Stiffness Measurement of Retinal Capillaries and Subendothelial Matrix using Atomic Force Microscopy.

Retinal capillary degeneration is a clinical hallmark of the early stages of diabetic retinopathy (DR). Our recent studies have revealed that diabetes-induced increase in retinal capillary stiffness plays a crucial and previously unrecognized causal role in inflammation-mediated degeneration of retinal capillaries. Retinal capillary stiffening results from overexpression of lysyl oxidase, an enzyme that crosslinks and stiffens the subendothelial matrix. Since tackling DR at the early stage is expected to prevent or slow down DR progression and associated vision loss, subendothelial matrix and capillary stiffness represent relevant and novel therapeutic targets for early DR management. Further, direct measurement of retinal capillary stiffness can serve as a crucial preclinical validation step for the development of new imaging techniques for non-invasive assessment of retinal capillary stiffness in animal and human subjects. With this view in mind, we here provide a detailed protocol for the isolation and stiffness measurement of mouse retinal capillaries and retinal subendothelial matrix using atomic force microscopy.

Mechanical Regulation of Retinal Vascular Inflammation and Degeneration in Diabetes.

Vascular inflammation is known to cause degeneration of retinal capillaries in early diabetic retinopathy (DR), a major microvascular complication of diabetes. Past studies investigating these diabetes-induced retinal vascular abnormalities have focused primarily on the role of molecular or biochemical cues. Here we show that retinal vascular inflammation and degeneration in diabetes are also mechanically regulated by the increase in retinal vascular stiffness caused by overexpression of the collagen-cross-linking enzyme lysyl oxidase (LOX). Treatment of diabetic mice with LOX inhibitor ß-aminopropionitrile (BAPN) prevented the increase in retinal capillary stiffness, vascular intracellular adhesion molecule-1 overexpression, and leukostasis. Consistent with these anti-inflammatory effects, BAPN treatment of diabetic mice blocked the upregulation of proapoptotic caspase-3 in retinal vessels, which concomitantly reduced retinal capillary degeneration, pericyte ghost formation, and the diabetes-induced loss of contrast sensitivity in these mice. Finally, our in vitro studies indicate that retinal capillary stiffening is sufficient to increase the adhesiveness and neutrophil elastase-induced death of retinal endothelial cells. By uncovering a link between LOX-dependent capillary stiffening and the development of retinal vascular and functional defects in diabetes, these findings offer a new insight into DR pathogenesis that has important translational potential.

Subendothelial Matrix Stiffening by Lysyl Oxidase Enhances RAGE-Mediated Retinal Endothelial Activation in Diabetes.

Endothelial cell (EC) activation is a crucial determinant of retinal vascular inflammation associated with diabetic retinopathy (DR), a major microvascular complication of diabetes. We previously showed that, similar to abnormal biochemical factors, aberrant mechanical cues in the form of lysyl oxidase (LOX)-dependent subendothelial matrix stiffening also contribute significantly to retinal EC activation in diabetes. Yet, how LOX is itself regulated and precisely how it mechanically controls retinal EC activation in diabetes is poorly understood. Here, we show that high-glucose-induced LOX upregulation in human retinal ECs (HRECs) is mediated by proinflammatory receptor for advanced glycation end products (RAGE). HRECs treated with methylglyoxal (MGO), an active precursor to the advanced glycation end product (AGE) MG-H1, exhibited LOX upregulation that was blocked by a RAGE inhibitor, thus confirming the ability of RAGE to promote LOX expression. Crucially, as a downstream effector of RAGE, LOX was found to mediate both the proinflammatory and matrix remodeling effects of AGE/RAGE, primarily through its ability to crosslink or stiffen matrix. Finally, using decellularized HREC-derived matrices and a mouse model of diabetes, we demonstrate that LOX-dependent matrix stiffening feeds back to enhance RAGE, thereby achieving its autoregulation and proinflammatory effects. Collectively, these findings provide fresh mechanistic insights into the regulation and proinflammatory role of LOX-dependent mechanical cues in diabetes while simultaneously implicating LOX as an alternative (downstream) target to block AGE/RAGE signaling in DR. ARTICLE HIGHLIGHTS: We investigated the regulation and proinflammatory role of retinal endothelial lysyl oxidase (LOX) in diabetes. Findings reveal that LOX is upregulated by advanced glycation end products (AGE) and receptor for AGE (RAGE) and mediates AGE/RAGE-induced retinal endothelial cell activation and subendothelial matrix remodeling. We also show that LOX-dependent subendothelial matrix stiffening feeds back to enhance retinal endothelial RAGE. These findings implicate LOX as a key proinflammatory factor and an alternative (downstream) target to block AGE/RAGE signaling in diabetic retinopathy.

Strengthening the immunization supply chain through EVM assessment: Comparing India's two successive national assessments.

Background: Effective immunization supply chain (iSC) is crucial for safe and timely transport of potent vaccines to the beneficiary. India's iSC, with a network of ~29,000 cold chain points (CCP), measures its quality standards using the World Health Organization-United Nations International Children's Emergency Fund (WHO-UNICEF) global tool on effective vaccine management (EVM). The two national EVM assessments (EVMA) were conducted in 2013 and 2018. This study helps to see the impact of policies and practices through EVMA in maintaining an efficient iSC for effective implementation of immunization program. Materials and Methods: We conducted a desk analysis using EVMA reports to summarize and compare the findings of the two studies. Cut-off of 80% was considered ideal for each category/criteria score. Results: Both EVMA 2013 and 2018 were conducted using Android-based global EVM tool, though across a wider sample of CCP. Maximum and minimum changes in scores were sub-national and lowest distribution (16% each) and national buffer stores (7%), respectively. Maximum and minimum improvements were seen in vaccine management (29%) and MIS and supportive functions (3%), respectively. The improvement was statistically significant for the overall scores (P = 0.02), primary (P = 0.01), subnational (P = 0.02), and lowest distribution stores (P = 0.03). Among the 36 recommendations of 2013 assessment, 78% and 22% were fully and partially implemented (or ongoing), respectively. Conclusion: Implementation of EVM recommendations has significantly led to improvement and continues to provide a benchmark for iSC and its processes. Follow-up assessments every 3-5 years can further help to evaluate iSC and ascertain the impact of recommendations.

Comparative immunogenicity of an mRNA/LNP and a DNA vaccine targeting HIV gag conserved elements in macaques.

Immunogenicity of HIV-1 mRNA vaccine regimens was analyzed in a non-human primate animal model. Rhesus macaques immunized with mRNA in lipid nanoparticle (mRNA/LNP) formulation expressing HIV-1 Gag and Gag conserved regions (CE) as immunogens developed robust, durable antibody responses but low adaptive T-cell responses. Augmentation of the dose resulted in modest increases in vaccine-induced cellular immunity, with no difference in humoral responses. The gag mRNA/lipid nanoparticle (LNP) vaccine provided suboptimal priming of T cell responses for a heterologous DNA booster vaccination regimen. In contrast, a single immunization with gag mRNA/LNP efficiently boosted both humoral and cellular responses in macaques previously primed by a gag DNA-based vaccine. These anamnestic cellular responses were mediated by activated CD8+ T cells with a phenotype of differentiated T-bet+ cytotoxic memory T lymphocytes. The heterologous prime/boost regimens combining DNA and mRNA/LNP vaccine modalities maximized vaccine-induced cellular and humoral immune responses. Analysis of cytokine responses revealed a transient systemic signature characterized by the release of type I interferon, IL-15 and IFN-related chemokines. The pro-inflammatory status induced by the mRNA/LNP vaccine was also characterized by IL-23 and IL-6, concomitant with the release of IL-17 family of cytokines. Overall, the strong boost of cellular and humoral immunity induced by the mRNA/LNP vaccine suggests that it could be useful as a prophylactic vaccine in heterologous prime/boost modality and in immune therapeutic interventions against HIV infection or other chronic human diseases.

Increased cell stiffness contributes to complement-mediated injury of choroidal endothelial cells in a monkey model of early age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the aging population. Yet no therapies exist for ~85% of all AMD patients who have the dry form that is marked by degeneration of the retinal pigmented epithelium (RPE) and underlying choroidal vasculature. As the choroidal vessels are crucial for RPE development and maintenance, understanding how they degenerate may lead to effective therapies for dry AMD. One likely causative factor for choroidal vascular loss is the cytolytic membrane attack complex (MAC) of the complement pathway that is abundant on choroidal vessels of humans with early dry AMD. To examine this possibility, we studied the effect of complement activation on choroidal endothelial cells (ECs) isolated from a rhesus monkey model of early AMD that, we report, exhibits MAC deposition and choriocapillaris endothelial loss similar to that seen in human early AMD. Treatment of choroidal ECs from AMD eyes with complement-competent normal human serum caused extensive actin cytoskeletal injury that was significantly less pronounced in choroidal ECs from young normal monkey eyes. We further show that ECs from AMD eyes are significantly stiffer than their younger counterparts and exhibit peripheral actin organization that is distinct from the longitudinal stress fibers in young ECs. Finally, these differences in complement susceptibility and mechanostructural properties were found to be regulated by the differential activity of the small GTPases Rac and Rho, because Rac inhibition in AMD cells led to simultaneous reduction in stiffness and complement susceptibility, while Rho inhibition in young cells exacerbated complement injury. Thus, by identifying cell stiffness and cytoskeletal regulators Rac and Rho as important determinants of complement susceptibility, the current findings offer a new mechanistic insight into choroidal vascular loss in early AMD that warrants further investigation for assessment of translational potential. © 2022 The Pathological Society of Great Britain and Ireland.

Sequential Analysis of Binding and Neutralizing Antibody in COVID-19 Convalescent Patients at 14 Months After SARS-CoV-2 Infection.

Durability of SARS-CoV-2 Spike antibody responses after infection provides information relevant to understanding protection against COVID-19 in humans. We report the results of a sequential evaluation of anti-SARS-CoV-2 antibodies in convalescent patients with a median follow-up of 14 months (range 12.4-15.4) post first symptom onset. We report persistence of antibodies for all four specificities tested [Spike, Spike Receptor Binding Domain (Spike-RBD), Nucleocapsid, Nucleocapsid RNA Binding Domain (N-RBD)]. Anti-Spike antibodies persist better than anti-Nucleocapsid antibodies. The durability analysis supports a bi-phasic antibody decay with longer half-lives of antibodies after 6 months and antibody persistence for up to 14 months. Patients infected with the Wuhan (WA1) strain maintained strong cross-reactive recognition of Alpha and Delta Spike-RBD but significantly reduced binding to Beta and Mu Spike-RBD. Sixty percent of convalescent patients with detectable WA1-specific NAb also showed strong neutralization of the Delta variant, the prevalent strain of the present pandemic. These data show that convalescent patients maintain functional antibody responses for more than one year after infection, suggesting a strong long-lasting response after symptomatic disease that may offer a prolonged protection against re-infection. One patient from this cohort showed strong increase of both Spike and Nucleocapsid antibodies at 14 months post-infection indicating SARS-CoV-2 re-exposure. These antibodies showed stronger cross-reactivity to a panel of Spike-RBD including Beta, Delta and Mu and neutralization of a panel of Spike variants including Beta and Gamma. This patient provides an example of strong anti-Spike recall immunity able to control infection at an asymptomatic level. Together, the antibodies from SARS-CoV-2 convalescent patients persist over 14 months and continue to maintain cross-reactivity to the current variants of concern and show strong functional properties.

Control of SARS-CoV-2 infection after Spike DNA or Spike DNA+Protein co-immunization in rhesus macaques.

The speed of development, versatility and efficacy of mRNA-based vaccines have been amply demonstrated in the case of SARS-CoV-2. DNA vaccines represent an important alternative since they induce both humoral and cellular immune responses in animal models and in human trials. We tested the immunogenicity and protective efficacy of DNA-based vaccine regimens expressing different prefusion-stabilized Wuhan-Hu-1 SARS-CoV-2 Spike antigens upon intramuscular injection followed by electroporation in rhesus macaques. Different Spike DNA vaccine regimens induced antibodies that potently neutralized SARS-CoV-2 in vitro and elicited robust T cell responses. The antibodies recognized and potently neutralized a panel of different Spike variants including Alpha, Delta, Epsilon, Eta and A.23.1, but to a lesser extent Beta and Gamma. The DNA-only vaccine regimens were compared to a regimen that included co-immunization of Spike DNA and protein in the same anatomical site, the latter of which showed significant higher antibody responses. All vaccine regimens led to control of SARS-CoV-2 intranasal/intratracheal challenge and absence of virus dissemination to the lower respiratory tract. Vaccine-induced binding and neutralizing antibody titers and antibody-dependent cellular phagocytosis inversely correlated with transient virus levels in the nasal mucosa. Importantly, the Spike DNA+Protein co-immunization regimen induced the highest binding and neutralizing antibodies and showed the strongest control against SARS-CoV-2 challenge in rhesus macaques.

Reactive oxygen species-mediated cytoplasmic stiffening impairs the phagocytic ability of the macrophage.

The phagocytic ability of macrophages empowers them to enforce innate immunity. RAW264.7, THP-1 and peripheral blood mononuclear cell-derived macrophages display considerable variability with regards to their phagocytic ability. We identify the underlying causes that attenuate the phagocytic abilities of a macrophage. Deformability of the cytoplasm and cortex influences the macrophage's phagocytic ability, and macrophages use the large cell-to-cell variability of their cytoplasmic stiffness to modulate their phagocytic ability. We find that the more-deformable macrophages have a higher phagocytic ability than those that are less deformable. Further, the subcellular spatial variability of cortex stiffness gives rise to more-deformable subdomains on the membrane for pathogen ingestion. We report a previously unknown negative-feedback loop that is triggered by the phagocytic oxidative burst. Macrophages utilize the excess reactive oxygen species to stiffen the cytoplasm, reducing their phagocytic propensity. In organisms, ageing or pathological conditions impair the phagocytic ability of macrophages. Our findings identify the targets that could potentially be utilized for restoring the phagocytic ability of the defunct macrophages.

Peptides derived from a short stretch of diphtheria toxin bind to heparin-binding epidermal growth factor-like growth factor.

Membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) is the receptor for diphtheria toxin (DT). Mutated or truncated, non-toxic DT has been used earlier for HB-EGF-targeted drug delivery and to modulate HB-EGF signaling. In the present work, we have synthesized a peptide corresponding to a 26 amino acid long stretch of the receptor-binding domain of DT. This region of DT makes multiple contacts with HB-EGF and has residues critical for binding to HB-EGF. We show that this peptide and two of its mutants bind to HB-EGF. We have also created recombinant proteins by fusing Maltose-binding Protein (MBP) with these peptides. These recombinant MBP-tagged peptides bind to HB-EGF with affinities in the range of 10-7 to 10-8 M. We have observed that these MBP-tagged peptides can modulate molecular signaling of HB-EGF. Therefore, this 26 amino acid long stretch of DT can be considered as an independent functional segment for binding to HB-EGF. Peptides corresponding to this region may be used for HB-EGF targeted cellular delivery of molecular cargo or to modulate HB-EGF signaling.

Hybrid bag of approaches to characterize selection criteria for cohort identification.

OBJECTIVE: The 2018 National NLP Clinical Challenge (2018 n2c2) focused on the task of cohort selection for clinical trials, where participating systems were tasked with analyzing longitudinal patient records to determine if the patients met or did not meet any of the 13 selection criteria. This article describes our participation in this shared task. MATERIALS AND METHODS: We followed a hybrid approach combining pattern-based, knowledge-intensive, and feature weighting techniques. After preprocessing the notes using publicly available natural language processing tools, we developed individual criterion-specific components that relied on collecting knowledge resources relevant for these criteria and pattern-based and weighting approaches to identify "met" and "not met" cases. RESULTS: As part of the 2018 n2c2 challenge, 3 runs were submitted. The overall micro-averaged F1 on the training set was 0.9444. On the test set, the micro-averaged F1 for the 3 submitted runs were 0.9075, 0.9065, and 0.9056. The best run was placed second in the overall challenge and all 3 runs were statistically similar to the top-ranked system. A reimplemented system achieved the best overall F1 of 0.9111 on the test set. DISCUSSION: We highlight the need for a focused resource-intensive effort to address the class imbalance in the cohort selection identification task. CONCLUSION: Our hybrid approach was able to identify all selection criteria with high F1 performance on both training and test sets. Based on our participation in the 2018 n2c2 task, we conclude that there is merit in continuing a focused criterion-specific analysis and developing appropriate knowledge resources to build a quality cohort selection system.

3D micro-environment regulates NF-κß dependent adhesion to induce monocyte differentiation.

Differentiation of monocytes entails their relocation from blood to the tissue, hence accompanied by an altered physicochemical micro-environment. While the mechanism by which the biochemical make-up of the micro-environment induces differentiation is known, the fluid-like to gel-like transition in the physical micro-environment is not well understood. Monocytes maintain non-adherent state to prevent differentiation. We establish that irrespective of the chemical makeup, a 3D gel-like micro-environment induces a positive-feedback loop of adhesion-MAPK-NF-κß activation to facilitate differentiation. In 2D fluid-like micro-environment, adhesion alone is capable of inducing differentiation via the same positive-feedback signaling. Chemical inducer treatment in fluid-like micro-environment, increases the propensity of monocyte adhesion via a brief pulse of p-MAPK. The adhesion subsequently elicit differentiation, establishing that adhesion is both necessary and sufficient to induce differentiation in 2D/3D micro-environment. MAPK, and NF-κß being key molecules of multiple signaling pathways, we hypothesize that biochemically inert 3D gel-like micro-environment would also influence other cellular functions.

Vaccine Safety and Surveillance for Adverse Events Following Immunization (AEFI) in India.

RATIONALE: Assured quality vaccines and safe immunization practices are pre-requisite to successful immunization programs. All vaccines go through stringent safety checks during pre-licensure stage. Adverse Events Following Immunization (AEFI) Surveillance program is an integral part of routine immunization program in India to monitor the vaccine safety in the post licensure phase. Indian AEFI Program: National AEFI surveillance relies on passive surveillance and reporting by the health functionaries and practitioners. Vigorous strengthening of AEFI surveillance has resulted in manifold rise in absolute number of AEFI reports across several reporting units in the country in the last decade. Establishment of National AEFI Secretariat, National Technical Collaborating Centre, and development of risk communication strategy as well as quality management certification are some of the unique aspects of this public health program. All serious AEFI reports undergo a systematic causality assessment as per WHO-algorithm by trained committees. National AEFI surveillance system has forged formal linkages with national pharmacovigilance program, the regulators, and professional bodies. Challenges: The number of the reported serious AEFIs are still far less than the expected numbers. Although the AEFI committees at the district and state levels have been established, a large proportion are far from functional. Way forward: As the national immunization program introduces newer vaccines for different age groups and coverage improves, the issues of vaccine hesitancy and confidence are likely to be raised more often and the AEFI surveillance program will have to assume greater responsibility to comprehensively respond to the community concerns and sustain public confidence in vaccines.

Diagnosis of limbal stem cell deficiency based on corneal epithelial thickness measured on anterior segment optical coherence tomography.

PURPOSE: The purpose of this study is to investigate the epithelial thickness in the cornea and limbus in limbal stem cell deficiency (LSCD) using anterior segment optical coherence tomography (AS-OCT). METHODS: This was a cross-sectional, comparative study. OCT images of 30 eyes of 19 patients with LSCD collected by AS-OCT were scanned. Corneal epithelial thickness was recorded at the central cornea and the superior, nasal, inferior, and temporal limbus. Measurment of the same region of 30 normal eyes served as control. Epithelial thickness in all locations was measured by 2 independent observers. RESULTS: The mean epithelial layer thickness was 61.3 ± 2.9 µ in the central cornea and 62.7 ± 4.3 µ in the limbus in the control. The epithelial thickness in LSCD patients was found to be 41.33 ± 2.8 µ. An average reduction of 22.2% in the central cornea and 32.15% in the limbus was found in patients with LSCD (P < 0.05). Epithelial thinning correlated with the severity of LSCD in both cornea and limbus. In eyes with sectoral LSCD, a similar degree of epithelial thinning was also detected in the clinically unaffected limbal regions. CONCLUSION: Both corneal and limbal epithelia become progressively thinner in LSCD. Epithelial thickness assessment using AS-OCT as a noninvasive tool could be used as a diagnostic measure of LSCD.

Receptor-Mediated Enhanced Cellular Delivery of Nanoparticles Using Recombinant Receptor-Binding Domain of Diphtheria Toxin.

Antibodies and peptides are often used to home nanoparticles (NPs) to specific cells. Here in this work, we have used recombinant receptor-binding domain of diphtheria toxin (RDT) as a homing molecule for NPs. Diphtheria toxin binds to heparin binding EGF-like growth factor (HB-EGF) through its receptor-binding domain. HB-EGF is often overexpressed as cell surface molecule in various types of cancer. We have prepared monodispersed, spherical PLGA NPs and coated these NPs with RDT. These NPs are characterized by FESEM and FT-IR spectroscopy. Using flow cytometry and fluorescence spectroscopy, we show that coating with RDT increases cellular uptake of PLGA NPs. We further show that RDT-coated nanoparticles are internalized through clathrin-dependent receptor-mediated endocytosis that can be reduced by specific inhibitor. These RDT-coated nanoparticles (RDT-NP) were further used for preferential delivery of Irinotecan, a chemotherapeutic agent, to cells overexpressing HB-EGF. We show that receptor-mediated enhanced uptake of RDT-NPs increases the potency of irinotecan in these cells.

Modulation of Amyloid Aggregates into Nontoxic Coaggregates by Hydroxyquinoline Appended Polyfluorene.

Inhibitory modulation toward de novo protein aggregation is likely to be a vital and promising therapeutic strategy for understanding the molecular etiology of amyloid related diseases such as Alzheimer's disease (AD). The building up of toxic oligomeric and fibrillar amyloid aggregates in the brain plays host to a downstream of events, causing damage to axons, dendrites, synapses, signaling, transmission, and finally cell death. Herein, we introduce a novel conjugated polymer (CP), hydroxyquinoline appended polyfluorene (PF-HQ), which has a typical "amyloid like" surface motif and exhibits inhibitory modulation effect on amyloid ß (Aß) aggregation. We delineate inhibitory effects of PF-HQ based on Thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), circular dichroism (CD), and Fourier transform infrared (FTIR) studies. The amyloid-like PF-HQ forms nano coaggregates by templating with toxic amyloid intermediates and displays improved inhibitory impacts toward Aß fibrillation and diminishes amyloid cytotoxicity. We have developed a CP based modulation strategy for the first time, which demonstrates beneficiary amyloid-like surface motif to interact efficiently with the protein, the pendant side groups to trap the toxic amyloid intermediates as well as optical signal to acquire the mechanistic insight.

Dynamic action of carotenoids in cardioprotection and maintenance of cardiac health.

Oxidative stress has been considered universally and undeniably implicated in the pathogenesis of all major diseases, including those of the cardiovascular system. Oxidative stress activate transcriptional messengers, such as nuclear factor-κB, tangibly contributing to endothelial dysfunction, the initiation and progression of atherosclerosis, irreversible damage after ischemic reperfusion, and even arrhythmia, such as atrial fibrillation. Evidence is rapidly accumulating to support the role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) as intracellular signaling molecules. Despite this connection between oxidative stress and cardiovascular disease (CVD), there are currently no recognized therapeutic interventions to address this important unmet need. Antioxidants that provide a broad, "upstream" approach via ROS/RNS quenching or free radical chain breaking seem an appropriate therapeutic option based on epidemiologic, dietary, and in vivo animal model data. Short-term dietary intervention trials suggest that diets rich in fruit and vegetable intake lead to improvements in coronary risk factors and reduce cardiovascular mortality. Carotenoids are such abundant, plant-derived, fat-soluble pigments that functions as antioxidants. They are stored in the liver or adipose tissue, and are lipid soluble by becoming incorporated into plasma lipoprotein particles during transport. For these reasons, carotenoids may represent one plausible mechanism by which fruits and vegetables reduce the risk of chronic diseases as cardiovascular disease (CVD). This review paper outlines the role of carotenoids in maintaining cardiac health and cardioprotection mediated by several mechanisms including redox signaling.

Multiple transfused thalassemia major: ocular manifestations in a hospital-based population.

PURPOSE: To study the ocular manifestations in multiple transfused beta-thalassemia major patients and assess the ocular side-effects of iron chelating agents. MATERIALS AND METHODS: In this prospective observational study, 45 multiple transfused beta-thalassemia major children between six months and 21 years of age were enrolled and assigned groups according to the treatment regimens suggested. Group A received only blood transfusions, Group B blood transfusions with subcutaneous desferrioxamine, Group C blood transfusions with desferrioxamine and oral deferriprone and Group D blood transfusions with deferriprone. Ocular status at the time of enrolment was documented. Subjects were observed quarterly for one year for changes in ocular status arising due to the disease process and due to iron chelation therapy. Children with hemoglobinopathies other than beta-thalassemia major, congenital ocular anomalies and anemia due to other causes were excluded. RESULTS: Ocular involvement was observed in 58% of patients. Lenticular opacities were the most common ocular finding (44%), followed by decreased visual acuity (33%). An increased occurrence of ocular changes was observed with increase of serum ferritin and serum iron levels as well as with higher number of blood transfusions received. Desferrioxamine seemed to have a protective influence on retinal pigment epithelium (RPE) mottling. Occurrence of lenticular opacities and RPE degeneration correlated positively with use of desferrioxamine and deferriprone respectively. Follow-up of patients for one year did not reveal any change in ocular status. CONCLUSION: Regular ocular examinations can aid in preventing, delaying or ameliorating the ocular complications of thalassemia.