed to activation-induced upregulation of CRAC channel expression. Orai and Stim family genes encode CRAC channel structural elements and regulatory proteins, respectively, but studies of their expression in T cells have led to controversial results. We re-examined Orai and Stim gene expression in resting, activated, and Jurkat T cells. Levels of Orai1 transcripts, encoding the human T cell CRAC channel subunit, were not significantly different between resting T and activated T cells. The total amount of all Orai transcripts was 2-fold higher in activated T cells than in resting T cells. Orai1 and total Orai transcript levels were significantly higher in Jurkat T cells than those in resting T cells. Stim expression did not vary significantly among cell types. Maximal whole-cell CRAC current amplitudes were 1.4-fold and 2.3-fold higher in activated and Jurkat T cells, respectively, than in resting T cells. Due to the small size of resting T cells, the surface CRAC channel density was 2.5-fold and 1.6-fold higher in resting T cells than in activated and Jurkat T cells, respectively. Predicted the rates of cytosolic Ca(2+) elevation calculated using the average values of CRAC channel currents and cell volumes showed that < 2-fold increase in the functional CRAC channel expression level cannot account for the enhanced rate of store-operated Ca(2+) entry in activated T cells compared with resting T cells.

SV2 acts via presynaptic calcium to regulate neurotransmitter release.

Synaptic vesicle 2 (SV2) proteins, critical for proper nervous system function, are implicated in human epilepsy, yet little is known about their function. We demonstrate, using direct approaches, that loss of the major SV2 isoform in a central nervous system nerve terminal is associated with an elevation in both resting and evoked presynaptic Ca(2+) signals. This increase is essential for the expression of the SV2B(-/-) secretory phenotype, characterized by changes in synaptic vesicle dynamics, synaptic plasticity, and synaptic strength. Short-term reproduction of the Ca(2+) phenotype in wild-type nerve terminals reproduces almost all aspects of the SV2B(-/-) secretory phenotype, while rescue of the Ca(2+) phenotype in SV2B(-/-) neurons relieves every facet of the SV2B(-/-) secretory phenotype. Thus, SV2 controls key aspects of synaptic functionality via its ability to regulate presynaptic Ca(2+), suggesting a potential new target for therapeutic intervention in the treatment of epilepsy.

Whole-cell recording of calcium release-activated calcium (CRAC) currents in human T lymphocytes.

In T lymphocytes, depletion of Ca(2+) from the intracellular Ca(2+) store leads to activation of plasmalemmal Ca(2+) channels, called Calcium Release-Activated Calcium (CRAC) channels. CRAC channels play important role in regulation of T cell proliferation and gene expression. Abnormal CRAC channel function in T cells has been linked to severe combined immunodeficiency and autoimmune diseases. Studying CRAC channel function in human T cells may uncover new molecular mechanisms regulating normal immune responses and unravel the causes of related human diseases. Electrophysiological recordings of membrane currents provide the most accurate assessment of functional channel properties and their regulation. Electrophysiological assessment of CRAC channel currents in Jurkat T cells, a human leukemia T cell line, was first performed more than 20 years ago, however, CRAC current measurements in normal human T cells remains a challenging task. The difficulties in recording CRAC channel currents in normal T cells are compounded by the fact that blood-derived T lymphocytes are much smaller in size than Jurkat T cells and, therefore, the endogenous whole-cell CRAC currents are very low in amplitude. Here, we give a step-by-step procedure that we routinely use to record the Ca(2+) or Na(+) currents via CRAC channels in resting human T cells isolated from the peripheral blood of healthy volunteers. The method described here was adopted from the procedures used for recording the CRAC currents in Jurkat T cells and activated human T cells.

Synaptotagmin-2 controls regulated exocytosis but not other secretory responses of mast cells.

Mast cell degranulation is a highly regulated, calcium-dependent process, which is important for the acute release of inflammatory mediators during the course of many pathological conditions. We previously found that Synaptotagmin-2, a calcium sensor in neuronal exocytosis, was expressed in a mast cell line. We postulated that this protein may be involved in the control of mast cell-regulated exocytosis, and we generated Synaptotagmin-2 knock-out mice to test our hypothesis. Mast cells from this mutant animal conferred an abnormally decreased passive cutaneous anaphylaxis reaction on mast cell-deficient mice that correlated with a specific defect in mast cell-regulated exocytosis, leaving constitutive exocytosis and nonexocytic mast cell effector responses intact. This defect was not secondary to abnormalities in the development, maturation, migration, morphology, synthesis, and storage of inflammatory mediators, or intracellular calcium transients of the mast cells. Unlike neurons, the lack of Synaptotagmin-2 in mast cells was not associated with increased spontaneous exocytosis.

Capacitance measurements in the mouse rod bipolar cell identify a pool of releasable synaptic vesicles.

The mouse is an important model system for understanding the molecular basis of neuronal signaling and diseases of synaptic communication. However, the best-characterized retinal ribbon-style synapses are those of nonmammalian vertebrates. To remedy this situation, we asked whether it would be feasible to track synaptic vesicle dynamics in the isolated mouse rod bipolar cell using time-resolved capacitance measurements. The results demonstrate that membrane depolarization triggered an increase in membrane capacitance that was Ca(2+) dependent and restricted to the synaptic compartment, consistent with exocytosis. The amplitude of the capacitance response recorded from the easily accessible soma of an intact mouse rod bipolar cell was identical to that recorded directly from the small synaptic terminal, suggesting that in the carefully selected cohort of cells presented here, axonal resistance was not a significant barrier to current flow. This supposition was supported by the analysis of passive membrane properties and a comparison of membrane capacitance measurements in cells with and without synaptic terminals and reinforced by the lack of an effect of sine-wave frequency (200-1,600 Hz) on the measured capacitance increase. The magnitude of the capacitance response increased with Ca(2+) entry until a plateau was reached at a spatially averaged intraterminal calcium of about 600 nM. We interpret this plateau, nominally 30 fF, as corresponding to a releasable pool of synaptic vesicles. The robustness of this measure suggests that capacitance measurements may be used in the mouse rod bipolar cell to compare pool size across treatment conditions.

SV2B regulates synaptotagmin 1 by direct interaction.

SV2 proteins are abundant synaptic vesicle proteins expressed in two major (SV2A and SV2B) and one minor (SV2C) isoform. SV2A and SV2B have been shown to be involved in the regulation of synaptic vesicle exocytosis. Previous studies found that SV2A, but not SV2B, can interact with the cytoplasmic domain of synaptotagmin 1, a Ca2+ sensor for synaptic vesicle exocytosis. To determine whether SV2B can interact with full-length synaptotagmin 1, we performed immunoprecipitations from brain protein extracts and found that SV2B interacts strongly with synaptotagmin 1 in a detergent-resistant, Ca2+ -independent manner. In contrast, an interaction between native SV2A and synaptotagmin 1 was not detectable under these conditions. The SV2B-synaptotagmin 1 complex also contained the synaptic t-SNARE proteins, syntaxin 1 and SNAP-25, suggesting that SV2B may participate in exocytosis by modulating the interaction of synaptotagmin 1 with t-SNARE proteins. Analysis of retinae in SV2B knock-out mice revealed a strong reduction in the level of synaptotagmin 1 in rod photoreceptor synapses, which are unique in that they express only the SV2B isoform. In contrast, other synaptic vesicle proteins were not affected by SV2B knock out, indicating a specific role for SV2B in the regulation of synaptotagmin 1 levels at certain synapses. These experiments suggest that the SV2B-synaptotagmin 1 complex is involved in the regulation of synaptotagmin 1 stability and/or trafficking. This study has demonstrated a new role of SV2B as a regulator of synaptotagmin 1 that is likely mediated by direct interaction of these two synaptic proteins.

Effects of PKA-mediated phosphorylation of Snapin on synaptic transmission in cultured hippocampal neurons.

Use-dependent activation of protein kinase A (PKA) modulates transmitter release, contributing to synaptic plasticity. Snapin, a PKA substrate in neurons, associates with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, and its phosphorylation leads to increased binding of synaptotagmin to the SNARE complex. We investigated the role of PKA-dependent phosphorylation of Snapin in hippocampal neurons. Overexpression of Snapin S50D, a mutant mimicking the phosphorylated state, resulted in a decreased number of readily releasable vesicles. In addition, both the release probability of individual vesicles and the depression rate during high-frequency stimulation were increased. Overexpression of Snapin S50A, a mutant that cannot be phosphorylated, did not alter the size of the pool or the probability of release. Furthermore, dialysis of Sp-cAMPS, a nonhydrolyzable analog of cAMP that will promote phosphorylation by PKA, also led to increased synaptic depression in cells overexpressing wild-type Snapin. These results establish Snapin as an important target of PKA in CNS synapses and indicate a role for Snapin in the plasticity of transmitter release.

Preconditioning-induced protection against cyanide-induced neurotoxicity is mediated by preserving mitochondrial function.

The central nervous system is one of the main target organs in cyanide toxicity. In this study, primary cultures of chick embryonic neurons were used to characterize sodium cyanide (NaCN)-induced cell death and to investigate the mechanism of NaCN-mediated preconditioning. After treatment of the cells with 1mM NaCN for 1h followed by a NaCN-free incubation period of 23 h, we observed features of apoptosis such as a reduction in nuclear size, chromatin condensation and nuclear fragmentation as evaluated by nuclear staining with Hoechst 33258 and electron microscopy. In addition, NaCN-induced neurotoxicity was reduced by the protein synthesis inhibitor cycloheximide (CHX) suggesting an active type of cell death. Most of the neurons with condensed chromatin and a shrunken nuclei also showed membrane damage at a late stage. Mitochondrial membrane potential as well as the protein levels of Bcl-2 and Bcl-x(L) decreased 15-60 min and 1-3 h after the exposure to NaCN (1mM, 1h), respectively. Preconditioning caused by incubating chick neurons with 100 microM NaCN for 30 min followed by a NaCN-free interval of 24h significantly protected the neurons against subsequent NaCN (1mM, 1h)-induced damage. Preconditioning prevented NaCN-induced decrease in the mitochondrial membrane potential as well as in the protein levels of Bcl-2 and Bcl-x(L) suggesting that preconditioning-induced neuroprotection is mediated by preserving mitochondrial function.