Jan-Feb 2025 (Vol 30 No 5)

Neutral pH - 7Minimal LacrimationLess number of instillations

Table of ContentsFrom the President’s DESK 07From the Desk of Managing Editor 08Neutral pH - 7Minimal LacrimationLess number of instillationsOcular Inflammation and Molecular Markers: “Untying the KnotsIntroduction to Ocular Inflammation and Molecular MarkersMolecular Markers in Various Ocular Inflammatory Diseases: Current PerspectivesOcular Surface Inflammation and Molecular Markers: A Comprehensive ReviewThe Continuous Fight of Inflammatory and Anti-Inflammatory Markers in Dry EyesPeripheral Ulcerative Keratitis - Triggers, Traits and Therapy 09 15 20 25 28Trabecular Meshwork Inflammation and Glaucoma: A Review of Implicated Molecular Pathways 41Proteins at the Frontline: Revolutionizing Uveitis Diagnosis with BiomarkersThe Role of Anti-Inflammatory Medications in the Treatment of UveitisJuvenile Idiopathic Arthritis and Associated Uveitis: A Comprehensive ReviewRetinal Angiogenesis: Role of Inflammatory MArkersOcular Tuberculosis - Shedding Light on Molecular Pathways of InflammationThe Crucial Role of Multimodal Imaging in Diagnosing and Managing Acute Vogt-KoyanagiHarada DiseaseOptic Neuritis: A Review of Clinical Presentations, The Current Status of Biomarkers and Specific TherapiesPaediatric Uveitis: Role of Targeted Treatment 44 52 58 64 68 72 78 55

04 05PresidentJoint Secretary Editor DJO Library OfficerVice President Secretary Treasurer Prof. Rohit SaxenaDr. R. P. SinghDr. Ankur SinghDr. Pranita SahayDr. Rajendra PrasadDr. Anu MalikDr. Rakesh GuptaDr. Jatinder SinghBhallaDr. Deepa SharmaDr. Ritin GoyalDr. AlkeshChaudharyDr. Ikeda LalDr. Siddharth MadanDr. Rahul Mayor Dr. Bhupesh SinghProf. Rajesh Sinha Dr. Prafulla Kumar Maharana Prof. Kirti SinghDOS Office BearersDOS Executive Committee (2024-2026)Executive MemberDOS Representatives to AIOS Ex-Officio MemberDOS Times - Volume 30, Number 5, January-February 2025 04 www.dosonline.org

04 DOS Times - Volume 30, Number 5, January-February 2025 05 www.dosonline.orgSection InchargesManaging EditorsThe Editorial BoardDr. Prafulla Kumar MaharanaEditor in ChiefDr. Prashant BawankuleOphthalmic QuizDr. Amit MehtaniWhat’s New?Dr. Arpan GandhiOphthalmology:Basic SciencesDr. Jatinder BaliInteresting Facts inOphthalmologyDr. Pranita SahayPG CornerDr. Neha GulianiOphthalmic HistoryDr. Arshi SinghDebates in OphthalmologyDr. Ritu Nagpal Dr. Anu MalikDr. Siddharth MadanLatest ArticlesDr. Deepali SinghalPhacoemulsification:From Basics to AdvancedDr. Ankur SinghDebates in OphthalmologyDr. Ashish MarkanDr. Ikeda LalOphthalmicImagingDr. Vineet SehgalDrugs inOphthalmologyDr. Digvijay SinghYO UpdatesDr. Karan BhatiaYO UpdatesDr. Amar PujariTools & Techniquesin Ophthalmology

DOS Times - Volume 30, Number 5, January-February 2025 06 www.dosonline.org 07Section EditorsRetina & UveaUvea & Ocular Inflammatory DisordersCornea & External Eye DiseasesOcular SurfaceDr. Abhishek JainDr. Avnindra GuptaDr. Daraius ShroffDr. Gagan BhatiaDr. Sandhya MakhijaDr. Tinku BaliDr. Anurag NarulaDr. Naginder VashishtDr. Parul JainDr. Sarita AggarwalDr. Supriya DharDr. Tulika ChauhanDr. Aastha SinghDr. Abhishek DaveDr. Rajat JainCataract & IOLDr. Aman MalikDr. Ritin GoyalDr. Shweta GaurRefractive SurgeryDr. Raghav MalikDr. Shilpi DiwanDr. Sridevi NairDr. Tushar GroverOculoplasty & AestheticsDr. Hina kauserDr. Poonam JainDr. Rwituja GroverDr. Sahil AgarwalGlaucomaDr. Annu JoonDr. Kiran BhanotDr. Suneeta DubeyNeuro-OphthalmologyStrabisumsCommunity OphthalmologyOcular OncologyComprehensive OphthalmologyDr. Rebika DhimanDr. Sumit MongaDr. Varshini ShankarDr. Anita GangerDr. Smita KapoorDr. Puneet JainDr. Shaloo BagejaDr. Sima DasDr. Sumit GroverDr. Harinder Singh SethiDr. R.K. DuveshDr. Utsav BansalDr. Suraj SenjamDr. Anil TanwarDr. Anuj MehtaDr. Ashish KumarDr. Ashok Grover Dr. Col. Ranjit GoenkaDr. G K DasDr. Jatinder Singh BhallaDr. Jeewan Singh TitiyalDr. Kirti SinghDr. M. VanathiDr. Mahipal SachdevDr. Manavdeep SinghDr. Namrata SharmaDr. OmprakashDr. Radhika TandonDr. Rishi MohanDr. Sanjay ChaudhryDr. Sanjay MishraDr. Sarita BeriDr. Subhash Dadeya Dr. Sudhank BhartiDr. Taru DeewanDr. Uma SridharDr. V P GuptaDr. Vinay GarodiaDr. Virender SangwanAdvisory Board

From The President’s Desk06 DOS Times - Volume 30, Number 5, January-February 2025 07 www.dosonline.orgProf. Rohit SaxenaDear Readers,It is with great enthusiasm, we present this special issue of DOS Times focused on Ocular Inflammation and Molecular Markers, aptly titled “Untying the Knots.” As clinicians, we often find ourselves at the crossroads of overlapping symptoms, elusive etiologies, and evolving treatment paradigms. With this issue, we aim to unravel some of these complexities through the lens of molecular science. From early diagnosis to precision therapy, the role of Molecular markers, once considered mere academic curiosities, is now rapidly expanding into mainstream clinical practice is rapidly expanding.Highlights include a deep dive into ocular tuberculosis, where molecular pathways are beginning to shed light on persistent diagnostic dilemmas. The importance of multimodal imaging in managing acute Vogt-Koyanagi-Harada disease and the evolving approach to pediatric uveitis with targeted immunomodulatory therapy underscore how molecular insights are transforming patient care. In addition, we explore the role of inflammatory markers in retinal angiogenesis, dry eye disease, allergic conjunctivitis, and even trabecular meshwork inflammation—each piece contributing to a broader understanding of inflammation’s reach across ocular tissues.The review of optic neuritis, which outlines the emerging role of biomarkers in distinguishing various etiologies and guiding therapy, especially in the era of neuro-immunological overlap. The article on juvenile idiopathic arthritisrelated uveitis and its long-term impact on vision offers a sobering reminder of the need for early molecular guidance and sustained care.Moreover, we delve into the therapeutic realm with discussions on anti-inflammatory medications, peripheral ulcerative keratitis, and the promise of protein-based diagnostics in uveitis, offering hope for a future where ocular inflammation is not just managed, but understood at its roots.The highlight of this year’s initiatives will be the landmark 75th Annual DOS Conference, set to take place from June 13th to 15th, 2025, at Bharat Mandapam – Convention Centre in New Delhi. This historic event marks the first time the conference will be held at this newly built, world-class venue, renowned for its state-of-the-art facilities, advanced technological capabilities, and a setting designed to foster learning and collaboration. We warmly invite you to be part of this milestone gathering, where your presence will add immense value to the discussions and contribute to the vibrant spirit of the occasion.As the curtain rises on this molecular era in ophthalmology, we hope this issue will serve as both an educational resource and a catalyst for further inquiry. With best wishes,Prof Rohit Saxena MD, PhDPresident, DOS

DOS Times - Volume 30, Number 5, January-February 2025 08 www.dosonline.orgFrom the EditorsDear Esteemed Readers,Welcome to this very special issue of our magazine, titled Ocular Inflammation and Molecular Markers: Untying the Knots, focussed on ocular inflammatory pathologies, which remain some of the most challenging situations the ophthalmologists encounter. Their causes are complex and a wide diversity of clinical presentations is encountered in clinics, often leading to significant delay in making an accurate diagnosis. With corticosteroids and immunosuppressants being commonly employed for acute relapses and reducing remissions, the risks associated with the long term use of these medications makes imperative that the disease be managed by a team of ophthalmologists, physicians, child care specialists and rheumatologists. An understanding into the underlying molecular pathways further helps in choosing the right kind of specific therapy for an individual patient. This issue, brings upon a collection of articles incorporating a wide coverage of ocular inflammations starting from ocular surface to corneal, limbus, sclera, trabecular meshwork as well as posterior segment of the eye. We have aimed to cover each particular disease from its basic concepts, key molecular pathways, available diagnostics to targeted and specific therapies. The articles have been written in simple languages, well supported by self-explanatory tables, flow charts, clinical photographs and images from diagnostic modalities. The articles have been authored by a range of specialist including fellows and senior residents in their training phase, early to experienced and fine specialist ophthalmologists with years of experience in this specialised field. Heartfelt thanks to all the authors for devoting their precious time in crafting out their work and sharing with us and to the entire editorial team for their continuing support. Thank you once again for being a part of this journey. We look forward to continuing this dialogue with each one of you in our future editions too.Dr. Ritu Nagpal, MD (AIIMS, New Delhi)Former Fellow; Cornea and Anterior segment, LVPEI HyderabadManaging Editor, DOS TimesDr. Prafulla Kumar Maharana Dr. Ritu Nagpal Dr. Anu Malik Dr. Ashish Markan

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 09 www.dosonline.orgIntroduction to Ocular Inflammation and Molecular MarkersSanjeev Kumar Nainiwal MD, DNB, MNAMS | Jyoti Nagar MBBS |Neha Kharwas MBBS | Dimple MBBS | Priyanka MBBS | Manisha Jhajharia MBBS | Versha Jeph MBBSDepartment of Ophthalmology, Sawai Man Singh Medical College & Hospital, Jaipur (Rajasthan), IndiaOcular inflammation, commonly referred to as uveitis, is a significant clinical condition characterized by inflammation of the eye structures, particularly the uveal tract (which includes the iris, ciliary body, and choroid). However, inflammation can also extend to the retina, optic nerve, and other ocular structures. The immune response within the eye plays a crucial role in uveitis. Ocular tissues are immunologically privileged, meaning they are relatively protected from systemic immune responses. However, this immune privilege can be disrupted due to infections, autoimmunity, or systemic inflammatory diseases, leading to the development of uveitis. The pathophysiology of uveitis involves complex interactions between innate and adaptive immune cells, cytokines, and various signaling molecules that mediate inflammatory responses.One of the key aspects of understanding and managing uveitis lies in the identification of molecular markers that can provide insights into the nature and severity of the disease. Molecular markers are biomarkers that can help identify the underlying immune mechanisms or pathological processes driving the inflammation. These markers are often detectable in the blood, aqueous humor, vitreous humor, or tears and can be used to monitor disease progression, determine the cause of the inflammation, and assess the response to therapy. Some molecular markers are associated with specific autoimmune or infectious diseases, making them essential tools for both diagnosis and management.Human leukocyte antigens (HLA) have proven to be of particular importance in uveitis. Specific HLA types are associated with various forms of uveitis, particularly those of autoimmune origin. Testing for these genetic markers can provide valuable information, supporting the diagnosis of specific disease processes and guiding therapeutic decisions.[1]The molecular landscape of uveitis is further complicated by the presence of uveitogenic proteins that can incite inflammation within the eye. These proteins are often components of the retina or other ocular structures and can trigger autoimmune responses in susceptible individuals. Some of the key uveitogenic proteins include rhodopsin, retinal arrestin, recoverin, phosducin, retinal pigment epithelium-derived protein, and interphotoreceptor retinoid-binding protein.[2] Understanding these proteins and their role in triggering immune responses is critical for advancing our knowledge of uveitis and for developing novel therapeutic targets aimed at modulating the immune response.In addition to genetic markers, cytokines and interleukins (IL) are central to the inflammatory process in uveitis. The practical application of cytokine assessment in serum and ocular fluids has become an essential tool for clinicians in the management of uveitis. Monitoring cytokine levels allows for better understanding of the inflammatory process in individual patients. Furthermore, the evaluation of cytokines can provide insight into disease activity, help predict disease outcomes, and guide treatment decisions.Despite the growing body of knowledge about the molecular markers involved in ocular inflammation, the complexity of uveitis necessitates a multifaceted approach to diagnosis and treatment. The interplay between genetic predisposition, immune response, cytokine production, and environmental factors contributes to the wide clinical spectrum of uveitis. Ongoing research into the molecular mechanisms of uveitis holds the potential to uncover new biomarkers and therapeutic targets, leading to more personalized and effective treatments for patients suffering from this debilitating condition.This review aims to explore the fundamentals of ocular immunology, with a focus on the role of interleukins, cytokines, and molecular markers in the pathogenesis

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 10 www.dosonline.orgof uveitis. (Table-1 & 2) By understanding the intricate immune mechanisms involved in uveitis and assessing the relevance of various biomarkers, clinicians can enhance their diagnostic accuracy and therapeutic strategies, ultimately improving outcomes for patients with ocular inflammation.Role of Cytokines and ChemokinesOcular inflammation, particularly uveitis, is immunemediated. CD4+ T cells and chemokines play crucial roles in disease progression.CD4+ T Cells in Ocular Inflammation: Central to the adaptive immune response. Differentiate into subtypes (e.g., Th1, Th2, Tregs) based on cytokine environment.• Th1 Cells: Secrete IFN-γ and IL-2, important in autoimmune uveitis (e.g., EAU).• Th2 Cells: Produce IL-4, less involved in uveitis.• Other Subtypes: iTregs, Th17, etc., contribute to immune balance.Chemokines in Ocular Inflammation: Regulate immune cell migration to inflamed tissues. Produced by endothelial cells in response to cytokines like TNF-α and IL-1. Types are:• Inflammatory Chemokines: CC and CXC chemokines recruit leukocytes (e.g., T cells, macrophages).• Immune Chemokines: Regulate adhesion and migration (e.g., CX3C).Key Chemokines in Ocular Inflammation are:• RANTES (CCL5): Attracts T cells, eosinophils, and basophils.• MCP-1 (CCL2): Recruits monocytes, contributing to chronic inflammation.• MIP-1: Attracts macrophages and T cells.• CXCL12: Recruits T cells.• Fractalkine (CX3C): Involved in immune cell recruitment.[3,4]IL Role in Ocular InflammmationTherapeutic TargetTherapies Indications EffectivenessIL-6 -Promotes Th17 differentiation and chronic ocular inflammation.IL-6 Receptor -Tocilizumab (Anti IL-6 receptor monoclonal antibody)-Sarilumab (IL-6 receptor inhibitor)-Refractory noninfectious uveitis, e.g., JIA associated uveitis- Other autoimmune uveitis-Reduces inflammation-Impoves visual outcomes, especially in JIA-associated uveitisIL-1 -IL 1 alpha and IL 1 beta activate neutrophils and macrophages, contributing to ocular tissue damage.IL-1 Receptor -Anakinra (IL-1 receptor antagonist)-Canakinumab (IL1beta inhibitor)-Behcet’s uveitis-Autoimmune Uveitis-Blau syndrome (autoinflammatory syndrome)-Reduces intraocular inflammation-Effective in controlling flare-ups.IL-17 -Promotes neutrophil recruitment and exacerbates autoimmune uveitis,scleritis, and dry eye disease.IL-17A -Secukinumab(IL-17A inhibitor)-Ixekizumab (IL-17A inhibitor)-HLA-B27-associated uveitis-Psoriatic arthritisrelated uveitis-Other cutoimmune ocular conditions.-Mixed results in noninfectious uveitis.-Effectiveness in spondylitis and psoriatic arthritisrelated uveitis.IL-12 &IL23-IL-23 promotes Th17 differentiation, and IL-12 regulates Th1 responses, implicated in chronic uveitis IL-12/Il-23 Receptor-Ustekinumab (IL-12/IL-23 inhibitor)-Guselkumab(IL-23 inhibitor)-Refractory Behcet’s uveitis-Sarcoidosis associated uveitis-Other systemic inflammatory diseases with ocular involvement.-Reduces flare-ups in systemic inflammation-Effective in chronic uveitisTable 1: Targeted Interleukin Inhibition in Ocular Inflammation.[5,6]

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 11 www.dosonline.orgIL Role in Ocular InflammmationTherapeutic TargetTherapies Indications EffectivenessIL-1 -IL-1 is a key driver of inflammation in dry eye diseases, contributing to ocular surface damage and discomfort.IL-1 -Lifitegrast (LFA-1 antagonist)-Cyclosporine (IL-2 modulator)-Refractory noninfectious uveitis, e.g., JIA associated uveitis- Other autoimmune uveitis-Reduces inflammation-Impoves visual outcomes, especially in JIA-associated uveitisIL-6 -IL-6 is involved in the inflammatory process of dry eye disease, particularly in the conjunctiva and lacrimal glands.IL-6 -Anti IL-6 therapies (e.g., Tocilizumab, Sarilumab)-Behcet’s uveitis-Autoimmune Uveitis-Blau syndrome (autoinflammatory syndrome)-Reduces intraocular inflammation-Effective in controlling flare-ups.Emerging Therapies -IL-10 modulates immune responses and reduces ocular inflammation IL-10 -IL-10 therapy -HLA-B27-associated uveitis-Psoriatic arthritisrelated uveitis-Other cutoimmune ocular conditions.-Mixed results in noninfectious uveitis.-Effectiveness in spondylitis and psoriatic arthritis-related uveitis.IL-22 -IL-22 is being explored for its potential in promoting wound healing of the cornea and oculae surface disorders.IL-22 -IL-22 modulation therapy-Corneal wound healing-Ocular surface disorders-Promoting healing and reducing inflammation in ocular surface diseases.Table 2: IL-Based Therapies in Dry Eye Disease and Ocular Surface Disorders.In addition to cytokines and chemokines, there are other molecular markers involved in ocular inflammation, especially in diseases like uveitis. These markers provide valuable information about disease progression, tissue damage, immune cell activation, and tissue remodeling. Some of these markers include:1. Soluble Intercellular Adhesion Molecule-1 (sICAM-1)• Role: sICAM-1 is a cell adhesion molecule that plays a significant role in immune cell migration and recruitment. It is involved in the interaction between leukocytes and endothelial cells, promoting inflammation.• Associated with Active uveitis, especially in autoimmune and viral-related uveitis.• Elevated Levels: Serum, aqueous humor.• Diagnostic Relevance: Elevated sICAM-1 levels are associated with the inflammatory activity and progression of uveitis.2. Vascular Endothelial Growth Factor (VEGF)• Role: VEGF is a key regulator of angiogenesis, which is the formation of new blood vessels. It is involved in vascular permeability and endothelial cell proliferation, which can contribute to ocular tissue damage and retinal inflammation in uveitis.• Associated Uveitis Types: Non-infectious uveitis, particularly in conditions like sarcoidosis, Behçet’s disease, and VKH.• Elevated Levels: Aqueous humor, vitreous humor.• Diagnostic Relevance: Elevated VEGF levels are indicative of neovascularization and blood-retinal barrier breakdown, common in uveitis-associated complications like cystoid macular edema (CME).

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 12 www.dosonline.org3. C-reactive Protein (CRP)• Role: CRP is an acute-phase protein produced by the liver in response to inflammation. It serves as a general marker of systemic inflammation.• Associated Uveitis Types: All forms of uveitis (especially systemic conditions with inflammatory components like juvenile idiopathic arthritisassociated uveitis, sarcoidosis).• Elevated Levels: Serum.• Diagnostic Relevance: CRP is used as a broad marker for inflammation and can help monitor disease activity in uveitis, especially in the presence of systemic inflammatory conditions.4. Matrix Metalloproteinases (MMPs)• Role: MMPs are enzymes that degrade components of the extracellular matrix, which are essential for tissue remodeling. In ocular inflammation, MMPs can contribute to tissue damage and the breakdown of the blood-retinal barrier.• Associated Uveitis Types: Chronic uveitis, especially in autoimmune diseases such as sarcoidosis, Behçet’s disease, and VKH.• Elevated Levels: Aqueous humor, vitreous humor, serum.• Diagnostic Relevance: Elevated MMPs, such as MMP2 and MMP-9, are associated with tissue remodeling and may indicate ongoing inflammation and damage in the ocular tissues.5. Heat Shock Proteins (HSPs)• Role: Heat shock proteins act as molecular chaperones, helping other proteins fold correctly and preventing cellular damage under stress. HSPs, particularly HSP60 and HSP70, are involved in immune responses and may trigger autoimmune reactions in uveitis.• Associated Uveitis Types: Autoimmune-related uveitis, including HLA-B27-associated uveitis and Behçet’s disease.• Elevated Levels: Serum, ocular fluids.• Diagnostic Relevance: Elevated HSP levels can indicate an autoimmune response, and they are often used as biomarkers for autoimmune-driven ocular inflammation.Application of Biomarkers in Therapeutics for UveitisBiomarkers play a crucial role in the management and treatment of uveitis by guiding therapeutic decisions, monitoring disease activity, and predicting treatment response. Their use extends to both diagnostic and therapeutic aspects, enhancing the effectiveness and precision of treatment regimens.1. Targeted Biologic Therapy:• TNF-α Inhibitors: Biomarkers like TNF-α, IL-1, and IL-6 are critical in identifying appropriate candidates for biologic therapies. For example, TNF inhibitors such as Infliximab and Adalimumab are commonly used to treat non-infectious uveitis (NIU), and their effectiveness is often guided by levels of TNF-α in the patient’s serum or ocular fluids.• Interleukin (IL) Inhibitors: IL-1 and IL-6 biomarkers help determine the use of IL-1 inhibitors (e.g., Anakinra and Canakinumab) or IL-6 inhibitors (e.g., Tocilizumab). These therapies are particularly effective in conditions like Behcet’s disease and juvenile idiopathic arthritis (JIA).• IL-17, IL-23 Inhibitors: Biomarkers that target IL17 and IL-23 pathways guide the use of inhibitors like Secukinumab and Guselkumab, which are effective in inflammatory conditions related to spondyloarthropathies and other forms of NIU.2. Personalized Treatment: Biomarkers such as serum cytokine levels, tear protein profiles, and immune cell markers are useful in tailoring individualized treatment regimens. For example, tear cytokine profiling has shown promise in predicting the onset of uveitis in children with JIA, offering early intervention opportunities.3. Monitoring Disease Activity and Response to Treatment: Biomarkers such as sICAM-1, VEGF, and C-reactive protein (CRP) are used to monitor disease progression and assess the therapeutic response. Elevated levels of these markers may indicate ongoing inflammation, macular edema, or other complications in uveitis, allowing for timely adjustments in therapy.4. Improved Clinical Outcomes: The use of biomarkers in conjunction with biologic therapies helps in achieving better clinical outcomes. For example, monitoring cytokines and inflammatory markers can guide adjustments in biologic therapy dosage, improving visual acuity, reducing flare-ups, and controlling complications like macular edema. Ocular inflammation can arise from various conditions besides uveitis. These conditions include dry eye disease, scleritis, keratitis, conjunctivitis, and retinal diseases. These condition often have underlying molecular

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 13 www.dosonline.orgbiomarker that help in their diagnosis, monitoring, and management (Table-3).In summary, Ocular inflammation encompasses a variety of conditions, including uveitis, dry eye disease, scleritis, keratitis, and retinal diseases, each driven by different immune mechanisms. The inflammatory response in these conditions often involves the activation of cytokines, chemokines, and enzymes that lead to immune cell infiltration, tissue damage, and remodeling. For instance, cytokines like TNF-α, IL-1β, and IL-6 play a critical role in driving inflammation in uveitis, while MMPs (matrix metalloproteinases) are implicated in tissue degradation in diseases like dry eye and scleritis.Molecular biomarkers have emerged as a promising tool for diagnosing and monitoring ocular inflammation. Recent advances in proteomics have led to the identification of specific biomarkers associated with different forms of ocular inflammation. These include IL-23, TIMP-1, and TNF-α in uveitis, which help differentiate between various subtypes and guide treatment decisions. Additionally, tear cytokine profiling and aqueous humor sampling offer non-invasive and accurate means to assess disease activity and predict the risk of inflammation.Ocular inflammatory conditionMolecular Biomarkers Treatment Importance1. Dry Eye Disease (DED) -Pro-inflammatory cytokines:IL-1beta, IL-6-MMP-9 (Matrix Metalloproteinase-9)-SICCA Scores: Elevated expression of SAA (Serum Amyloid A), TIMP-1 (Tissue Inhibitor of Metalloproteinase)-Autoimmune Biomarkers: SS-A(Ro), SSB(La), acetylcholine receptor antibodies-Anti-inflammatory treatments: Cyclosporine A (Restasis), Lifitegrast, corticosteroids-Lubrication: Artifical tears, punctal plugs-Biologic therapies: Rituximab in autoimmune related DED2. Scleritis -Rheumatoid Factor (RF)-Anti-CCP (Cyclic Citrullinated Peptide)-MMP-2, MMP-3, MMP-9-Immunosuppressive: Methotrexate, coticosteroids-Biologic therapies: Rituximab, Abatacept3. Keratitis -Toll like receptors (TLRs)-IFN-gamma- Elevated in viral and autoimmune keratitis-Pro inflammatory cytokines: IL-6, IL-1 beta, TNF-alpha -Antibiotics/Antivirals-Corticosterois-Immunosuppressive agents: In autoimmune keratitis (e.g., cyclosporine, methotrexate)4. Ocular Rosacea -IL-8: Elevated in tear and ocular surface-MMPs-Topical antibiotics-Oral tetracyclines-Corticosteroids-Lifitegrast: For inflammation controlTable 3: Ocular biomarkers in conditions besides uveitis.The identification of molecular markers allows for more precise and targeted therapies, improving treatment outcomes and reducing unnecessary systemic side effects. As research progresses, targeted therapies such as IL-6 inhibitors, JAK inhibitors, and mTOR inhibitors show promise in managing ocular inflammatory diseases. Personalized approaches using molecular diagnostics will likely improve patient outcomes by tailoring treatment to individual biomarker profiles.References1. Scharf Y, Zonis S. Histocompatibility antigens (HLA) and uveitis. Surv Ophthalmol. 1980;24:220–8. doi: 10.1016/0039-6257(80)90043-0. [DOI].2. Indian J Ophthalmol.2020Aug 20;68(9):1750-1763. Interleukins and cytokine biomarkers in uveitis.doi: 10.4103/ijo.IJO_564_20.3. Baggiolini M, Dahinden CA. CC chemokines in allergic inflammation. Immunol Today. 1994;15:127–33. doi: 10.1016/0167-5699(94)90156-2. [DOI].4. Cocchi F, DeVico AL, Garzino-Demo A, Arya SK,

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 14 www.dosonline.orgGallo RC, Lusso P. Identification of RANTES, MIP1a, and MIP- 1b as the major HIV-suppressive factors produced by CD8+Tcells. Nature. 1995;270:1811–5. doi: 10.1126/science.270.5243.1811.5. Horai R, Caspi RR. Cytokines in autoimmune uveitis. J Interferon Cytokine Res. 2011;31:733–44. doi: 10.1089/jir.2011.0042.6. Ahn JK, Yu HG, Chung H, Park YG. Intraocular cytokine environment in active Behcet uveitis. Am J Ophthalmol. 2006;142:429–34. doi: 10.1016/j.ajo.2006.04.016.Sanjeev Kumar Nainiwal MD, DNB, MNAMS Senior Professor Ophthalmology Sawai Man Singh Medical College & Hospital, Jaipur RajasthanCorresponding Author:

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 15 www.dosonline.orgMolecular Markers in Various OcularInflammatory Diseases: Current PerspectivesVipin Rana[1] MS, DNB, MNAMS, FICO, MRCSEd, FAICO, MCh | Ranjit Goenka[1] MS | Amit Nandan Tripathi[1] MBBS |Ashish Markan[2] MD, MCh, FRCS | Manasi Tripathi[3] MD, FRCS 1. Department of Ophthalmology, Command Hospital (Eastern Command), Alipore, Kolkata, West Bengal, India2. Department of Ophthalmology, Dr. R. P. Centre for Ophthalmic Sciences, AIIMS, New Delhi3. Department of Ophthalmology, I Clinix, New Delhi, IndiaAbstract: Ocular inflammation comprises diverse diseases involving the uvea, sclera, cornea, retina, and orbit. Due to heterogeneous etiologies including autoimmune, infectious, and systemic inflammatory processes, these conditions pose significant diagnostic and therapeutic challenges. Conventional clinical methods, while foundational, often lack specificity and precision. The emergence of molecular biomarkers, including cytokines, chemokines, autoantibodies, genetic, and proteomic markers, has significantly advanced our understanding of ocular inflammatory diseases, providing avenues for improved diagnostic accuracy, treatment monitoring, personalized therapeutic strategies, and prognostication. This review extensively explores current molecular biomarkers across various ocular inflammatory conditions, emphasizing their clinical relevance, associated challenges, and promising future perspectives.Keywords: Ocular inflammation, Molecular biomarkers, Cytokines, Autoimmune ocular disease, Diagnostic biomarkersIntroductionOcular inflammation significantly impacts visual health globally, encompassing conditions such as uveitis, dry eye disease (DED), ocular graft-versus-host disease (oGVHD), scleritis, keratitis, autoimmune retinopathy (AIR), idiopathic orbital inflammatory syndrome (IOIS), and sarcoidosis-related ocular inflammation.[1,2,3]These diseases, if not promptly identified and effectively managed, can lead to severe and irreversible visual impairment.[1] Although traditional diagnostic approaches-including clinical examination, imaging, and basic laboratory tests-remain essential, they often fail to identify precise underlying pathogenic mechanisms or predict treatment responses reliably.[4] The advent and utilization of molecular biomarkers have significantly enhanced clinical management by offering more objective, quantifiable indicators of disease activity, severity, therapeutic efficacy, and prognosis. This comprehensive review will systematically detail the current knowledge surrounding these biomarkers, their specific clinical utility in ocular inflammatory diseases, the challenges encountered in their routine clinical application, and their future potential facilitated by emerging technologies.Molecular Biomarkers in Ocular InflammationMolecular biomarkers are measurable indicators reflecting biological processes, disease states, and responses to therapy. In ocular inflammation, these biomarkers can be categorized into several key groups:Cytokines and ChemokinesCytokines and chemokines are critical mediators in inflammatory responses, orchestrating immune activity by modulating cell signaling pathways.[2]Among cytokines, pro-inflammatory types such as interleukin-6 (IL-6) are essential in driving acute and chronic inflammatory responses through immune cell activation and increased vascular permeability.[4] Tumour Necrosis Factor-alpha (TNF-α) significantly contributes to tissue damage and leukocyte infiltration, especially in conditions like uveitis and scleritis.[5] Interferon-gamma (IFN-γ) facilitates macrophage and T cell activation, predominantly in autoimmune uveitis, while interleukin-1 beta (IL-1β) enhances vascular permeability and recruits leukocytes to inflamed ocular tissues.[4]Conversely, anti-inflammatory cytokines such as

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 16 www.dosonline.orginterleukin-10 (IL-10) and Transforming Growth Factorbeta (TGF-β) play crucial roles in moderating excessive inflammation. IL-10 inhibits pro-inflammatory cytokine production, whereas TGF-β maintains immune tolerance, reducing inflammatory damage.[6]Chemokines also play pivotal roles, notably CXCL8 (IL-8), which actively recruits neutrophils during acute inflammatory responses.[4] CCL2, or Monocyte Chemoattractant Protein-1, facilitates the movement and infiltration of monocytes and macrophages into inflamed ocular tissues.[6]Cellular Markers and Immune Cell ProfilingRecent advancements in immunophenotyping techniques allow detailed analysis of immune cell subsets participating in ocular inflammation. CD4+ T cells, specifically Th1 and Th17 subsets, are central in autoimmune uveitis, driving chronic inflammatory responses.[3] Regulatory T cells (Tregs) function critically in promoting immune tolerance and reducing excessive inflammatory reactions.[2] Additionally, macrophages and dendritic cells are key contributors to antigen presentation and initiating immune responses, while B cells and associated autoantibodies mediate antibody-driven inflammatory ocular disorders.[4]Genetic and Epigenetic BiomarkersGenetic susceptibility significantly influences ocular inflammatory conditions. Advances in genomics and epigenomics have identified numerous genetic markers associated with specific diseases.[7] Human Leukocyte Antigen (HLA) associations are particularly notable; for instance, HLA-B27 strongly correlates with anterior uveitis, particularly in patients with spondyloarthropathies. HLA-A29 represents the primary genetic risk factor for birdshot chorioretinopathy.[7] Additionally, microRNAs (miRNAs) like miR-146a and miR-155 regulate inflammatory pathways, emerging as promising biomarkers for autoimmune uveitis. miR-21 also plays a role in modulating immune responses within ocular inflammatory conditions.[8]Autoantibodies and Protein BiomarkersAutoantibodies provide important diagnostic and prognostic insights in ocular inflammation. Anti-retinal antibodies are characteristic of autoimmune retinopathies and certain uveitic conditions, while anti-phospholipid antibodies correlate with vasculitic manifestations in retinal inflammation.[9] Specific protein biomarkers, such as calprotectin, are elevated in conditions like uveitis, indicating neutrophil-driven inflammation.[10] Serum amyloid A, another systemic inflammatory marker, correlates with ocular involvement in systemic inflammatory conditions.[11]Tear and Aqueous Humour BiomarkersNon-invasive sampling of tears and aqueous humor offers valuable, localized insight into ocular inflammatory processes. Matrix Metalloproteinase-9 (MMP-9) serves as an indicator of corneal epithelial dysfunction commonly observed in dry eye disease and ocular surface inflammation. Vascular Endothelial Growth Factor (VEGF) is associated with neovascularization and is significantly elevated in inflammatory ocular diseases with pathological angiogenesis. Additionally, Intercellular Adhesion Molecule-1 (ICAM-1) expression relates directly to leukocyte migration and activity, playing a crucial role in ocular surface inflammation and immune cell recruitment.[2]Molecular Biomarkers in Specific Ocular DiseasesUveitis Uveitis encompasses inflammatory diseases of the uveal tract, classified anatomically as anterior, intermediate, posterior, or panuveitis, and etiologically into infectious or non-infectious categories.[12] The pathogenesis involves complex dysregulation of innate immunity, characterized by activation of Toll-like receptors (TLRs) and inflammasomes, resulting in cytokine release (IL-1β, IL-6, TNF-α).[13] Adaptive immunity predominantly engages Th1 and Th17 pathways, raising IFN-γ and IL-17 levels, respectively.[14] Reduced regulatory T cell (Treg) function and the generation of retinal-specific autoantibodies further exacerbate autoimmune inflammation.[15]Clinically, biomarkers are critical for precise diagnosis and management. Elevated IL-10 in vitreous fluid differentiates intraocular lymphoma from chronic posterior uveitis.[16] High TNF-α and IL-6 concentrations in aqueous humour signal active non-infectious inflammation, guiding targeted anti-TNF therapies like adalimumab or infliximab.[5] Elevated chemokines CXCL9 and CXCL10 specifically suggest autoimmune etiologies, including Behçet’s disease and Vogt-Koyanagi-Harada syndrome, aiding clinicians in initiating early immunosuppressive treatment.[17] Pathogen-specific cytokines, notably IL-8 and IFN-γ, facilitate rapid identification of infectious uveitis, ensuring timely antimicrobial interventions.Practically, ophthalmologists rely on multiplex cytokine assays from aqueous and vitreous samples for differential diagnosis.[4] For instance, elevated IL-10 levels combined with cytological analysis confirm intraocular lymphoma diagnosis, distinguishing it from chronic uveitis.[16]

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 17 www.dosonline.orgMonitoring cytokine changes during therapy provides objective evidence of treatment efficacy, guiding timely adjustments to corticosteroids or biologics.Dry Eye Disease (DED)Dry Eye Disease is characterized by tear film instability and chronic inflammation involving pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, as well as elevated MMP-9 levels.[18] Clinically, tear osmolarity distinguishes evaporative from aqueous-deficient DED, allowing targeted therapeutic approaches.[19] Elevated tear MMP9, detectable via point-of-care assays like InflammaDry, confirms inflammatory activity and practically guides initiation and monitoring of treatments such as cyclosporine or lifitegrast.[20] Serial measurement of tear biomarkers helps clinicians monitor therapeutic effectiveness, enabling precise management and improvement in patient quality of life.Ocular Graft-versus-Host Disease (oGVHD)Ocular GVHD is a post-transplant complication characterized by severe dry eye, conjunctival inflammation, and corneal epithelial damage. It arises from donor-derived T lymphocytes attacking host ocular tissues, leading to elevated pro-inflammatory cytokines (IL-6, TNF-α, IL-17), oxidative stress markers, and increased expression of matrix metalloproteinase-9 (MMP-9). Dysfunction in lacrimal glands, reflected by elevated B-cell activating factor (BAFF) and epidermal growth factor (EGF), further exacerbates tear film instability and ocular surface damage.[21]Practically, clinicians use biomarkers like IL-6, TNF-α, IL-17, and MMP-9 (InflammaDry test) for early disease detection, severity assessment, and therapeutic monitoring.[21,22] Regular biomarker evaluations help clinicians objectively tailor therapies such as corticosteroids, immunomodulators (cyclosporine, tacrolimus), and JAK inhibitors, improving patient outcomes by reducing inflammation, preserving visual function, and enhancing ocular surface comfort.[22]ScleritisScleritis frequently associates with systemic autoimmune disorders like rheumatoid arthritis and granulomatosis with polyangiitis.[23] Elevated biomarkers TNF-α, IL-6, ANCA, rheumatoid factor, and anti-citrullinated protein antibodies (ACPA) offer valuable diagnostic clues. Clinically, these biomarkers enable early identification of underlying systemic inflammation, prompting systemic evaluation and guiding initiation of immunosuppressive treatments.[24] Regular monitoring of these markers is practical for assessing systemic and ocular disease activity, facilitating timely therapeutic interventions.KeratitisKeratitis involves corneal inflammation, infectious or immune-mediated, with biomarkers IL-1β, IL-17, VEGF, and MMP-9 playing critical roles.[2] Elevated VEGF guides clinicians toward anti-angiogenic treatments in neovascular keratitis. Elevated MMP-9 practically aids clinicians in identifying active corneal inflammation, directing targeted anti-inflammatory or immunomodulatory therapy. Measurement of cytokines such as IL-17 provides practical differentiation of immune-mediated keratitis from infectious cases, enabling targeted antimicrobial or immunosuppressive treatment.[25]Autoimmune Retinopathy (AIR) AIR results from retinal degeneration driven by autoantibodies like anti-retinal antibodies (ARA) and inflammatory mediators such as CXCL10.[3] Clinically, detecting ARAs provides diagnostic clarity, differentiating AIR from hereditary retinal diseases. Elevated CXCL10 levels indicate active retinal inflammation and T-cell mediated injury, assisting clinicians practically in initiating timely immunosuppressive treatments.[26] Monitoring these biomarkers during therapy enables effective management and may help stabilize visual function.Idiopathic Orbital Inflammatory Syndrome (IOIS)IOIS features chronic idiopathic orbital inflammation, characterized by elevated biomarkers IL-6, TNF-α, TGF-β, and MMP-2/MMP-9. Practically, clinicians use these biomarkers to determine disease activity and the risk of fibrosis and tissue remodeling.[27] Early detection of elevated fibrosis markers guides clinicians towards aggressive therapeutic strategies, including corticosteroids and targeted immunosuppressants, effectively minimizing orbital structural damage and preserving ocular function.Sarcoidosis-Related Ocular InflammationSarcoidosis-related ocular inflammation involves systemic biomarkers like angiotensin-converting enzyme (ACE), serum lysozyme, IL-12, IL-18, TNF-α, and S100 proteins. Elevated ACE and serum lysozyme levels help confirm systemic sarcoidosis, aiding in early systemic disease identification and guiding systemic immunosuppressive treatments.[28] Serial measurement of cytokines like IL-12, IL-18, and TNF-α helps clinicians monitor granulomatous inflammation activity, enabling precise adjustments in therapy to reduce ocular and systemic complications.

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 18 www.dosonline.orgFuture Perspectives and ChallengesDespite the significant clinical potential of molecular biomarkers, their widespread adoption faces several critical challenges. A major barrier remains the standardization of biomarker assays, as variability in sensitivity and specificity across laboratories and populations can significantly impact reliability. Achieving consensus on standardized protocols and assay validation processes is imperative to ensure reproducibility and comparability of biomarker results across clinical settings.Integration of multi-omics approaches-encompassing genomics, proteomics, and metabolomics-also represents a promising yet complex challenge. These comprehensive strategies could substantially enhance biomarker discovery, improving diagnostic precision and therapeutic personalization. However, the integration of diverse data streams requires substantial advancements in bioinformatics and computational methodologies, emphasizing the need for continued research and interdisciplinary collaboration.Another critical hurdle is the cost and accessibility of advanced molecular diagnostics. Currently, many molecular biomarker assays remain expensive, limiting their availability in routine clinical practice, particularly in resource-limited settings. Reducing costs through technological innovations and developing streamlined, affordable testing methods will be essential to facilitate broader clinical implementation and benefit a more extensive patient population.ConclusionThe integration of molecular biomarkers into the clinical management of ocular inflammatory diseases marks a transformative advancement, significantly enhancing diagnostic precision, therapeutic monitoring, personalized care, and prognostic accuracy. While significant challenges remain, particularly concerning assay standardization, multi-omics integration, and accessibility, ongoing research and technological innovations hold substantial promise to overcome these barriers. Continued advancement in biomarker research will undoubtedly enable more precise, effective, and personalized ophthalmic care, ultimately improving clinical outcomes and quality of life for patients suffering from ocular inflammatory conditions.References1. Bansal R, Gupta A. Protein Biomarkers in Uveitis. Front Immunol. 2020 Dec 3;11:610428. doi: 10.3389/fimmu.2020.610428.2. Tamhane M, Cabrera-Ghayouri S, Abelian G, Viswanath V. Review of Biomarkers in Ocular Matrices: Challenges and Opportunities. Pharm Res. 2019 Jan 23;36(3):40. doi: 10.1007/s11095-019-2569-8.3. Grange L, Dalal M, Nussenblatt RB, Sen HN. Autoimmune retinopathy. Am J Ophthalmol. 2014 Feb;157(2):266-272.e1. doi: 10.1016/j.ajo.2013.09.019. Epub 2013 Sep 29.4. Wakefield D, Lloyd A. The role of cytokines in the pathogenesis of inflammatory eye disease. Cytokine. 1992 Jan;4(1):1-5. doi: 10.1016/1043-4666(92)90028-p.5. Jiang Q, Li Z, Tao T, Duan R, Wang X, Su W. TNF-α in Uveitis: From Bench to Clinic. Front Pharmacol. 2021 Nov 2;12:740057. doi: 10.3389/fphar.2021.740057. 6. Al-Qahtani AA, Alhamlan FS, Al-Qahtani AA. ProInflammatory and Anti-Inflammatory Interleukins in Infectious Diseases: A Comprehensive Review. Tropical Medicine and Infectious Disease. 2024; 9(1):13. doi:10.3390/tropicalmed9010013.7. Thorsby E, Lie BA. HLA associated genetic predisposition to autoimmune diseases: Genes involved and possible mechanisms. Transpl Immunol. 2005 Aug;14(3-4):175-82. doi: 10.1016/j.trim.2005.03.021..8. Wei Y, Li N, Zhao L, Yang C, Ma B, Li X, Wei R and Nian H (2020) MicroRNAs and AutoimmuneMediated Eye Diseases. Front. Cell Dev. Biol. 8:818. doi: 10.3389/fcell.2020.00818.9. Cifuentes-González C, Uribe-Reina P, Reyes-Guanes J, Muñoz-Ortiz J, Muñoz-Vargas PT, Rojas-Carabali W, Nova-Florián DV, De-Los-Ríos AS, MantillaHernández RD, de-la-Torre A. Ocular Manifestations Related to Antibodies Positivity and Inflammatory Biomarkers in a Rheumatological Cohort. Clin Ophthalmol. 2022 Aug 9;16:2477-2490. doi: 10.2147/OPTH.S361243.10. Gazim CC, Borba AA, Castro GKP, Simioni J, Gehlen ML, Ramos Júnior O, Skare TL. Fecal calprotectin levels in acute anterior uveitis in patients with spondyloarthritis. Arq Bras Oftalmol. 2023 JanFeb;86(1):33-37. doi: 10.5935/0004-2749.20230008.11. Sorić Hosman I, Kos I, Lamot L. Serum Amyloid A in Inflammatory Rheumatic Diseases: A Compendious Review of a Renowned Biomarker. Front Immunol. 2021 Feb 19;11:631299. doi: 10.3389/fimmu.2020.12. Chen SC, Sheu SJ. Recent advances in managing and

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 19 www.dosonline.orgunderstanding uveitis. F1000Res. 2017 Mar 16;6:280. doi: 10.12688/f1000research.10587.1.13. Pratap DS, Lim LL, Wang JJ, Mackey DA, Kearns LS, Stawell RJ; Wellcome Trust Case Control Consortium 2; Burdon KP, Mitchell P, Craig JE, Hall AJ, Hewitt AW. The role of toll-like receptor variants in acute anterior uveitis. Mol Vis. 2011;17:2970-7. Epub 2011 Nov 16.14. Luger D, Silver PB, Tang J, Cua D, Chen Z, Iwakura Y, Bowman EP, Sgambellone NM, Chan CC, Caspi RR. Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category. J Exp Med. 2008 Apr 14;205(4):799-810. doi: 10.1084/jem.20071258. 15. Lee AYJ and Foulsham W (2022) Regulatory T Cells: Therapeutic Opportunities in Uveitis. Front. Ophthalmol. 2:901144. doi: 10.3389/fopht.2022.901144.16. Cassoux N, Giron A, Bodaghi B, Tran TH, Baudet S, Davy F, Chan CC, Lehoang P, Merle-Béral H. IL-10 measurement in aqueous humor for screening patients with suspicion of primary intraocular lymphoma. Invest Ophthalmol Vis Sci. 2007 Jul;48(7):3253-9. doi: 10.1167/iovs.06-0031.17. Huang Y, Yu H, Cao Q, Deng J, Huang X, Kijlstra A, Yang P. The Association of Chemokine Gene Polymorphisms with VKH and Behcet’s Disease in a Chinese Han Population. Biomed Res Int. 2017;2017:1274960. doi: 10.1155/2017/1274960.18. Enríquez-de-Salamanca A, Bonini S, Calonge M. Molecular and cellular biomarkers in dry eye disease and ocular allergy. Curr Opin Allergy Clin Immunol. 2012 Oct;12(5):523-33. doi: 10.1097/ACI.0b013e328357b488. 19. Lemp MA, Bron AJ, Baudouin C, Benítez Del Castillo JM, Geffen D, Tauber J, Foulks GN, Pepose JS, Sullivan BD. Tear osmolarity in the diagnosis and management of dry eye disease. Am J Ophthalmol. 2011 May;151(5):792-798.e1. doi: 10.1016/j.ajo.2010.10.032.20. Lanza NL, Valenzuela F, Perez VL, Galor A. The Matrix Metalloproteinase 9 Point-of-Care Test in Dry Eye. Ocul Surf. 2016 Apr;14(2):189-95. doi: 10.1016/j.jtos.2015.10.004.21. Bohlen J, Gomez C, Zhou J, Martinez Guasch F, Wandvik C, Sunshine SB. Molecular Biomarkers in Ocular Graft-versus-Host Disease: A Systematic Review. Biomolecules. 2024 Jan 12;14(1):102. doi: 10.3390/biom14010102.22. Cheng X, Huang R, Huang S, Fan W, Yuan R, Wang X, Zhang X. Recent advances in ocular graft-versus-host disease. Front Immunol. 2023 Jan 25;14:1092108. doi: 10.3389/fimmu.2023.1092108.23. Smith JR, Mackensen F, Rosenbaum JT. Therapy insight: scleritis and its relationship to systemic autoimmune disease. Nat Clin Pract Rheumatol. 2007 Apr;3(4):219-26. doi: 10.1038/ncprheum0454. 24. Das UN. Molecular pathobiology of scleritis and its therapeutic implications. Int J Ophthalmol. 2020 Jan 18;13(1):163-175. doi: 10.18240/ijo.2020.01.23.25. Suryawanshi A, Veiga-Parga T, Reddy PB, Rajasagi NK, Rouse BT. IL-17A differentially regulates corneal vascular endothelial growth factor (VEGF)-A and soluble VEGF receptor 1 expression and promotes corneal angiogenesis after herpes simplex virus infection. J Immunol. 2012 Apr 1;188(7):3434-46. doi: 10.4049/jimmunol.1102602.26. Detrick B, Gangaputra S, Palsgrove DN, Heaney CD, Hooks JJ, Nida Sen H. Elevated serum levels of IL-6 and CXCL9 in autoimmune retinopathy (AIR) patients. J Neuroimmunol. 2018 Mar 15;316:74-79. doi: 10.1016/j.jneuroim.2017.12.014.27. Fang Y, Shen B, Dai Q, Xie Q, Wu W, Wang M. Orbital inflammatory pseudotumor: new advances in diagnosis, pathogenesis, and treatment. Eur J Med Res. 2023 Oct 4;28(1):395. doi: 10.1186/s40001-023-01330-0.28. Kraaijvanger R, Janssen Bonás M, Vorselaars ADM, Veltkamp M. Biomarkers in the Diagnosis and Prognosis of Sarcoidosis: Current Use and Future Prospects. Front Immunol. 2020 Jul 14;11:1443. doi: 10.3389/fimmu.2020.01443. Vipin Rana MS, DNB, MNAMS, FICO,MRCSEd, FAICO, MCh Associate Professor & Vitreoretinal SurgeonDepartment of Ophthalmology, Command Hospital, Kolkata, C/O 56 APO, IndiaCorresponding Author:

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 20 www.dosonline.orgOcular Surface Inflammation and Molecular Markers: A Comprehensive ReviewAastha Garg MS | Abha Gour DOMSDepartment of Cornea & Anterior Segment, Dr Shroffs Charity Eye Hospital, New DelhiIntroductionThe ocular surface comprises the cornea, conjunctiva, the limbus, meibomian glands, and the ocular adnexa. The epithelium of the ocular surface structures supports the tear film and helps maintain the homeostasis for a healthy ocular surface.[1] Inflammation of the ocular surface is a pathological condition causing dry eye disease (DED). The clinical subtypes of DED include meibomian gland disease (MGD); autoimmune conditions like Sjögren’s syndrome (SS) and Steven–Johnson syndrome (SJS); and DED induced by medical or surgical intervention.[2] Another cause of ocular surface inflammation (OSI) includes allergic conjunctivitis i.e. atopic keratoconjunctivitis (AKC) and vernal keratoconjunctivitis (VKC) caused by the involvement of innate immunity. Recently, analysis of the molecular markers in tear film represents an innovative approach as a non-invasive diagnostic and prognostic tool.[3] Impression cytology, on the other hand, is a minimally invasive technique and is considered the gold standard for analyzing cell structure and morphology.[4]Advantage of Tears as a SampleTear film sampling offers a promising approach to objectively measuring ocular surface inflammation. Sample collection is non-invasive, and the tears have a molecular-rich composition comprising many proteins, lipids, metabolites, and electrolytes that reflect the physiological and pathological states of the ocular surface. The simplicity of sample collection also allows for repeated sampling, hence assisting in evaluating the disease progression and the response to medication.[5]Sample collection techniques, including the Schirmer strip-based dry method, capillary tube-based method, and buffer wash method, allow for efficient and cost-effective sampling. Analytical techniques like mass spectrometry and antibody microarrays enable precise quantification of tear biomarkers, enhancing the reliability of tear analysis in clinical diagnostics. (Figure-1)Figure 1: Depicting the techiques of tear collection and analysisPathophysiology of Ocular Surface InflammationThe tear film stability is affected by osmotic, mechanical, and inflammatory damage. Increased stress to the ocular surface epithelial cells causes an increase in the tear osmolarity, activating intracellular signaling pathways that trigger the production of proinflammatory cytokines, such as interleukin (IL)-1, tumor necrosis factor (TNF), and IL-6. This resulting proinflammatory environment promotes the activation and maturation of immature antigen-presenting cells (iAPCs). The subsequently activated and matured antigen-presenting cells (mAPCs) cause the priming of the naive T cells to CD4+ helper T cells and Th 17 cells in the lymphoid tissue, which on infiltrating the ocular surface, secrete additional proinflammatory cytokines such as interferon-gamma (IFNy) and IL-17 respectively which further facilitate the production of proinflammatory cytokines, chemokines, matrix metalloproteinases (eg, MMP-3 and MMP-9), cell adhesion molecules (CAM), and pro-lymphangiogenic molecules (vascular endothelial growth factor [VEGF] D and VEGF-C).[1] This immune activation disrupts the

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 21 www.dosonline.orgocular surface barrier, reduces goblet cell density, and impairs tear film stability, starting a vicious cycle of chronic inflammation and progressive ocular damage.Molecular Markers in Tear FluidTear fluid contains a diverse array of molecular markers that reflect the inflammatory milieu of the ocular surface. Proinflammatory cytokines, such as IL-1β, IL-6, and TNF-α, are consistently elevated in OSI and serve as biomarkers of disease severity. These cytokines promote epithelial apoptosis, enhance leukocyte recruitment, and stimulate the production of MMPs, which degrade the extracellular matrix and compromise epithelial integrity. Chemokines such as CCL2 and CXCL8 play a crucial role in immune cell trafficking, further exacerbating inflammation. The CXCL12/CXCR4 axis, which regulates leukocyte migration, has been implicated in chronic OSI and tissue remodeling. MMP-9, the most frequently studied tear biomarker, is elevated in DED, MGD, and allergic conjunctivitis. Its upregulation correlates with corneal epithelial damage and disease severity, making it a valuable diagnostic and prognostic indicator.Biomarkers of Ocular Surface Inflammation in Different Disease Conditions1. Dry Eye Disease (DED)a. Analysis of the Tear Fluid: Cytokines: Regulate tissue growth, migration, differentiation, and development • Upregulated cytokines: as discussed above, mature APCs secrete TNFα, IL-6, IL-1. IL 1β is involved in cellular apoptosis and pain hypersensitivity and IL-17 induces the production of IL-6. • Dysregulated cytokines: IL 2, IL 4, IL 9, IL 10, and IFN-γ • IL-10 is a protective factor and has shown varied expression patterns across different studies. It can increase, decrease or show no change in its expression. Chemokines: cause migration of immune cells• CXCL8/IL-8, CCL5/RANTES, IP-10/CXCL10, MCP1/CCL2, MIP1α/CCL3, CX3CL1 are usually overexpressed. RANTES promotes the movement of mature antigen-presenting cells (APCs) and leukocytes to inflamed areas. MCP-1 plays a key role in guiding the migration and infiltration of monocytes. IL-8, a well-known proinflammatory chemokine produced by mononuclear macrophages, epithelial cells, and fibroblasts, contributes to fibrosis, neovascularization, and endothelial dysfunction, and its levels are positively associated with the severity of various ocular diseases. Soluble receptors, cell adhesion molecules and enzymes• ICAMs & MMPs are elevated in corneal epithelium, conjunctival cells, and lacrimal glands. ICAM-LFA1 interaction aids immune cell recruitment and cytokine activation.• sTNFR1 (soluble TNF receptor 1), which is expressed by corneal epithelium and stromal fibroblast, has been seen to be significantly higher in such patients. It usually reduces TNFα signaling by removing surface TNFR1. In DED, both TNFR1 and sTNFR1 are upregulated which paradoxically promotes inflammation and cell death.• MMPs and ECM remodeling: MMPs, especially MMP9 are upregulated due to inflammation causing degradation of epithelial tight junction proteins, contributing to corneal barrier dysfunction and worsening DED symptoms.Neurotrophic Factors:• Neuropeptides like serotonin, calcitonin generelated peptide (CGRP), nerve growth factor (NGF), and substance P are involved in sensory signaling and tissue repair. Elevated serotonin in DED tears correlates with symptom severity and may enhance corneal sensitivity. NGF, often increased, supports tear secretion and epithelial healing, while reduced CGRP is linked to lacrimal gland dysfunction. b. Impression Cytology: Impression cytology (IC) is used to detect morphological changes in ocular surface diseases (OSDs), especially dry eye disease (DED), such as goblet cell loss and epithelial cell enlargement. In DED, IC helps identify squamous metaplasia, marked by goblet cell loss, increased keratinization, and epithelial cell changes reflecting altered cell differentiation due to reduced tear production. IC can quantify inflammatory markers like HLA-DR, which is upregulated in DED and correlates with disease severity. Studies show IC is a reliable method for detecting HLA-DR expression, making it helpful in diagnosing inflammation and monitoring therapeutic outcomes in DED, including treatments like cyclosporine, corticosteroids, and omega fatty acids. ICAM-1, like HLA-DR, is upregulated in ocular surface inflammation and may serve as a biomarker for DED, with increased expression correlating negatively with goblet cell count

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 22 www.dosonline.organd tear production. Mucin genes such as MUC1and MUC5AC are significantly reduced in DED.[4]2. Allergic Conjunctivitis: It is inflammatory response of ocular surface involving early and late phase immune response. Early phase involves the allergen interacting with the mast cells, causing their degranulation and further causing the release of histamines and cytokines (IL-4 mainly). This in turn promotes T-cell activation and IgE production. The late phase is subsequently set off which is characterized by infiltration of immune cells into the conjunctiva (mainly eosinophils, basophils and Th-2 lymphocytes). Chemokines play a key role in this late phase reaction by guiding the leukocyte migration into the sites of inflammation. a. Analysis of Tear Fluid:• Cytokines: IL-4 as discussed above is central to Th2 immune response. It causes the B cell class switching to IgE, primes mast cells and increases the vascular permeability of VCAM-1. IL-4 and IL-13 induce fibroblast proliferation which contributes significantly to papillae formation and mucin secretion. IL-5 causes proliferation, maturation and recruitment of eosinophils to the ocular surface. IL-6 trans-signalling promotes inflammation, vascular permeability and mast cell proliferation. Cytokines such as IL-9, IL-10, IL-13, IL-25, IL-31, IL-33, and TSLP (Thymic Stromal Lymphopoietin) also play a critical role in allergic conjunctivitis. While, IL-9 and IL-33 enhances mast cell activity and promotes goblet cell hyperplasia, IL10 counteracts inflammation by suppressing Th2 response and mast cell degranulation. IL-31 is responsible for mediating itch and TSLP stimulates dendritic cells, mast cells, and sensory nerves, intensifying Th2 inflammation and pruritus.[6]• Chemokines: CXCL8/IL-8 attracts neutrophils, basophils, and T cells. CCL11/Eotaxin-1 is a eosinophil chemoattractant and is highly expressed in chronic vernal keratoconjunctivits (VKC). CCL5/RANTES, CCL2/MCP-1, CCL7/MCP-3 cause recruitment of monocytes and eosinophils. CCL24/Eotaxin-2, CCL26/Eotaxin-3 are mainly seen to be upregulated in VKC and is involved in eosinophil activation and chemotaxis.[7]• Proteomic of Tear Fluid:Biomarker Type Biomarker role Protease-Activated Receptor-2 (PAR-2) Receptor protein Barrier integrity/inflammatory marker Heat Shock Protein-70 (HSP-70) Stress-response protein Stress inflammatory marker Eosinophil Cationic Protein (ECP) Eaosinophile- derived granule Eosinophilic inflammatory marker b. Impression Cytology: In atopic keratoconjunctivitis (AKC), impression cytology has shown inflammatory cell infiltration and epithelial changes like eosinophil and neutrophil invasion linked to squamous metaplasia and corneal damage. In VKC, elevated mRNA levels of eotaxin-1,2,3, CCL-20 and IL-8 have been seen.[4] 3. Meibomian Gland Dysfunction (MGD)Pathophysiology and Biomarker Profile:MGD is a chronic inflammatory condition characterized by abnormal meibum secretion, gland obstruction, and evaporative tear loss. The resulting tear film instability triggers ocular surface inflammation and damage.• Lipid Mediators:Lipid mediators play a major role in MGD pathophysiology. Proinflammatory lipid mediators, including prostaglandins (PGE2, PGD2) and leukotrienes (LTB4, LTC4), are elevated in tear fluid. These mediators promote inflammation, increase vascular permeability, and contribute to ocular pain.• Cytokines and Chemokines:Tear fluid of MGD patients shows increased levels of IL-1β, IL-6, and TNF-α, reflecting chronic inflammation. Chemokines such as CCL2 and CXCL12 are upregulated, promoting immune cell infiltration and tissue remodeling.• Matrix Metalloproteinases:MMP-9 levels are significantly elevated in MGD, correlating with meibomian gland obstruction and ocular surface damage. MMP-9 degrades the extracellular matrix, contributing to tissue remodeling and glandular dysfunction.

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 23 www.dosonline.org4. ConjunctivochalasisPathophysiology and Biomarker Profile:Conjunctivochalasis (CCh) is a chronic ocular surface condition characterized by redundant and loose conjunctival tissue. It leads to tear film instability, ocular irritation, and chronic inflammation.• Cytokines and Chemokines:Tear cytokine profiles in conjunctivochalasis patients show elevated IL-1β and IL-6 levels, indicating chronic inflammation. These cytokines contribute to fibroblast activation, tissue remodeling, and conjunctival laxity.• Matrix Metalloproteinases:Increased MMP-9 expression is associated with tissue degradation and conjunctival laxity in CCh. MMP-9 activity leads to extracellular matrix breakdown, exacerbating the condition.5. Ocular RosaceaPathophysiology and Biomarker Profile:Ocular rosacea is a chronic inflammatory condition associated with meibomian gland dysfunction, telangiectasia, and recurrent eyelids and ocular surface inflammation.• Cytokines and Chemokines:Patients with ocular rosacea exhibit elevated IL-1β, IL-6, and TNF-α tear levels, reflecting chronic inflammation. CXCL1 and CXCL8 are also increased, promoting neutrophil recruitment and tissue damage.• Lipid Mediators:Tear fluid in ocular rosacea contains increased prostaglandins and leukotrienes, contributing to chronic inflammation, vasodilation, and ocular redness.6. Infectious KeratitisPathophysiology and Biomarker Profile:Infectious keratitis is an acute inflammatory condition caused by bacterial, viral, fungal, or parasitic pathogens, leading to corneal inflammation and tissue damage.• Cytokines and Chemokines:Tear cytokine profiles in infectious keratitis show elevated IL-1β, IL-6, and TNF-α, which promote immune cell infiltration and inflammatory tissue damage. CXCL8 (IL-8) is upregulated, recruiting neutrophils to the infection site.• Matrix Metalloproteinases:Increased MMP-9 activity in tear fluid contributes to corneal tissue breakdown and ulceration. Elevated MMP-9 levels are associated with disease severity and poor clinical outcomes.7. Stevens-Johnson Syndrome (SJS) and Ocular Cicatricial Pemphigoid (OCP)Pathophysiology and Biomarker Profile:SJS and OCP are severe immune-mediated disorders characterized by chronic inflammation, conjunctival scarring, and ocular surface damage.• Cytokines and Chemokines:Tear fluid in SJS and OCP patients contains elevated levels of IL-1β, IL-6, and TNF-α, indicating chronic inflammation. Increased CXCL12 and CCL2 expression promotes fibroblast activation and conjunctival fibrosis.• Matrix Metalloproteinases:MMP-9 and MMP-3 levels are elevated, contributing to extracellular matrix degradation and fibrosis. These biomarkers correlate with disease severity and progression.Clinical Applications of Tear BiomarkersTear biomarkers have emerged as valuable clinical tools for diagnosing, monitoring, and managing OSI. MMP-9, in particular, is used as a clinical biomarker for DED, with its elevated levels indicating ocular surface damage and disease severity. Quantifying tear cytokines, such as IL-6 and TNF-α, offers insights into the effectiveness of anti-inflammatory therapies. Biomarker profiling also allows for personalized medicine approaches, enabling clinicians to tailor treatments based on individual inflammatory profiles. For example, patients with elevated MMP-9 levels may benefit from corticosteroid or cyclosporine therapy, while those with increased Th17-associated cytokines may respond better to IL-17 inhibitors. Furthermore, tear biomarkers are increasingly being incorporated into clinical trials as objective metrics for evaluating treatment efficacy and monitoring disease progression.Emerging Biomarkers and Future DirectionsAdvancements in proteomics and metabolomics are expanding the repertoire of tear biomarkers for OSI. Heat shock proteins (HSPs), which are upregulated in response to cellular stress, are being investigated as potential biomarkers of OSI. Soluble receptors, including sICAM-1 and sTNFR, have shown promise in reflecting chronic inflammation and immune activation. MicroRNAs (miRNAs) in tear fluid are also emerging

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 24 www.dosonline.orgas diagnostic biomarkers, with their expression profiles correlating with disease severity and treatment response. Future research should focus on large-scale validation of these novel biomarkers, as well as the development of point-of-care diagnostic tests for rapid biomarker detection in clinical settings.ConclusionDifferent ocular surface diseases exhibit distinct biomarker profiles, making tear fluid analysis a valuable tool for diagnosis and disease monitoring. Elevated MMP-9, IL-1β, and TNF-α levels are common in chronic inflammatory diseases such as DED and MGD, while allergic conjunctivitis is associated with Th2 cytokines (IL-4, IL-5, IL-13). Lipid mediators play a prominent role in MGD and allergic conditions, while tear cytokines and MMPs serve as reliable biomarkers of inflammation and disease severity across multiple ocular surface diseases. The ongoing identification of novel biomarkers holds promise for personalized medicine approaches in ocular surface disease management.References1. Stevenson W, Chauhan SK, Dana R. Dry eye disease: an immune-mediated ocular surface disorder. Arch Ophthalmol 2012;130(1):90–100. 2. Kumar NR, Praveen M, Narasimhan R, Khamar P, D’Souza S, Sinha-Roy A, et al. Tear biomarkers in dry eye disease: Progress in the last decade. Indian J Ophthalmol 2023;71(4):1190–202. 3. Di Zazzo A, Micera A, De Piano M, Cortes M, Bonini S. Tears and ocular surface disorders: Usefulness of biomarkers. Journal of Cellular Physiology 2019;234(7):9982–93. 4. Hagan S. Biomarkers of Ocular Surface Disease Using Impression Cytology. Biomarkers in Medicine 2017;11(12):1135–47. 5. Can tear analysis drive a clinical decision? – A real‐world experience | Request PDF. ResearchGate [Internet] 2025 [cited 2025 Apr 9];Available from: https://www.researchgate.net/publication/388182188_Can_tear_analysis_drive_a_clinical_decision_-_A_real-world_experience.6. Chigbu DI, Karbach NJ, Abu SL, Hehar NK. Cytokines in Allergic Conjunctivitis: Unraveling Their Pathophysiological Roles. Life (Basel) 2024;14(3):350. 7. Erdinest N, London N, Solomon A. Chemokines in allergic conjunctivitis. Curr Opin Allergy Clin Immunol 2020;20(5):516–27.Abha Gour DOMSDepartment of Cornea & Anterior SegmentDr Shroff’s Charity Eye Hospital, New DelhiCorresponding Author:

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 25 www.dosonline.orgThe Continuous Fight of Inflammatory and Anti-Inflammatory Markers in Dry EyesMansi Jand DNB | Sonu Beniwal MBBS, DNB | Bharti Arya MBBS, MSDepartment of Ophthalmology, Medanta, Medicty, GurgaonDry eye is a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. It is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface according to Dry eye workshop (DEWS-II).Baudouin et al.[1] proposed the concept of a ‘‘vicious cycle of inflammation’’ as a core driver in dry eye and breaking the cycle was reported to be an important step in the treatment of dry eye.Proinflammatory CytokinesThey are present in tears and may have a key role in the pathogenesis of several corneal diseases, including dry eye disease.[3] There is increased level of proinflammatory cytokines, such as IL-1, IL-6, and IL-8, and decreased epidermal growth factor (EGF) levels in eyes with Sjogren’s syndrome.[4] Hyperosmolar stress also has a direct proinflammatory effect on the ocular surface that increases the tear cytokine levels.[5]Anti-Inflammatory TherapyBased on the concept that inflammation is a key component of the pathogenesis of dry eye, the efficacy of a number of anti-inflammatory agents for treatment of dry eye disease has been evaluated.CyclosporineThe potential of cyclosporine-A (CsA) for treating dry eye disease was initially recognized in dogs that develop spontaneous Keratoconjunctivitis sicca.[6] The therapeutic efficacy of CsA for human Keratoconjunctivitis sicca was then documented in several small, single centre, randomized, double-masked clinical trials.[7,8]In a Phase 2 clinical trial, four concentrations of CsA (0.05%, 0.1%, 0.2%, or 0.4%) administered twice daily to both eyes of 129 patients for 12 weeks was compared to vehicle treatment of 33 patients.[9] CsA was found to significantly decrease conjunctival rose Bengal staining, superficial punctate keratitis, and ocular irritation symptoms (sandy or gritty feeling, dryness, and itching) in a subset of 90 patients with moderate-to-severe Keratoconjunctivitis sicca. There was no clear dose response; CsA 0.1% produced the most consistent improvement in objective endpoints, whereas CsA 0.05% gave the most consistent improvement in patient symptoms (Level I).CorticosteroidsThey act as an effective anti-inflammatory therapy in dry eye disease. In a 4-week, double-masked, randomized study in 64 patients with Keratoconjunctivitis sicca and delayed tear clearance, loteprednol etabonate 0.5% ophthalmic suspension (Lotemax [Bausch and Lomb, Rochester, NY]), q.i.d., was found to be more effective than its vehicle in improving signs and symptoms of dry eyes.[10]Examples are-1% non-preserved Methylprednisolone, .0.5% loteprednol etabonate, 0.1% FML, .0.01% dexamethasone.TetracyclinesProperties of Tetracyclines and their derivatives are antibacterial Properties, anti-Inflammatory Properties and anti-angiogenic Properties.Clinical Applications of Tetracycline • Acne Rosacea, including its ocular manifestations, is an inflammatory disorder, occurring mainly in adults, with peak severity in the third and fourth decades.• Chronic Posterior Blepharitis: Meibomianitis, Meibomian Gland Dysfunction Chronic blepharitis is typically characterized by inflammation of the eyelids. Use of tetracycline in patients with meibomianitis has been shown to decrease lipase production by tetracy-

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 26 www.dosonline.orgcline sensitive as well as resistant strains of staphylococci. This decrease in lipase production is associated with clinical improvement[11]TacrolimusIt is a macrolide produced by Streptomyces tsukubaensis, was discovered in 1984 in Japan while searching for new immunosuppressive and cancer chemotherapeutic agents. Like cyclosporine, it blocks T-lymphocyte activity, but its immunosuppressive potential is higher than that of cyclosporine.In an open-label, prospective study with 14 patients with GVHD with severe DED and intolerance to topical cyclosporine, patients were instructed to instilled 0.03% topical tacrolimus once a day for three months.[1] Dry eye symptoms and signs improved.LubricinLubricin (proteoglycan-4) is a lubricating, mucin like glycoprotein that was first identified in synovial fluid.[13]More recently, lubricin has been discovered on the ocular surface and in the meibomian glands.[14] It is a highly effective friction reducing boundary lubricant, at both synthetic and tissue surfaces, functions synergistically with Hyaluronic acid.Currently, no lubricin-based lubricants are commercially avail able.Recombinant Human Nerve Growth Factor (RH-NGF)NGF is involved in the regulation of growth, proliferation and survival of neurons and is found naturally in tears. NGF has been reported to have trophic effects on the ocular surface, through activation of tropomyosin receptor kinase A and p75 neurotrophin receptor.[15]Tavilermide (MIM-D3) is a small-molecule NGF peptidomimetic that increases tear and mucin-like fluids and reduces corneal fluorescein staining.LifitegrastIt is a small molecule integrin antagonist, engineered to mimic ICAM-1’s binding domain to LFA-1 and believed to act as a competitive antagonist to block binding between LFA-1 and ICAM 1, resulting in inhibition of T cell migration into target tissues, reduction of cytokine release, and reduction of further T cell recruitment.[16-18]Role of Anti-Inflammatory Therapy in Dry EyesDry eye is associated with ocular surface inflammation compromising tear secretion and causing ocular surface disease and irritation symptoms. Ocular surface inflammation should be assumed in patients with an unstable tear film and ocular surface epithelial disease that is detected by staining with diagnostic dyes. Anti-inflammatory therapy can be considered for patients using artificial tears who continue to have clinically detectable ocular surface disease, particularly if inflammatory signs (for example, conjunctival redness) and irritation symptoms are present. Among the treatment options, topical corticosteroids appear to have the most rapid onset of action (2 to 4 weeks). They can be used concomitantly with CsA, a drug which requires several weeks to produce a clinical therapeutic effect and up to 6 months for maximum improvement.Figure 1: Inflammatory vicious cycle in dry eye. The inflammatory vicious cycle associated with dry eye that consists of tear instability, tear hyperosmolarity, apoptosis of the conjunctival/corneal cells, neurogenic inflammation and cytokine release (adapted from Pflugfelder et al. 2017).[2] The cycle can be initiated and amplified by intrinsic and extrinsic factors.Figure 2: Inflammatory markers in dry eyes and their target molecules.

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 27 www.dosonline.orgReferences1. Baudouin C. A new approach for better comprehension of diseases of the ocular surface [in French]. J Fr Ophtalmol. 2007;30:239–246.2. Pflugfelder SC, de Paiva CS. The pathophysiology of dry eye disease: what we know and future directions for research. Ophthalmology 2017;124:S4–S13.3. VanDerMeid KR, Su SP, Krenzer KL, Ward KW, Zhang JZ. A method to extract cytokines and matrix metalloproteinases from Schirmer strips and analyze using Luminex. Mol Vis. 2011;17:1056–1063.4. Pflugfelder SC, Jones D, Ji Z, Afonso A, Monroy D. Altered cytokine balance in the tear fluid and conjunctiva of patients with Sjogren’s syndrome keratoconjunctivitis sicca. Curr Eye Res. 1999;19:201–211.5. Luo L, Li DQ, Corrales RM, Pflugfelder SC. Hyperosmolar saline is a proinflammatory stress on the mouse ocular surface. Eye Contact Lens. 2005;31:186–193.6. Kaswan rl, Salisbury Ma, Ward Da. Spontaneous canine keratoconjunctivitis sicca. a useful model for human keratoconjunctivitis sicca: treatment with cyclosporine eye drops. Arch Ophthalmol 1989;107:1210-16 (BS2) .7. Gunduz K, Ozdemir O. Topical cyclosporin treatment of keratoconjuncti vitis sicca in secondary Sjogren’s syndrome. Acta Ophthalmol 1994;72:38 42 (CS2).8. laibovitz ra, Solch S, andrianao J. pilot trial of cyclosporin 1% ophThalmic ointment in the treatment of keratoconjunctivitis sicca. Cornea 1993;12:315-23 (CS1).9. Stevenson D, Tauber J, reis Bl. efficacy and safety of cyclosporin a ophthalmic emulsion in the treatment of moderate-to-severe dry eye disease. a dose-ranging, randomized trial. Ophthalmology 2000;107:967-74 (CS1).10. Pflugfelder Sc, Maskin Sl, anderson B, et al. a randomized, double masked, placebo-controlled, multicenter comparison of loteprednol etabonate ophthalmic suspension, 0.5%, and placebo for treatment of keratoconjunctivitis sicca in patients with delayed tear clearance. Am J Ophthalmol 2004;138:444-57 (CS1).11. Dougherty JM, Mcculley Jp, Silvany re, et al. The role of tetracycline in chronic blepharitis. Invest Ophthalmol Vis Sci 1991;32:2970–5 (CS2).12. Ocular surface and lacrimal gland in f lammation has been identified in dry eye that plays a role in the pathogenesis of KCS. Antiinflammatory therapy has efficacy for treating KCS.13. Swann DA, Sotman S, Dixon M, Brooks C. The isolation and partial charac terization of the major glycoprotein (LGP-I) from the articular lubricating fraction from bovine synovial fluid.14. Biochem J 1977;161(3):473e85. [618] Schmidt TA, Sullivan DA, Knop E, Richards SM, Knop N, Liu S, et al. Tran scription, translation, and function of lubricin, a boundary lubricant, at the ocular surface. JAMA Ophthalmol 2013;131(6):766e76.15. LF. Trk receptors: mediators of neurotrophin action. Curr Opin Neurobiol 2001;11(3):272e80.16. Science 1999;285(5425):221e7. [674] Zhong M, Gadek TR, Bui M, Shen W, Burnier J, Barr KJ, et al. Discovery and Development of Potent LFA-1/ICAM1 Antagonist SAR 1118 as an Ophthalmic Solution for Treating Dry Eye. ACS Med Chem Lett 2012;3(3): 203e6. 17. Sun Y, Zhang R, Gadek TR, O’Neill CA, Pearlman E. Corneal inflammation is inhibited by the LFA-1 antagonist, lifitegrast (SAR 1118). J Ocul Pharmacol Ther 2013;29(4):395e402. 18. Perez VL, Pflugfelder SC, Zhang S, Shojaei A, Haque R. Lifitegrast, a Novel Integrin Antagonist for Treatment of Dry Eye Disease. Ocul Surf 2016;14(2): 207e15.Mansi Jand DNB3 Year Resident, Medanta, Medicty, GurgaonCorresponding Author:

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 28 www.dosonline.orgPeripheral Ulcerative Keratitis - Triggers, Traits and Therapy Prajakta Dandekar[1] MD, FAICO | Talluri Ronnie Abhishek[2] MD | Somasheila I Murthy[1,3] MD1. Cornea and Anterior Segment Service, Shantilal Shanghvi Eye Institute, Wadala East, Mumbai, Maharashtra 2. Saroja N Rao Center for Uveitis, L V Prasad Eye Institute, Kode Venkatadri Chowdary Campus, Tadigadapa, Vijayawada, Andhra Pradesh3. Shantilal Shanghvi Cornea Institute, LV Prasad Eye Institute, Kallam Anji Reddy Campus, Hyderabad, TelanganaIntroductionPeripheral ulcerative keratitis (PUK) is characterised by inflammatory progressive peripheral corneal thinning and ulceration. Despite being a rare disease, its clinical importance lies in the fact that it can lead to tissue destruction and severe visual loss. The incidence of PUK is estimated at approximately three per million annually, with no significant gender predilection.[1,2] The condition may also be associated with immune-mediated serious systemic diseases such as rheumatoid arthritis (RA),[3] granulomatosis with polyangiitis (GPA), systemic lupus erythematosus (SLE), polyarteritis nodosa and inflammatory bowel disease[4,5] although idiopathic forms like Mooren’s ulcer also occur. It could even be the first presentation of a life-threatening disease and the ocular features can precede systemic disease by decades.[6]Triggers: Etio-Pathogenesis of PUKa. Immune Mediated Mechanisms: Both cell-mediated and humoral immunity, in conjunction with the corneal tissue-destroying action of metalloproteinases (MMPs), are implicated in the pathogenesis of PUK.[7] The peripheral cornea is preferentially involved as the collagen fibrils are less densely packed.[8,9] Unlike the rest of the cornea, the periphery lacks immune privilege due to its contiguity with the limbus which contains a rich vascular and lymphatic network. The circulating immune cells and inflammatory mediators such as cytokines and immune complexes can thus access the peripheral corneal stroma leading to inflammation and tissue damage. These immune complexes recruit neutrophils and macrophages which release proteolytic enzymes such as matrix metalloproteinases (MMP) and collagenases that further contribute to corneal ulceration and thinning. Elevated MMP activity has been strongly associated with corneal perforation in PUK.[10,11,12] Figure-1 outlines the pathways involved in the immunopathogenesis of PUK.[7,13]Figure 1: Pathogenesis of PUK.b. Mooren’s Ulcer: Mooren’s ulcer was first described by Bowman and then McKenzie named it as “ulcus roden” of the cornea in 1854. This is a specific sub-types of PUK. Mooren’s ulcer is a rare, painful, chronic, and progressive PUK of presumed autoimmune origin, characterized by crescentshaped corneal ulceration starting at the periphery and progressing circumferentially and centrally. (Figure-2) Importantly, it occurs without any associated systemic vasculitis, which helps differentiate it from other causes of PUK like those associated with RA or GPA.[14] The cases are often named as Mooren’s after ruling out all causes of PUK, calling it the diagnosis of exclusion but in real time it is type of PUK with autoantigens generated against the corneal stromal keratocyte. Watson and colleagues[15]classified it into three types-1. Unilateral: often mentioned

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 29 www.dosonline.orgas benign since it typically is seen in older individuals and is associated with slower progression and better prognosis. 2. Bilateral in young patients: described as malignant, presents in an aggressive fashion with poor response to therapy and high chances of recurrence. 3. Bilateral in old patients (in the late 5th decade and later): which is again benign and has good prognosis with fewer chances of recurrence. As per the clinical criteria described by them, unilateral or bilateral PUK with crescenteric ulceration, progressive peripherally, with classic overhanging edges, with the trailing edge showing healing with pannus formation and no evidence of scleritis can be considered as Mooren’s ulcer and does not need any other evaluation for systemic disease. This disease can progress relentlessly till the antigenic stimulus from the stromal keratocytes abates. Sainz de la Maza M et al,[16] in their study noted that patients with associated scleritis had the poorest outcomes as compared to patients with PUK alone. This type of scleritis was found to have greater stromal lysis due to occlusive vasculitis, which was detected on fluorescein angiography studies.[17,18]Figure 2: Slit lamp image in a 54 year old male diagnosed with unilateral Mooren’s ulcer in the left eye with peripheral corneal thinning from 2-7 o’clock, with corneal perforation and iris prolapse.Figure 3: Slit lamp photograph in a patient showing 360 degrees stromal thinning with peripheral ulcerative keratitis in a case of human immunodeficiency syndrome.c. Infectious Causes of PUK: The association of systemic infections such as tuberculosis (TB), syphilis, human immunodeficiency syndrome, and hepatitis B and C virus infections has been reported in various studies in the development of patient to develop PUK. Arora et al,[19] report a case of PUK with chronic malabsorption syndrome secondary to giardiasis and miliary TB. Gupta N et al,[20] in their case of necrotizing scleritis with PUK in Sweet’s syndrome culture positive for Mycobacterial tuberculosis and found good response after initiation of anti-tubercular therapy (ATT) whereas the case managed by Anitha et al[21] had PUK with an association of systemic tuberculosis where the smear and culture scrapings were negative and no improvement was noted after initiation of ATT. The management in such cases is tricky since non-infective aetiology needs oral steroids which have disastrous outcomes if given in cases of systemic tuberculosis. In syphilis, the keratitis is caused by direct invasion by the spirochete[22] or by an immune reaction against the spirochaetal antigens.[23] Vignesh AP et al,[24] in their case with positive flourescent treponemal antibody absorption tests, had good response with topical steroids and intramuscular injection of 2.4 million units benzathine penicillin G, given once weekly for 3 consecutive weeks.Soni,[25] Gharoi,[26] and Tavassoli[27] et al in their cases reported PUK as the initial presentation of Human immunodeficiency virus (HIV) and recommend testing for it in unidentified causes of PUK. HIV vasculitides likely results in immune complex deposition at the limbus.[28] (Figure-3).Wei DW et al,[29] reported a case of PUK secondary to hepatitis B virus (HBV)-associated cryoglobulinemia and vasculitis treated with oral Tenofovir and prednisone, cyclophosphamide, and mycophenolate mofetil for immunosuppression. Coelho P et al,[30] managed PUK in high viral load of HCV related cryoglobulinemia in a case with IV illicit drug abuse.Sharma et al[31] in a prospective interventional study over 18 months found local infections to be a cause for PUK

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 30 www.dosonline.orgFigure 4: Slit lamp photograph of a 41 year old female patient, known case of Rheumatoid arthritis on low dose steroids and immunosuppression for 15 years. Right eye showing PUK from 3-7 o’clock with no lucid interval.Figure 5: Slit lamp image of the left eye in a 35 year old male with complaints of recurrent whitening in peripheral cornea with intact epithelium alternating in right and in the left eye. His rheumatology work up was negative. Patient was diagnosed as Marginal keratitis and treated with lid scrubs along with topical loteprednol 0.5% eye drops. in 19.7% patients. Praidou et al,[32] found a case of bilateral Herpes simplex virus keratitis masquerading as PUK and managed with oral Valacyclovir and topical steroids. Gupta V et al,[33] reported unilateral peripheral ulceration, positive for PCR for Herpes Zoster virus, in a patient with immunocompromised seropositive rheumatoid arthritis without any precedent rash on the forehead. Kate et al,[34]have reported a misdiagnosed case of pythium insidiosum presenting as PUK which was confirmed only after the corneal button was sent for histopathological analysis. PUK may present as a paraneoplastic phenomenon in systemic malignancies especially haematology related hence ophthalmology visit for any case of red eye should be mandatory for early initiation of treatment.[35-38]d. Post-Trauma: Ocular trauma or prior surgeries may act as local triggers for PUK by disrupting immune privilege. Mondino et al,[39]reported peripheral corneal melt seven months after penetrating keratoplasty. Burkholder et al,[40] reported a case of PUK one week post LASIK, in a patient of post infectious glomerulonephropathy. Kiire et al,[41] and Akpek et al[42]reported a case each of PUK in a patient of ocular cicatricial pemphigoid and rheumatoid arthritis respectively following uncomplicated cataract surgery. Penbe et al,[43]report a case of PUK triggered by an inactive COVID 19 vaccine who needed a penetrating keratoplasty for tectonic stability. Traitsa. Symptoms of PUKPUK is associated with pain throughout the course of the disease. Pain out of proportion to the ulcer is a characteristic of Mooren’s ulcer which also is differentiated from other forms of PUK by its absence of scleral involvement.[44,45] In acute cases, decreased vision may be due to inflammation and vary from mild to severe while in chronic PUK it is due to corneal astigmatism and corneal opacity. Severe decrease in vision occurs in perforation.[46] Ciliary congestion in PUK and necrotising scleritis is due to small vessel vasculitis in the intrascleral anterior ciliary arteries and perilimbal arteries.[47,48] Photophobia is induced by pupillary constriction and ciliary spasm due to inflammations of the anterior segment or due to stimulation of the terminal fibres of the trigeminal nerve in the cornea.[49] Reflex lacrimation is seen in PUK. b. SignsThe ulceration starts as a grey-white infiltrate in the peripheral cornea followed by epithelial defect and stromal thinning.[50] (Figure-4)The ulcer initially involves superficial one-third of the cornea and, gradually going on to involve the deeper stroma due to lysis and may progresses to perforation.[51] True PUK has an epithelial defect associated with subepithelial infiltrate where as non-ulcerative peripheral corneal thinning has intact epithelium.[52]Associated limbitis, episcleritis and scleritis suggest systemic disease association. Severe epithelial and stromal necrosis are hallmarks of active PUK,[53] occurs due to the neutrophil degranulation, lysosomal enzymes and oxygen free radicals.[54] Viral associated PUK can have associated

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 31 www.dosonline.orgepitheliopathy, keratitis, endothelitis or limbitis. Angiogenesis or neovascularization may be observed within the peripheral cornea in long standing cases which is a sign of chronic inflammation trying to heal.[55] Severe and long-standing cases which progress to corneal perforation present with plugging of the perforation by the iris tissue. Iritis and hypopyon are less often associated with non-infectious PUK, but can occur in the setting of severe inflammation such as in Mooren’s ulcer, especially when not under therapy (Figure-6). Figure 6: Slit lamp image of a 60 year old female patient with Rheumatoid arthritis associated PUK with severe corneal thinning from 9-11o’clock and hypopyon.Figure 8: Slit lamp photograph of the right eye in a 80 year old male diagnosed as Mooren’s ulcer showing peripheral corneal thinning from 3-8 clock hours.Figure 9: Slit lamp photographs of the left eye in the same patient (Figure-8) showing epithelised prolapsed iris with peripheral ulcer from 3-10 o’clock, hazy central island of cornea and a total cataract.Figure 7: Slit lamp image of a 59 year old female patient who initially presented as as geographic ulcer and was treated with steroids+antivirals. The patient had an initial resolution followed by worsening. Smear showed Gram positive cocci, so a diagnosis of Herpes simplex virus keratitis with PUK associated with secondary infection was made and started on Fortified Cefazoline eye drops and planned for cyanoacrylate glue and BCL.Secondary infections may develop in severe cases, and in perforated or near-perforated PUK (Figure-7).c. Traits of Non-Infectious PUKConstitutional symptoms, musculoskeletal, gastrointestinal, cardiovascular, respiratory, skin, and neurological manifestations occur due to multisystem involvement.[56] Recurrence of symptoms is commonly seen in non-infectious PUK. Bilaterality is usually seen in non-infectious aetiology. (Figure-8,9)Associated keratoconjunctivitis sicca causing epithelial breakdown is noted in cases associated with rheumatoid arthritis.[57] Superficial punctate keratitis can be noted in a majority of patients with systemic lupus erythromatosis (SLE) suggesting immune mechanisms along with dry eye as the causes of corneal involvement.[58] Certain PUK which are due to granulomatous inflammation can

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 32 www.dosonline.orgFigure 10: Slit lamp photograph of a patient with c-ANCA associated PUK who presented as the initial manifestation of GPA. The picture shows mild conjunctival congestion with active ulceration from 4-8 clock hrs and superior conjunctivalization of the cornea, suggesting some old insult.Figure 12: Slit lamp image of the left eye of the same patient as Figure-11, showing active scleritis with scleral thinning in down gaze. Patient was managed with IV Methyl prednisolone followed by immunosuppression in concordance with the rheumatologist. Figure 11: Slit lamp image in downgaze showing scleral thinning in the right eye of a c-ANCA positive patient.present with involvement of orbit and adnexal structures. PUK may be the initial manifestation in granulomatosis polyangiitis (GPA) (Figure-10,11,12) while it occurs late in rheumatoid arthritis.[59]Associated retinal artery occlusions, hypertensive retinopathy in associated systemic diseases and retino-choroidal infiltration in malignancies is seend. Traits of Infectious PUKSpread of infection from peripheral cornea with limbal erythema, oedema and infiltrate is a feature. Increase in pain with spread of infection to sclera occurs and the spread occurs from cornea to sclera or from sclera to cornea (example: gram negative bacteria like Pseudomonas aeruginosa).[60,61] History of contact lens usage may be present which can cause reduced oxygen to corneal surface and causes microtrauma predisposing to microbial keratitis[62] In infectious PUK, a stormy rapid presentation is noted, and other features such as conjunctival membranes and hypopyon and corneal infiltrates beyond the periphery are noted. Infectious aetiology is usually unilateral except in ophthalmia neonatorum. Associated corneal involvement as mentioned earlier, or posterior segment involvement such as retinal necrosis is seen in viral etiology. Ring-shaped infiltrate in Acanthamoeba infections and wreath-like pattern can be noted in Nocardia keratitis. Phlyctenulosis or associated posterior segment choroidal granuloma or retinal vasculitis with clinical predictors like sub-vascular lesions, occlusive vasculitis, focal vascular tortuosity is seen tubercular aetiology.[63] The different types of PUK and its presentations are listed in Table-1. PUK with Systemic Autoimmune Disease PUK Without Systemic/Local Disease-Mooren’s PUK with Systemic InfectionPUK like Picture with Local Infection Aetiology Rheumatoid arthritis (RA) Granulomatous polyangiitis, No systemic disease Tuberculosis, Syphilis HIV Hepatitis and Hepatitis B and C cryoglobulinemiaPseudomonas aeruginosa, Pythium, Nocardia, Herpes viral keratitis

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 33 www.dosonline.orgDifferential diagnosis is crucial to distinguish PUK from other conditions that mimic its presentation. Marginal keratitis, Terrien’s marginal degeneration, Mooren’s ulcer, and infectious keratitis are among the conditions that need to be considered in the differential diagnosis.[64]e. Investigations: Laboratory investigations include autoimmune panels such as rheumatoid factor and anti-cyclic citrullinated peptides (Anti-CCP) are investigated as a marker for RA.[65] While there may be seronegative RA with negative rheumatoid factor, anti-CCP antibodies are more specific and are found to be positive in 50-80 % of RA patients[66]Anti-neutrophil cytoplasmic antibodies (c-ANCA) has a specificity of 90% in biopsy proven GPA, sensitivity is 100% in active generalised disease.[67] The specific investigations are mentioned in a tabular form in Table-2. Infectious workup involves corneal cultures for bacterial, fungal, or viral pathogens to rule out infectious causes.[68]High index of suspicion is needed to diagnose infectious PUK with Systemic Autoimmune Disease PUK Without Systemic/Local Disease-Mooren’s PUK with Systemic InfectionPUK like Picture with Local Infection scleroderma, Systemic lupus erythematosus, polyarthritis nodosa, inflammatory bowel disease History and Features Patients may have features of joint deformities (RA), features of respiratory infection/renal issues (GPA), skin rash (Scleroderma) malar rash (SLE), diarrhoea/constipation (IBD) No systemic features Associated with fever, weight loss, jaundice, skin rash depending on the systemic disease and its characteristic featuresAssociated with history of trauma/CL use or surgeryUlcer CharacteristicsUlcers are peripheral and spread circumferentially with or without scleral involvement. Associated with dry eye, non-healing epithelial defects, repeated sterile perforations, keratinisation, conjunctival cicatrizing changes Ulcers are peripheral and spread circumferentially without scleral involvement and have characteristic feature of overhanging edge with associated stromal melt Ulcers are quiet often associated with the scleral involvement and may have features of conjunctival involvement in the form of phlycten or iris and ciliary body inflammation –presenting as associated uveitis, retinal vasculitis/ granulomatous lesionstuberculomas Ulcers may present in peripheral manner but often have central spread (Ring infiltrate/wreath laying pattern) with hypopyon and scleral involvement may or may not be present. Most of the cases are unilateralRheumatoid factor, Anti citrullinated protein antibodiesRheumatoid arthritisAngiotensin-converting enzyme SarcoidosisHRCT Chest, Mantoux test, QuantiFERON TbTuberculosisAnti-neutrophil cytoplasmic antibodies (cANCA)GPAAntinuclear antibodies Systemic lupus erythematosusTable 1: Differentiating types of peripheral ulcerative keratitis.scleritis associated with PUK in cases of Nocardia,[69] Pythium[70] presenting as PUK and Herpes Simplex Virus[71]presenting as marginal keratitis before labelling them as autoimmune aetiology and if misdiagnosed can lead to worsening due to steroids and delayed treatment. Imaging techniques like anterior segment optical coherence tomography (AS-OCT) can quantify stromal thinning and monitor disease progression based on epithelial and stromal reflectivity.[72]

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 34 www.dosonline.orgTPHA SyphilisHIV serology HIVHepatitis B and C Hepatitis and cryoglobulinaemiaTherapya. Medical Management: The management of peripheral ulcerative keratitis (PUK) requires a multidisciplinary approach at halting the progression, treating the underlying cause and restoring the globe integrity. The tailored approach consists of combining immunosuppression and surgical intervention, guided by disease aetiology and severity.For non-infectious autoimmune cases, systemic corticosteroids remain first-line therapy, with pulse intravenous methylprednisolone (0.5-1g/day for 3 days) recommended, followed by oral prednisolone (1mg/kg/day) tapered over months.[73,74] Steroids act in six to eight hours, controlling the disease till the exact autoimmune aetiology is assessed. They act as a bridge giving time to the disease modifying antirheumatic drugs (DMARDs) or other immunomodulators (IMTs) which typically takes two weeks to three months for the onset and complete action.[75,76]Steroid-sparing agents are initiated concurrently, with methotrexate (7.5-25mg/week) preferred for rheumatoid arthritis-associated cases.[77-81] The second choice of immunomodulatory therapy includes Azathioprine (1-2.5mg/kg/day)[82-84] and cyclosporine.[85] Mycophenolate mofetil (MMF) (1gm twice daily) can be used if there are side effects with other IMT drugs.[86] Reliable biomarkers to monitor efficacy of IMTs are awaited.[87] In a retrospective study by Ruiz-Lozano et al,[88] patients of RA, exposed late to IMTs and retarded control of inflammation had more chances of recurrence of PUK which increased the chances of vision loss. Cyclophosphamide is reserved for recalcitrant cases or those that show rapid progression, bilateral severe cases, perforations and pre-operatively. After the initiation of biologicals, steroid dependency drastically reduced over a period of one year in a study by Dominguez Casasa et al.[89] Rituximab, a chimeral monoclonal antibody that binds to CD20 sites has been tried in cases of PUK with RA with two doses of 1gm each given two weeks apart, and repeated after three to six months (Rheumatology protocol) and a dose of 375mg/m2 body area given weekly over 8 weeks (Foster protocol).[90] Lower doses given as maintenance have shown to be decrease the remission rates. Combining MTX with Rituximab may give protection against hypogammaglobulinemia.[91]The adverse effects reported are recurrent pneumoniae and tachyphylaxis.Pahor et al states that ocular manifestations have been the first sign in most of the patients of GPA and hence all such cases should be investigated for it.[92] PUK with adjacent necrotizing scleritis is often a manifestation of vasculitis in GPA.[93] Systemic immunosuppression is necessary, with corticosteroids like prednisolone being the first-line agents. Watkins et al,[94] recommends use of oral steroids (1mg/kg/day) and oral cyclophosphamide (2mg/kg/day) for treatment of severe cases associated with Antinuclear cytoplasmic antibody positive vasculitis such as GPA. Ahmed et al[95] have shown comparison of cyclophosphamide and Rituximab in GPA and reported remission in all cases. Stone et al,[96] conducted a non-inferiority trial for comparing Rituximab to cyclophosphamide and found Rituximab to be noninferior to Cyclophosphamide in treating GPA and more efficacious in achieving remission in a relapsing disease. Infliximab is given in the loading dose of 3-5mg/kg as baseline and then repeated two, six and eight weekly or zero, two and six weeks followed by maintenance regiment. Usually the improvement is noted between one week to 6 months.[97,98] Two cases of infection have been reported in the literature.[99]Antimicrobial therapy targets infectious etiologies, necessitating culture-guided treatment with prophylactic antibiotics during epithelial defect. b. Surgical ManagementCyanoacrylate glue application prevents stromal lysis and is applied over areas showing stromal loss and thinning. The glue is applied in the crater if the thinning is >50 percent. Resolution of ulcer has been reported from 42% to 83% of cases.[100-102] The advantage being that it can be used for both infective and non-infective cases (Figure-13).Conjunctival Resection: The source of collagenase and other enzymes which destroy the stroma in PUK are based in the limbal conjunctiva. Hence peritomy in the area near the melt 2mm at the sides and 4mm away from the limbus is recommended.[103,104] Genvert G et al,[105] states use of conjunctival resection (to remove the antibody producing inflammatory cells by eliminating the necrotic tissue) and cryotherapy (intracellular ice crystals form and disrupt the cell membrane) for refractory cases of GPA (Figure-14,15).

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 35 www.dosonline.orgFigure 13: PUK managed with Tissue adhesive and BCL.Figure 16: Slit lamp image of the patient in Figure-6, post corneal patch graft and amniotic membrane transplant.Figure 14: Slit lamp photograph of the right eye (Figure-8) after treatment with conjunctival resection and cyanoacrylate application in conjunction with oral Methotrexate.Figure 15: Slit lamp photograph of the left eye (Figure-9) after treatment with conjunctival resection and cyanoacrylate application in conjunction with oral Methotrexate.Figure 17: Slit lamp photograph of the left eye (Figure-9) after treatment with conjunctival resection and cyanoacrylate application in conjunction with oral Methotrexate.Other techniques: Amniotic membrane graft provides collagen and adds to the volume of the corneal stroma.[106,107] It can be used in cases of thinning as a inlay graft or can be also used as a sandwich technique in cases of perforation. It is usually applied with fibrin glue. It can also be applied with the help of 10-0 Nylon sutures (Figure-16).Jianjun Gu et al,[108] have reported 78 % of resolution out of six cases of GPA, with the aid of immunosuppressants and surgical treatment in the form of scleral patch graft or lamellar keratoplasty. Lamellar keratoplasties have limited success due to the underlying dry eye and corneal hypoesthesia. Banana/C-shaped corneal patch grafts are also used in cases in which AMG/tenons cannot be used and therapeutic penetrating keratoplasty grafts needed will be large sized.[109-111] (Figure-17)

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 36 www.dosonline.orgPrognosisThe prognosis of PUK depends on timely intervention and effective control of systemic disease. Advances in immunomodulatory therapy have significantly improved outcomes, though untreated PUK carries a high risk of severe visual impairment or even enucleation in up to 10% of cases.[112] Future directions in PUK management include emerging therapies targeting specific pathways like MMP inhibition, which hold promise for reducing tissue destruction. Greater integration of biologics into treatment protocols could revolutionize the management of autoimmune-related PUK, offering new avenues for therapy. Early recognition and comprehensive management are critical to preserving vision and mitigating systemic complications associated with PUK.In conclusion, PUK is a complex condition requiring a comprehensive approach that addresses both local ocular manifestations and systemic autoimmune or infectious triggers. The integration of advanced diagnostic techniques and targeted therapeutic strategies has improved outcomes for patients with PUK. Continued research into the pathogenesis and treatment modalities will be essential for further enhancing patient care and visual preservation in this challenging condition.References1. Tauber J, Sainz de la Maza M, Hoang-Xuan T, Foster CS. An analysis of therapeutic decision making regarding immunosuppressive chemotherapy for peripheral ulcerative keratitis. Cornea 1990;9:66–73. 2. McKibbin M, Isaacs JD, Morrell AJ. Incidence of corneal melting in association with systemic disease in the Yorkshire Region, 1995-7. Br J Ophthalmol 1999;83:941– 943.3. Gregory JK, Foster CS. Peripheral ulcerative keratitis in the collagen vascular diseases. Int Ophthalmol Clin. 1996;36:21–30.4. Kate A, Basu S. Systemic Immunosuppression in Cornea and Ocular Surface Disorders: A Ready Reckoner for Ophthalmologists. Semin Ophthalmol. 2022 Apr 03;37(3):330-344.5. Ladas JG, Mondino BJ. Systemic disorders associated with peripheral corneal ulceration. Curr Opin Ophthalmol. 2000;11:468–471. 6. M. Artifoni, P. R. Rothschild, A. Brézin, L. Guillevin, and X. Puéchal,“Ocular inflammatory diseases associated with rheumatoid arthritis,” Nature Reviews Rheumatology, vol. 10, no. 2, pp. 108–116, 2014.7. Gomes BF, Santhiago MR. Biology of peripheral ulcerative keratitis. Exp Eye Res. 2021 Mar;204:108458. 8. Boote C, Dennis S, Newton RH, Puri H, Meek KM. Collagen fibrils appear more closely packed in the prepupillary cornea: optical and biomechanical implications. Invest Ophthalmol Vis Sci. 2003 Jul;44(7):2941-8.9. Müller LJ, Pels E, Schurmans LR, Vrensen GF. A new three- dimensional model of the organization of proteoglycans and collagen fibrils in the human corneal stroma. Exp Eye Res 2004;78:493–501.10. Messmer EM, Foster CS. Vasculitic peripheral ulcerative keratitis. Surv Ophthalmol 1999;43:379-396.11. Smith VA, Rishmawi H, Hussein H, Easty DL. Tear film MMP accumulation and corneal disease. Br J Ophthalmol. 2001;85:147–153.12. Smith VA, Hoh HB, Easty DL. Role of ocular matrix metalloproteinases in peripheral ulcerative keratitis. Br J Ophthalmol. 1999;83:1376–1383.13. Hassanpour K, ElSheikh RH, Arabi A, Frank CR, Elhusseiny AM, Eleiwa TK, Arami S, Djalilian AR, Kheirkhah A. Peripheral ulcerative keratitis: a review. Journal of Ophthalmic & Vision Research. 2022 Apr 29;17(2):252.14. Chi H, Hao W, Qi X, Zhang T, Dong Y, Gao H, et al. A proteomic approach towards understanding the pathogenesis of Mooren’s ulcer. Exp Eye Res. 2021;205:108509. 15. Watson PG. Management of Mooren’s ulceration. Eye (Lond). 1997;11 ( Pt 3):349-56.16. Sainz de la Maza M, Foster CS, Jabbur NS, Baltatzis S. Ocular characteristics and disease associations in scleritis-associated peripheral keratopathy. Arch Ophthalmol. 2002 Jan;120(1):15-9. 17. Watson PG, Bovey E. Anterior segment fluorescein angiography in the diagnosis of scleral inflammation. Ophthalmology 1985;92:111.18. Watson PG. Vascular changes in peripheral corneal destructive disease. Eye 1990;4:65–73.19. Arora T, Sharma N, Shashni A, Titiyal JS. Peripheral ulcerative keratitis associated with chronic malabsorption syndrome and miliary tuberculosis in a child. Oman J Ophthalmol. 2015 Sep-Dec;8(3):205-7. 20. Gupta N, Chawla B, Venkatesh P, Tandon R. Necrotizing scleritis and peripheral ulcerative

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 37 www.dosonline.orgkeratitis in a case of Sweet’s syndrome found culturepositive for Mycobacterium tuberculosis. Ann Trop Med Parasitol. 2008;102:557–60.21. Anitha, Venugopal; Ghorpade, Aditya1; Ravindran, Meenakshi2. A modified Tenons sling annular graft for advanced peripheral ulcerative keratitis with an hourglass cornea. Indian Journal of Ophthalmology 70(2):p 655-657, February 2022.22. Radolf JD, Hazlett KRO, Lukehart SA. Pathogenesis of syphilis. In: Radolf JD, Lukehart SA, editors. Pathogenic Treponemes: Cellular and Molecular Biology. Norfolk, UK: Caister Academic Press; 2006. pp. 197–236.23. Merchant TE, Lass JH, Roat MI, Skelnik DL, Glonek T. P-31 NMR analysis of phospholipids from cultured human corneal epithelial, fibroblast and endothe- lial cells. Curr Eye Res. 1990;9:1167–1176. 24. Vignesh AP, Srinivasan R, Vijitha S. Ocular syphilis masquerading as bilateral peripheral ulcerative keratitis. Taiwan J Ophthalmol. 2016 OctDec;6(4):204-205.25. Soni ND, Ingole AB, Murade SM. An unusual case of peripheral ulcerative keratitis as a presenting feature in an otherwise healthy patient with undiagnosed human immunodeficiency virus infection and low CD4 counts. Indian J Ophthalmol 2013;61:138–9.26. Gharai S, Venkatesh P, Tandon R, et al. Peripheral ulcerative keratitis and central retinal vein occlusion as the initial manifestation of HIV infection. Ocul Immunol Inflamm 2007;15:407–9. 27. Tavassoli S, Gunn D, Tole D, Darcy K. Peripheral ulcerative keratitis with corneal melt as the primary presentation in a case of human immunodeficiency virus. BMJ Case Rep. 2019 Feb 22;12(2):e226936.28. Chetty R. Vasculitides associated with HIV infection. J Clin Pathol 2001;54:275–8.29. Wei DW, Pagnoux C, Chan CC. Peripheral Ulcerative Keratitis Secondary to Chronic Hepatitis B Infection. Cornea. 2017 Apr;36(4):515-517.30. Coelho P, Menezes C, Gonçalves R, Rodrigues P, Seara E. Peripheral Ulcerative Keratitis Associated with HCV-Related Cryoglobulinemia. Case Rep Ophthalmol Med. 2017;2017:9461937.31. Sharma N, Sinha G, Shekhar H, Titiyal JS, Agarwal T, Chawla B, Tandon R, Vajpayee RB. Demographic profile, clinical features and outcome of peripheral ulcerative keratitis: a prospective study. Br J Ophthalmol. 2015 Nov;99(11):1503-8. 32. Praidou, Anna MD, MSc, PhD; Androudi, Sofia MD, PhD; Kanonidou, Evgenia MD, PhD; Konidaris, Vassilios MD, PhD; Alexandridis, Alexandros MD, PhD; Brazitikos, Periklis MD, PhD. Bilateral Herpes Simplex Keratitis Presenting as Peripheral Ulcerative Keratitis. Cornea 31(5):p 570-571, May 2012. 33. Gupta V, Pal H, Das S, Pathuri DS, Vathulya M. Varicella Zoster Reactivation Manifesting as Serpiginous Peripheral Keratitis and Disciform Keratitis Occurring After Necrotizing Fasciitis in an Immunocompromised Male: A Case Report. Cureus. 2023 Jun 22;15(6):e40787.34. Kate A, Bagga B, Ahirwar LK, Mishra DK, Sharma S. Unusual Presentation of Pythium Keratitis as Peripheral Ulcerative Keratitis: Clinical Dilemma. Ocul Immunol Inflamm. 2022 Oct-Nov;30(7-8):2023-2026.35. Malecha MA, Holland EJ. Peripheral keratitis associated with chronic myelomonocytic leukaemia. Cornea. (2002) 21:723–4 10.36. Sainz de la Maza M, Foster CS. Peripheral ulcerative keratitis and malignancies. Cornea. (1994) 13:364–7. 10.37. Chawla B, Agarwal P, Tandon R, Titiyal JS. Peripheral ulcerative keratitis with bilateral optic nerve involvement as an initial presentation of acute lymphocytic leukemia in an adult. Int Ophthalmol. (2009) 29:53–5. 38. Morjaria R, Barge T, Mordant D, Elston J. Peripheral ulcerative keratitis as a complication of acute myeloid leukaemia. BMJ Case Rep. (2014) 2014:bcr2014206399. 39. Mondino BJ, Hofbauer JD, Foos RY. Mooren’s ulcer after penetrating keratoplasty. Am J Ophthalmol. 1987 Jan 15;103(1):53-6.40. Burkholder BM, Kuo IC. Peripheral Ulcerative Keratitis following Laser in situ Keratomileusis. Case Rep Ophthalmol. 2016 Jan 8;7(1):9-15.41. Kiire CA, Srinivasan S, Inglis A. Peripheral ulcerative keratitis after cataract surgery in a patient with ocular cicatricial pemphigoid. Cornea. 2011;30:1176–1178. 42. Akpek EK, Demetriades AM, Gottsch JD. Peripheral ulcerative keratitis after clear corneal cataract extraction(1). J Cataract Refract Surg. 2000 Sep;26(9):1424-7. 43. Penbe A. Peripheral Ulcerative Keratitis Secondary to the Inactive COVID-19 Vaccine-CoronaVac. Ocul Immunol Inflamm. 2023 Apr;31(3):536-540.44. Alhassan MB, Rabiu M, Agbabiaka IO. Interventions

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 38 www.dosonline.orgfor Mooren’s ulcer. Cochrane Database Syst Rev. 2014 Jan 22;2014(1):CD006131. 45. Srinivasan M, Zegans ME, Zelefsky JR, et al. Clinical characteristics of Mooren’s ulcer in South India. Br J Ophthalmol. 2007;91:570–575.46. Gupta Y, Kishore A, Kumari P, Balakrishnan N, Lomi N, Gupta N, Vanathi M, Tandon R. Peripheral ulcerative keratitis. Survey of ophthalmology. 2021 Nov 1;66(6):977-98.47. Austin P, Green WR, Sallyer DC, Walsh FB, Kleinfelter HT. Peripheral corneal degeneration and occlusive vasculitis in Wegener’s granulomatosis. Am J Ophthalmol. 1978;85:311–7. 48. Frayer WC. The histopathology of perilimbal ulceration in Wegener’s granulomatosis. Arch Oph thalmol. 1960;64:58–64.49. Sihota R. Parsons’ Diseases of the Eye.-. RELX India Pvt. Ltd.; 2020.50. Tandon R, Galor A, Sangwan V, Manotosh R. Peripheral Ulcerative Keratitis. Berlin, Heidelberg/New York, NY: Springer; 2017.51. Keenan JD, Mandel MR, Margolis TP. Peripheral ulcerative keratitis associated with vasculitis manifesting asymmetrically as fuchs superficial marginal keratitis and terrien marginal degeneration. Cornea. 2011;30(7):825–7.52. Shiuey Y, Foster CS. Peripheral ulcerative keratitis and collagen vascular disease. International ophthalmology clinics. 1998 Jan 1;38(1):21-32.53. Byrd LB, Gurnani B, Martin N. Corneal ulcer. InStatPearls [Internet] 2024 Feb 12. StatPearls Publishing.54. Philip S. Immune complex-mediated vasculitis, Chapter 21. In: John H, Kippel J, et al. editors. Primer in rheumatic diseases, Berlin: Springer; 2008. p. 427–34.55. Ellenberg D, Azar DT, Hallak JA, Tobaigy F, Han KY, Jain S, Zhou Z, Chang JH. Novel aspects of corneal angiogenic and lymphangiogenic privilege. Progress in retinal and eye research. 2010 May 1;29(3):208-48.56. Mathur A, Ashar J, Sangwan V. Mooren’s ulcer in children. Br J Ophthalmol. 2012;96:796–800.57. Hemady R, Chu W, Foster CS. Keratoconjunctivitis sicca and corneal ulcers. Cornea. 1990;9:170–3.58. Spaeth GL. Corneal staining in systemic lupus erythematosus. N Engl J Med. 1967;276:1168–71.59. Fauci AS, Haynes BF, Katz P, Wolff SM. Wege ner’s granulomatosis: prospective clinical and therapeutic experience with 85 patients for 21 years. Ann Intern Med. 1983;98:76–85.60. Raber IM, Laibson PR, Kurz GH, Bernardino VB. Pseudomonas corneoscleral ulcers. Am J Ophthal mol. 1981;92:353–62.61. Codère F, Brownstein S, Jackson WB. Pseudomonas aeruginosa scleritis. Am J Ophthalmol. 1981;91: 706–10.62. Dart JK, Stapleton F, Minassian D. Contact lenses and other risk factors in microbial keratitis. Lancet. 1991;338:650–3.63. Kaza H, Tyagi M, Pathengay A, Basu S. Clinical predictors of tubercular retinal vasculitis in a highendemic country. Retina. 2021 Feb 1;41(2):438-44.64. Sabhapandit S, Murthy SI. Clinical Syndromes, Classifications, and Differential Diagnosis. Peripheral Ulcerative Keratitis: A Comprehensive Guide. 2017:61-80.65. Lewena et al, Diagnostic Pitfalls in Immunology Testing Maher. Clinics in Laboratory Medicine, Volume 39, Issue 4, 567 - 578.66. Willemze A, Trouw LA, Toes RE, Huizinga TW. The influence of ACPA status and characteristics on the course of RA. Nature Reviews Rheumatology. 2012 Mar;8(3):144-52.67. Pagnoux C. Updates in ANCA-associated vasculitis. European journal of rheumatology. 2016 Jan 29;3(3):122.68. Hofling-Lima AL, Müller EG. Infectious Causes. InPeripheral Ulcerative Keratitis: A Comprehensive Guide 2017 Mar 10 (pp. 81-92). Cham: Springer International Publishing69. Rao SK, Madhavan HN, Sitalakshmi G, Padmanabhan P. Nocardia asteroides keratitis: report of seven patients and literature review. Indian journal of ophthalmology. 2000 Jul 1;48(3):217-21.70. Vishwakarma P, Mohanty A, Kaur A, Das S, Priyadarshini SR, Mitra S, Mittal R, Sahu SK. Pythium keratitis: Clinical profile, laboratory diagnosis, treatment, and histopathology features posttreatment at a tertiary eye care center in Eastern India. Indian Journal of Ophthalmology. 2021 Jun 1;69(6):1544-52.71. Chranioti A, Malamas A, Metallidis S, Mataftsi A, Chalvatzis N, Ziakas N. Bilateral herpes simplex virus-related peripheral ulcerative keratitis leading to

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 39 www.dosonline.orgcorneal perforation in a patient with primary herpes simplex virus infection. Journal of Ophthalmic & Vision Research. 2019 Jan;14(1):93.72. Garg A, De Rojas J, Mathews P, Hazan A, Lin J, Trief D, Suh LH. Using anterior segment optical coherence tomography to monitor disease progression in peripheral ulcerative keratitis. Case Reports in Ophthalmological Medicine. 2018;2018(1):3705753.73. Świerczyńska M, Tronina A, Mrukwa-Kominek E. Peripheral Ulcerative Keratitis Associated with Autoimmune Diseases. InKeratitis-Current Perspectives 2023 Sep 13. IntechOpen.74. Maleki A, Valerio T, Massoudi Y, Ruggeri ML, Foster CS, Anesi SD. Updates on Systemic Immunomodulation in Peripheral Ulcerative Keratitis. Journal of Clinical & Translational Ophthalmology. 2024 Oct 23;2(4):131-9.75. Hoes JN, Jacobs JW, Buttgereit F, Bijlsma JW. Current view of glucocorticoid co-therapy with DMARDs in rheumatoid arthritis. Nature Reviews Rheumatology. 2010 Dec;6(12):693-702.76. Bornstein C, Craig M, Tin D. Practice guidelines for pharmacists: the pharmacological management of rheumatoid arthritis with traditional and biologic disease-modifying antirheumatic drugs. Canadian Pharmacists Journal/Revue des Pharmaciens du Canada. 2014 Mar;147(2):97-109.77. Friedman B, Cronstein B. Methotrexate mechanism in treatment of rheumatoid arthritis. Joint bone spine. 2019 May 1;86(3):301-7.78. Kremer JM, Alarcón GS, Lightfoot Jr RW, Willkens RF, Furst DE, Williams HJ, Dent PB, Weinblatt ME. Methotrexate for rheumatoid arthritis. Arthritis & Rheumatism: Official Journal of the American College of Rheumatology. 1994 Mar;37(3):316-28.79. Cutolo M, Sulli A, Pizzorni C, Seriolo B, Straub RH. Anti-inflammatory mechanisms of methotrexate in rheumatoid arthritis. Annals of the rheumatic diseases. 2001 Aug 1;60(8):729-35.80. Borchers AT, Keen CL, Cheema GS, Gershwin ME. The use of methotrexate in rheumatoid arthritis. InSeminars in arthritis and rheumatism 2004 Aug 1 (Vol. 34, No. 1, pp. 465-483). WB Saunders.81. Dwosh IL, Stein HB, Urowitz MB, Smythe HA, Ogryzlo MA, Hunter T. Azathioprine in early rheumatoid arthritis. Arthritis & Rheumatism: Official Journal of the American College of Rheumatology. 1977 Mar;20(2):685-92.82. Suarez‐Almazor ME, Spooner C, Belseck E, Cochrane Musculoskeletal Group. Azathioprine for treating rheumatoid arthritis. Cochrane Database of Systematic Reviews. 1996 Sep 1;2010(1).83. Squirrell DM, Winfield J, Amos RS. Peripheral ulcerative keratitiscorneal melt’and rheumatoid arthritis: a case series. Rheumatology. 1999 Dec 1;38(12):1245-8.84. Jifi-Bahlool H, Saadeh C, O’Conner J. Peripheral ulcerative keratitis in the setting of rheumatoid arthritis: treatment with immunosuppressive therapy. InSeminars in arthritis and rheumatism 1995 Aug 1 (Vol. 25, No. 1, pp. 67-73). WB Saunders.85. Watanabe R, Ishii T, Yoshida M, Takada N, Yokokura S, Shirota Y, Fujii H, Harigae H. Ulcerative keratitis in patients with rheumatoid arthritis in the modern biologic era: a series of eight cases and literature review. International Journal of Rheumatic Diseases. 2017 Feb;20(2):225-30.86. Korsten, P.; Bahlmann, D.; Patschan, S.A. Rapid healing of peripheral ulcerative keratitis in rheumatoid arthritis with prednisone, methotrexate and adalimumab combination therapy. Rheumatology 2017, 56, 1094.87. Mesa Navas MA, Velásquez Franco CJ, Gómez Suárez IC, Montoya Ramírez JC. Corneal melt as a complication of a peripheral ulcerative keratitis in a patient with rheumatoid arthritis Rev Colomb Reumatol Engl Ed. 2021;28:69–75. 88. Ruiz-Lozano RE, Ramos-Davila EM, Garza-Garza LA, Gutierrez-Juarez K, Hernandez-Camarena JC, Rodriguez-Garcia A. Rheumatoid arthritis-associated peripheral ulcerative keratitis outcomes after early immunosuppressive therapy. Br J Ophthalmol. 2023 Sep;107(9):1246-1252. 89. Dominguez-Casas, L.C.; Sánchez-Bilbao, L.; CalvoRío, V.; Maíz, O.; Blanco, A.; Beltrán, E.; MartínezCosta, L.; Demetrío-Pablo, R.; del Buergo, M.; Rubio-Romero, E.; et al. Biologic therapy in severe and refractory peripheral ulcerative keratitis (PUK). Multicenter study of 34 patients. Semin. Arthritis Rheum. 2020, 50, 608–615.90. C.S. Foster, P.Y. Chang, A.R. Ahmed Combination of rituximab and intravenous immunoglobulin for recalcitrant ocular cicatricial pemphigoid: a preliminary report Ophthalmology, 117 (5) (2010), pp. 861-869.91. G. Boleto, J. Avouac, J. Wipff, M. Forien, M. Dougados,

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 40 www.dosonline.orgC. Roux, et al. Predictors of hypogammaglobulinemia during rituximab maintenance therapy in rheumatoid arthritis: a 12-year longitudinal multi-center study Semin Arthritis Rheum, 48 (2) (2018), pp. 149-154.92. Pahor D, Gracner B, Gracner T, Pahor A.Ocular symptoms as the initial signs of Wegener’s granulomatosis. Klin Monbl Augenheilkd. 2009 May;226(5):409-13. 93. B. Kubaisi, S. K. Abu, and C. S. Foster,“Granulomatosis with polyangiitis (Wegener’s disease): an updated review of ocular disease manifestations,” Intractable Rare Diseases Research, vol. 5, no. 2, pp. 61–69, 2016.94. A. S. Watkins, J. H. Kempen, D. Choi et al.,“Ocular disease in patients with ANCA-positive vasculitis,” Journal of Ocular Biology Diseases and Informatics, vol. 3, no. 1, pp. 12–19, 2009.95. A. Ahmed, C.S. Foster Cyclophosphamide or rituximab treatment of scleritis and uveitis for patients with granulomatosis with polyangiitis Ophthalmic Res, 61 (1) (2019), pp. 44-50.96. Stone JH, Merkel PA, Spiera R; RAVE-ITN Research Group. Rituximab versus cyclophosphamide for ANCA-associated vasculitis. N Engl J Med. 2010 Jul 15;363(3):221-32. 97. A. Perdriger Infliximab in the treatment of rheumatoid arthritis Biol Targets Ther, 3 (2009), pp. 183-191.98. P.E. Lipsky, D.M. van der Heijde, E.W. St Clair et al. Infliximab and methotrexate in the treatment of rheumatoid arthritis. Anti-tumor necrosis factor trial in rheumatoid arthritis with concomitant therapy study group N Engl J Med, 343 (22) (2000), pp. 1594-1602.99. W.J. Sandborn, S.B. Hanauer Infliximab in the treatment of Crohn’s disease: a user’s guide for clinicians Am J Gastroenterol, 97 (12) (2002), pp. 2962-2972.100.Refojo MF, Dohlman CH, Ahmad B, et al. Evaluation of adhesives for corneal surgery. Arch Ophthalmol. 1968;80(5):645–656. 101.Webster RG, Slansky HH, Refojo MF, et al. The use of adhesive for the closure of corneal perforations: report of two cases. Arch Ophthalmol. 1968;80:705–709.102.Leahey AB, Gottsch JD, Stark WJ. Clinical experience with N-butyl cyanoacrylate (Nexacryl) tissue adhesive. Ophthalmology. 1993;100:173–180. 103.Brown S. Mooren’s ulcer. Treatment by conjunctival excision. Br J Ophthalmol. 1975;59:675–682.104.Bartly J, Mondino BJ. Inflammatory diseases of the peripheral cornea. Ophthalmology. 1988;95:463–472. 105.Genvert G, Sakauye C, Arentsen J. Treatment of marginal corneal ulcer with cryotherapy and conjunctival recession or resection. Cornea. 1984-1985;3:256–261.106.Solomon A, Meller D, Prabhasawat P. Amniotic membrane grafts for nontraumatic corneal perforations, descemetoceles, and deep ulcers. Ophthalmology. 2002;109:694–703.107.Touhami A, Grueterich M, Tseng SCG. The role of NGF signaling in human limbal epithelium expanded by amniotic membrane culture. Invest Ophthalmol Vis Sci. 2002;43:987–994.108.Gu J, Zhou S, Ding R, Aizezi W, Jiang A, Chen J. Necrotizing scleritis and peripheral ulcerative keratitis associated with Wegener’s granulomatosis. Ophthalmol Ther. 2013 Dec;2(2):99-111.109.Parmar GS, Ghodke B, Bose S, Meena AK. Stencilingbased ‘prick and Print’ technique for harvesting shaped corneal grafts for management of peripheral corneal perforations. Cornea. 2019;38(1):105–109.110.Gao H, Wang H, Echegaray, JJ, et al. Partial lamellar keratoplasty for peripheral corneal disease using a graft from the glycerin-preserved corneoscleral rim.Graefe’s Arch Clin Exp Ophthalmol. 2014;252:963–968.111.Lin H.-C, Lee Y.-S, Chia J.-H. Management of rheumatoid arthritis–related peripheral ulcerative keratitis using glycerol-preserved corneas. AsiaPacific J Ophthalmol. 2013;2(5):291–294.112.Spalton DJ, Graham EM, Page NG, Sanders MD. Ocular changes in limited forms of Wegener’s granulomatosis. Br J Ophthalmol. 1981; 65: 553-563. Somasheila I Murthy MDMedical Director, Shantilal Shanghvi Eye Institute, RJ Gaikwad Marg, Barkat Ali Naka, Wadala East, Mumbai, Maharashtra 400037Corresponding Author:

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 41 www.dosonline.orgTrabecular Meshwork Inflammation and Glaucoma: A Review of Implicated Molecular PathwaysMonika Arora[1] MS | Ragib Khan[1] | Ashima Varshney[1] MS | Anu Malik[2] MS1. Sitapur Eye Hospital, U.P2. Department of Ophthalmology, Dr. R. P. Centre for Ophthalmic Sciences, AIIMS, New DelhiAbstract: Glaucoma is a progressive optic neuropathy characterized by retinal ganglion cell (RGC) degeneration and optic nerve damage, often associated with elevated intraocular pressure (IOP). Increasing evidence suggests that trabecular meshwork (TM) inflammation plays a critical role in disease pathogenesis by disrupting aqueous humor outflow. This review explores the molecular pathways involved in TM inflammation, including cytokine-mediated responses, oxidative stress-induced damage, extracellular matrix (ECM) remodelling, TGF-β signalling, and NF-KB activation. These mechanisms contribute to TM dysfunction, increased IOP, and subsequent glaucomatous damage. Understanding these pathways offers new therapeutic opportunities aimed at preserving TM function and reducing disease progression.Key words: Trabecular meshwork inflammation, Glaucoma pathogenesis, NF-kB signaling, Oxidative stress in glaucoma, Extracellular Matrix Remodelling.IntroductionGlaucoma is a leading cause of irreversible blindness worldwide, with primary open-angle glaucoma (POAG) being the most common form. TM dysfunction is a key factor in POAG, leading to increased aqueous humor outflow resistance and IOP elevation. While traditional treatments focus on lowering IOP, recent research highlights the role of inflammation in TM pathology. Inflammatory cytokines, oxidative stress, ECM dysregulation, and pro-fibrotic signaling contribute to TM dysfunction and chronic IOP elevation. This review delves into the molecular pathways of TM inflammation, emphasizing their role in glaucoma progression and potential therapeutic targets.Diagram below provides a detailed view of how oxidative stress affects the trabecular meshwork, highlighting the role of antioxidants in mitigating damage.

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 42 www.dosonline.orgDiscussion1. Role of Pro-inflammatory Cytokines in TM DysfunctionInflammatory cytokines contribute to TM dysfunction by altering cell structure, promoting fibrosis, and reducing aqueous humor outflow. The key cytokines involved are TNF-α which induces TM cell apoptosis and increases ECM deposition, IL-1β and IL-6 promote inflammatory signalling and reduce TM cell viability, MCP-1 enhances immune cell infiltration into the TM.2. Oxidative Stress and TM DegenerationTM cells are highly susceptible to oxidative damage due to continuous exposure to aqueous humor dynamics. Oxidative stress results from an imbalance between reactive oxygen species (ROS) production and antioxidant defences. Oxidative stress plays a crucial role in TM degeneration. Reactive oxygen species (ROS) induce mitochondrial dysfunction, apoptosis, and senescence in TM cells. The upregulation of oxidative stress markers, such as superoxide dismutase (SOD) and glutathione peroxidase, suggests an adaptive response to prolonged oxidative damage.3. Extracellular Matrix Remodeling and Fibrosis in the TMECM remodeling in the TM plays a crucial role in aqueous humor outflow resistance. In glaucoma, excessive ECM deposition reduces TM permeability due to upregulation of MMP inhibitors thereby decreasing ECM turnover and finally TM fibrotic changes occur4. TGF-β Signaling and Its Role in TM FibrosisTGF-β is a major regulator of fibrosis and ECM production in the TM. It is significantly upregulated in glaucomatous eyes. Increased TGF-β activity in glaucomatous eyes promotes fibrotic changes, including excessive deposition 5. NF-kB Signalling and Chronic Inflammation in TMNF-kB is a central regulator of inflammation and is persistently activated in glaucomatous TM cells. NFkB is a Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells. It is a key transcription factor involved in regulating inflammation, immune responses, cell survival, and oxidative stress, making it highly relevant in conditions like glaucoma and trabecular meshwork dysfunction. Consequences of NF-κB activation in TM leads to Induction of pro-apoptotic genes and TM cell loss finally increase IOP. Targeting NF-κB could help in reducing TM inflammation and preserving aqueous humor dynamics.6. Potential Therapeutic TargetsGiven the role of inflammation in TM dysfunction, targeting these molecular pathways presents a promising strategy for glaucoma treatment. Anti-inflammatory agents, antioxidants, and TGF-β inhibitors are under investigation to mitigate TM damage and improve aqueous humor dynamics.of fibronectin and collagen, reducing TM permeability and elevating IOP.Figure 1: Cytokine induced TM dysfunction.Figure 2: TGF-β Signaling in TM fibrosis.Figure 3: NF-kβ Activation in TM Dysfunction.

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 43 www.dosonline.orgConclusionTrabecular meshwork inflammation plays a crucial role in glaucoma pathogenesis by altering cellular homeostasis, promoting fibrosis, and increasing aqueous humor outflow resistance. Key molecular pathways, including cytokine signaling, oxidative stress, ECM remodeling, TGF-β activation, and NF-κB-mediated inflammation, contribute to TM dysfunction. Targeting these pathways through anti-inflammatory agents, antioxidants, and fibrosis inhibitors holds promise for novel glaucoma treatments. Future research should focus on developing targeted therapies that restore TM function, improve aqueous humor dynamics, and prevent disease progression.References1. Wang Y, Wang S, Zhang Q, Cui X, Li W. Role of inflammatory cytokines in trabecular meshwork dysfunction and glaucoma pathogenesis. J Ocul Pharmacol Ther. 2023; 39(2): 140-152.2. Fan W, Li X, Wang W, Mo JS, Li J, Zhang J. NF-κB activation mediates inflammatory cytokine expression in trabecular meshwork cells under oxidative stress. Exp Eye Res. 2023; 226: 109275.3. Liton PB, Luna C, Challa P, Epstein DL, Gonzalez P. Involvement of inflammatory pathways in trabecular meshwork degeneration in glaucoma. Mol Vis. 2022; 28: 344-358.4. Zhou L, Li X, Lu S, Wang C, Zhang H. NF-κB signaling in trabecular meshwork inflammation and fibrosis: A potential target for glaucoma therapy. Front Cell Neurosci. 2021; 15: 597898.5. Clark AF, Matsumoto Y, Podos SM. Role of TGF-β and NF-κB activation in extracellular matrix remodeling in the trabecular meshwork. Exp Eye Res. 2020; 145: 124-132.6. Kumar A, Johnson M, Sharma S. Inflammatory and oxidative stress pathways regulating extracellular matrix degradation in trabecular meshwork cells. J Glaucoma. 2019; 28(5): 417-424.Monika Arora MSAssistant ProfessorGlaucoma Services Sitapur Eye Hospital, U.PCorresponding Author:7. Li G, Luna C, Liton PB. The role of oxidative stress and inflammatory mediators in chronic trabecular meshwork damage in glaucoma. Mol Vis. 2018; 25: 389-400.8. Meyers EE, Kuchtey J, Kuchtey RW. NF-κB as a central mediator of ocular hypertension-induced trabecular meshwork inflammation. Invest Ophthalmol Vis Sci. 2017; 58(4): 1982-1991.9. Vohra R, Aldrich A, Van Winkle TJ, Crean T, Keller KE. The interplay between cytokines and oxidative stress in trabecular meshwork dysfunction in glaucoma. Neurobiol Dis. 2016; 66: 95-104.10. Chen Y, Wang X, Zhang S, Li H. Anti-inflammatory strategies for protecting trabecular meshwork function in glaucoma. Prog Retin Eye Res. 2015; 36: 105-123.

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 44 www.dosonline.orgProteins at the Frontline: Revolutionizing Uveitis Diagnosis with BiomarkersAnu Sehrawat MS | Shweta VermaMS | Harinder Singh SethiMD, DNB, FRCSDepartment of Ophthalmology, VMMC and Safdarjung Hospital, New DelhiAbstract: Proteomics has emerged as a powerful tool for understanding ocular diseases, particularly uveitis, by identifying specific protein biomarkers that aid in diagnosis, prognosis, and therapeutic development. The Human Eye Proteome Project (HEPP) has significantly contributed to mapping the eye’s proteome, leading to discoveries in aqueous humor, vitreous humor, tears, and corneal tissues. Advances in mass spectrometry-based proteomics have facilitated the identification of differentially expressed proteins in various uveitis subtypes, including Behçet’s disease, Vogt-Koyanagi-Harada syndrome, juvenile idiopathic arthritis-associated uveitis, and birdshot retinochoroidopathy. Specific biomarkers, such as Amphiregulin, EMILIN1, and IFN-related proteins, have been linked to inflammatory pathways, offering potential therapeutic targets. Proteomic analysis has also revealed cytokine profiles that differentiate infectious from non-infectious uveitis. Emerging research suggests that proteomics-guided therapies, including bortezomib, sirolimus, and ivermectin, may hold promise for uveitis treatment. Additionally, proteomics is aiding in gene therapy approaches and personalized medicine by linking disease-specific protein signatures to targeted drug discovery. While challenges remain in translating proteomic findings into clinical applications, ongoing research continues to refine biomarker panels and advance novel therapeutic strategies for uveitis management.IntroductionThe human eye is a multi-compartment organ with distinct and specialized characteristics that correspond to its numerous roles. The pathophysiology of many common eye ailments remains vaguely understood, despite improvements in ocular imaging and treatments over the past ten years. Understanding the pathophysiology, diagnosis, and therapy of eye disorders can be greatly aided by proteomics.[1] By characterizing the diversity and quantity of proteins-including their isoforms and post translational modifications (PTMs)-in both health and normal aging against those that occur with disease or during treatment, proteomics can offer fresh insights into the pathophysiology of ocular disease.[2]The Human Eye Proteome Project (HEPP), established in 2012 as part of the Biology/Disease-driven Human Proteome Project (B/D-HPP), aims to advance proteomic research related to ocular diseases through the application of cutting-edge protein measurements.[3]Uveitis has been found to be the cause of 10% of legal blindness in the United States.[4] The precise visual morbidity associated with uveitis in India remains undetermined, however it seems to be comparatively higher than in developed nations.[4]Over the past few years, Mass Spectrometry (MS) based proteomics has become widely used in laboratory medicine due to its capability for identifying and quantifying biomolecules from a variety of biological components. The use of MS based Proteomics technology is growing and is frequently employed.This review’s objectives are to: (i) provide an overview of the advancements made in human eye proteomic studies; (ii) to emphasise the necessity of finding novel biomarkers for uveitis; and (iii) demonstrate how the significant developments in proteomics can be employed to learn more about the pathophysiology of eye disorders.[2]Innovations in Ocular Proteomics: Mapping The Human Eye’s ProteomeOnly a portion of the proteomes of the various eye tissues and related fluids have been fully described. The majority of research has focused on the more approachable parts of

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 45 www.dosonline.orgthe eye, such as the vitreous humor, aqueous humor, and tears.TearThe thin (3-40μm) layer of tears that covers the eye’s surface epithelium is a complex extracellular fluid that may be readily and non-invasively analyzed for proteins that are helpful in tracking ocular conditions.[5]Schirmer’s strip and fire-polished glass capillary tubes are the two most widely used collection techniques.[6] Over 1500 distinct proteins make up the average 3–7μg/μL protein concentration in tears, with lysozyme, lipocalin, immunoglobulins, lactoferrin, IgA, and proline-rich proteins accounting for 90% of this concentration.[7]A huge number of proteins have been identified in the acute anterior uveitis eye. More recent studies like Eidet et al. demonstrated that when compared to healthy controls, the acute phase response protein Serpin Family A Member 3 (SERPINA3) was elevated in the healthy eye of acute anterior uveitis patients.[6]The main constraint of proteomic research in tear is that the detection of less abundant proteins is obscured by the presence of large abundance proteins.CorneaThe corneal layers differentiation into corneal epithelium, stroma and endothelium alongwith contemporary mass spectrometry allowed identification and quantification of 3250 distinct proteins as individual layers can be involved in various pathologies.[8] The predominant proteins comprised aquaporin-1, serotransferrin, collagen Type I and III, immunoglobulins, albumin, complement components 3 and 9, and Tissue inhibitors of metalloproteinases. Proteomics has been studied more extensively in Fuch’s endothelial dystrophy and keratoconus as compared to uveitis.Aqueous HumourLee et al. detected 1683 proteins in Aqueous Humour samples and developed a database.[9] Albumin, Serotransferrin, Immunoglobulin gamma-1 heavy chain, Hemopexin, Complement C3 and Alpha-1-antitrypsin were the most abundant proteins. A study conducted on Fuchs uveitis syndrome (FUS) showed that the complement C1q and secretogranin-1 were highly elevated.[10]Ciliary BodyProteomic study of normal human ciliary body using LC-MS/MS has revealed 2,815 proteins.[11] Additionally, 211 proteins of these were identified in the analysis of aqueous humor.LensCrystallins are the primary component of lens proteins and offer a valuable chance to investigate post-translational modifications due to their longevity.[12]Vitreous HumorThe vitreous, a highly hydrated viscous substance is rich in structural (collagen types II, IX and XI) as well as nonstructural proteins.[13] About 2,062 proteins have been identified in vitreous humor.[14] Kuiper et al. devised a triad of interleukin (IL)-10, IL-21, and angiotensin converting enzyme (ACE) that could correctly classify patients of non-infectious uveitis, age-related macular degeneration, idiopathic, primary vitreoretinal lymphoma and rhegmatogenous retinal detachment with 79.4% sensitivity and 92.5% specificity.[15]Non Infectious UveitisAnkylosing SpondylitisAnkylosing spondylitis (AS) is an inflammatory condition marked by both joint and systemic findings. The predominant extra-articular manifestation of ankylosing spondylitis is Non-infectious sterile uveitis. The investigation of human protein microarrays from serum of patients with AS demonstrated higher serum levels of Prefoldin 5 (PFDN5) and anti-PFDN5 antibodies in AS patients with uveitis as compared to diminished levels seen in AS patients without uveitis.[16] MiR-223-3p, miR-155, miR-182, let-7e, miRNA-1, miR-9-3, miR-23a, miR-146a, mir-29a-3p, miR-196a2, and miR-143 are the microRNAs (miRs) that have the best likelihood of identifying the onset of uveitis.[17]Lee et al. showed that miR-3620-3p was strongly correlated with history of uveitis and established it as a promising biomarker for Ankylosing Spondylitis.[18]Sarcoid UveitisUveitis is the most prevalent ocular symptom of sarcoidosis, a multisystem, chronic inflammatory disease marked by noncaseating granulomas.[19] The vitreous humor of eyes with sarcoidosis has been found to include greater quantities of certain cytokines, such as IL-1, IL-6, IL-8, Interferon (IFN-γ), and TNF-α, as well as chemokines, such as MIP-1α and MIP-1β.[20]Behcets DiseaseBehcet’s disease (BD) is a chronic, multisystem autoinflammatory condition marked by recurring oral and genital ulcers, uveitis, skin lesions, and neurological, gastrointestinal and vascular symptoms.[21] Proteomic analysis demonstrated that Amphiregulin overexpression,

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 46 www.dosonline.orgIFN signaling, and the ErbB signaling pathway are specific to behcets disease.[22]Amphiregulin, an epidermal growth factor ligand is essential for tissue healing, homeostasis maintenance, host resistance and tolerance, local inflammation control, and immunological suppression of the tumor microenvironment.[23]Lee et al identified the protein biomarkers EMILIN1 and LYZ may have a role in the pathophysiology of BD as well as the control of infection and inflammation, (M Lee et al., 2006) EMILIN1, an extracellular matrix glycoprotein associated with elastic fibers can inhibit the migration of macrophages as well as the production of inflammatory factors, such as TNF-α and IL-1β.[25] Whereas LYZ is an antimicrobial protein present in neutrophils and macrophages which hydrolyze peptidoglycans.[26]Vogt Koyanagi Hrada Syndrome (VKH)VKH is a debilitating ocular condition resulting from immune system dysfunctions that target melanocytes, presenting as changes in skin and hair, auditory anomalies, and central nervous system involvement.[27]It is frequently misdiagnosed. Platelet Derived Growth Factor (PDGF) signaling system, TGF-β signaling pathway and complement cascade are the major mediators.[28] Li X et al. confirmed the correlation of STXBP1 (Syntaxinbinding protein 1) and APOH (Beta-2-glycoprotein) biomarker with VKH in their proteomic analysis by ELISA.[29] The primary biological functions of APOH include disrupting the protein C pathway and removing liposomes and apoptotic bodies.[30] STXBP1 plays a role in vesicle transport, neurotransmitter release, type II IFN response, and mast cell granule release. The STXBP1 gene’s association with immune cells such CD4+ T cells, macrophages, and dendritic cells implies it regulates immunological response.[31]Juvenile Idiopathic Arthritis-Associated Uveitis ( JIA-U)Ten to twenty percent of children with juvenile idiopathic arthritis generally JIA-U, and as many as fifty percent of those children experience irreversible visual sequalae.[32]Aqueous humor and serum protein assessment revealed S100 proteins, cytokines, and chemokines as biomarkers for uveitis.[33] Children with JIA-U have shown considerably higher levels of S100A12 and IL-8 (mediates neutrophil activation) in the tears of patients with active uveitis[34] and MCP-1 (chemotactically recruits monocytes and basophils) in their non-inflamed uveitic tears on proteomic analysis.[35]Fuch’s Uveitis Syndrome (FUS)FUS is a chronic, usually unilateral variant of mild anterior uveitis, distinguished by the clinical hallmark of heterochromia. On comparative proteomic analysis of aqueous humour of FUS uveitis with Idiopathic Anterior Uveitis (IAU) by Muhaya et al., they discovered that FUS had lower amounts of IL-12 and higher levels of IFNγ.[36] Compared to other uveitic entities like Behcet’s disease and Vogt-Koyanagi-Harada (VKH) syndrome, a recent study in Chinese patients found significantly greater levels of IFN-γ, MCP-1, macrophage inflammatory protein (MIP)-1β, and TNF-α in AH samples of FUS patients.[37] The results of the multivariate analysis demonstrated a strong correlation between MIP-1β and FUS.Birdshot RetinochoroidopathyMultiple hypopigmented chorioretinal lesions are a hallmark of birdshot retinochoroidopathy (BSRC), which frequently results in retinal and optic disc atrophy, which compromises vision and the visual field. It shows strong correlation with HLA-A29. More recently, it is described as a T Cell, especially Th17 system mediated etiopathogenesis.[38] Kuiper et al. identified Relatively increased levels of TNF-α, IL-1β, IL-17, IL-6 and IL-2 have been identified in BSRC patients. The Th17 cellmediated inflammation theory was further strengthened by detection of elevated serum levels of interleukin IL-21, IL-23, and TGF-β1 by proteomics in BSRC patients with active disease.[39]Infectious UveitisToxoplasmosisOcular toxoplasmosis, a chronic necrotizing retinitis caused by Toxoplasma gondii, can cause sequalae such retinal detachment, choroidal neovascularization, and glaucoma.[40] Autoimmune processes involving retinal immune-dominant membrane antigens can cause it. During this process, the blood levels of IgE, IgA, IgM and IgG antibodies turns positive in accordance with the stages of infection. The immunological response involves activation of macrophages, NK cells, and T-cells, resulting in the release of cytokines such IL-4, IL-5, IL-10, IL-12, IL-27, and TNF-α. Several cytokines induce IFN-γ production in CD8+ T and NK cells. CD4+ Th 2 cells generate IL-4, IL-5, and IL-10 to reduce inflammation.[41] Toxoplasma retinochoroiditis is linked to IL-10 gene polymorphism.

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 47 www.dosonline.orgTuberculosis(TB)Tuberculosis is caused by Mycobacterium tuberculosis which causes cell mediated delayed type hypersensitivity (type IV reaction).[42] After reaching at the local lymph nodes, mycobacteria are presented to CD4+ cells. The release of IFN-γ by these cells can reactivate macrophages. After becoming epithelioid cells, macrophages create granulomas. Reactivation of primary TB can cause secondary TB if fresh bacteria invade the lesion. After anti-tubercular therapy, cytokines and chemokines investigation in TB-related uveitis showed autoimmune processes rather than active TB.[43] Proteomic profile of vitreous discovered that the protein that was shown to be most significantly increased in the TB uveitis group when compared to controls was insulin-like growth factor 2 messenger RNA binding protein 3 (IGF2BP3).[44]Viral DiseaseCell-mediated immunity significantly contributes to viral illnesses in which infected cells in the uveal and ocular tissues exhibit viral-specific antigens on their surfaces and induce targeted tissue destruction, as seen in acute retinal necrosis (ARN) and Cytomegalovirus (CMV) retinitis. Samples of ocular fluids from patients who had viral uveitis showed the presence of cytokines, including IL6, IL-10, and IFN-γ. Similarly, Interleukins 2, 6, 10 and IFN-γ were discovered to be linked to herpes group virus isolates in ARN. Soluble tumor necrosis factor receptor 1 (sTNFR1) and CC Chemokine (I-309) have also been correlated with herpes virus uveitidis.Fungal UveitisFungal keratouveitis exhibits higher levels of IL-1 β, IL6, IL-8, and IFN-γ. Balamurugan et al. described that the CD4+ cells trafficking and their modulation by effector cytokines during invasive aspergillosis will be pivotal for targeted immunotherapy.[45] A systemic aspergillosis can cause type I and III hypersensitivity. Candida’s ability to stimulate interphotoreceptor retinoid-binding proteinmediated Card 9, a signaling molecule of subgroup C-type lectin receptor (CLR1), may help defend the host.[46]Protein Biomarkers in Masquerades SyndromesUveal MelanomaUveal melanoma (UM) is the leading primary eye tumour.[47] Aqueous cytokines in uveal melanoma are identical to those in uveitic eyes. Tumor cells and normal tissue release the proinflammatory cytokines IL-6 and Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), which promote tumor growth. T-cell infiltration in tumor cells was suggested to be determined by elevated cytokines.[48] Proinflammatory cytokines like GM-CSF boost macrophage major histocompatibility complex (MHC) class II expression. This increases TRAIL (TNF-related apoptosis-inducing ligand) release and stimulates CD4+ T helper cell antigen presentation.LymphomaPrimary intraocular lymphoma (PIOL) is an infrequent ocular neoplasm but a great mimickerof uveitis. A strong correlation of optic nerve infiltration and poor tumor differentiation with high levels of survivin and transforming growth factor β1 was identified.[49] Takeda A et al. found that compared to controls, Fibroblast growth factor (FGF2), Hepatocyte growth factor (HGF), and Beta nerve growth factor (βNGF) were considerably enhanced, but IP-10 levels were low in lymphoma patients. The RAS-MAPK/P13K-Akt/STAT signaling pathway promotes tumor development, metastasis, and VEGF overexpression. Also, IL-6, IL-7, and IL-8 levels are much greater.[50]Retinal Detachment (RD)In eyes affected by rhegmatogenous RD due to uveal melanoma, Chemokine ligand 19 (CCL-19) was found to be a potential indicator for the early development of proliferative vitreoretinopathy alterations.[51] In vitreous samples of 24 retinal detachment patients, Kiang et al. found elevated levels of IL-6, IL-8, MDC (CCL22), fractalkine, and MCP-1 (CCL2), and suggested that microglial cells play an important role in inflammatory response.[52]Retinitis Pigmentosa (RP)Proteomic analysis of aqueous samples of RP showed increased levels of matrix metalloproteinases like MMP-2, MMP-3, MMP-7, MMP-8, TSP-2 (thrombospondin), and PAI-1 (Plasminogen activator inhibitor) alongwith decreased levels of BMP-4 (Bone morphogenetic protein). RP eye aqueous contained higher MCP-1, IL-8, HGF, and IL-6. RP eyes developed posterior subcapsular cataracts, capsule contraction syndrome and mimic uveitis due to elevated proinflammatory cytokines.Type of uveitis Differentially Expressed ProteinsAnkylosing Spondylitis Prefoldin 5 (PFDN5) and anti-PFDN5 antibodies

Special Issue - Ocular Inflammation and Molecular Markers: “Untying the Knots”DOS Times - Volume 30, Number 5, January-February 2025 48 www.dosonline.orgType of uveitis Differentially Expressed ProteinsSarcoid Uveitis IL-1, IL-6, IL-8, IFN-γ, TNF-α, MIP-1α and MIP-1βBehcets Disease Amphiregulin overexpression, CTDP1, Alpha-1-acid glycoprotein 1, EMILIN1 and LYZVogt Koyanagi Hrada Syndrome (VKH) STXBP1, APOH and CD18Juvenile Idiopathic Arthritis-Associated Uveitis (JIA-U)S100 protein, Transthyretin (TTT), MCP-1 and IL-8Fuch's Uveitis Syndrome (FUS) MIP-1β, IFN-γ, MCP-1 and TNF-αBirdshot Retinochoroidopathy IL-21, IL-23 and TGF-β1Toxoplasmosis IL-12, IL-27 and TNF-α, IL-10 gene polymorphismTuberculosis (TB) insulin-like growth factor 2 messenger RNA binding protein 3 (IGF2BP3), IFN-γHerpes Virus Uveitis Soluble tumor necrosis factor receptor 1 (sTNFR1) and CC Chemokine (I-309), IL-6, IL-10, and IFN-γFungal Uveitis of IL-1 β, IL-6, interphotoreceptor retinoid-binding protein-mediated Card 9Table 1: Differentially Expressed Proteins In Uveitis.Recent Progress in Proteomics and its Clinical PotentialInitial proteomic investigations of the eye employed the conventional protein separation technique of twodimensional electrophoresis (2DE) in conjunction with ion trap or MALDI-TOF mass spectrometry. Over the past few years, Mass Spectrometry (MS) based proteomics has become widely used in laboratory medicine due to its capability for identifying and quantifying biomolecules from a variety of biological components. The use of MS based Proteomics technology is growing and is frequently employed for early diagnosis, prognosis and monitoring of response to therapy. Protein biomarkers in BD patients’ serum and tears offered important clues in distinguishing the disease from another autoimmune disorder (VKH disease), aided in disease staging using serum biomarkers, and directed monitoring of uveitis treatment using tear biomarkers, in the absence of clinical or diagnostic biomarkers.Moreover, by identifying a protein or cytokine profile shared by several varieties of autoimmune uveitis, a single treatment can be used to address all of these conditions. Proteomics can associate diseases with therapeutics via proteins and identify suitable medications by assessing the interconnections of disease-drug linkages. In order to develop biomarker panels and medications for autoimmune uveitis using proteomic analysis, Li et al. assessed the prediction of prospective therapeutic treatments such as ivermectin and sirolimus for VKH.[53] Additionally, vorinostat, bortezomib and everolimus are under trial in uveitis patients and animal models.[54] Similarly, proteomic also help to identify the drugs to be avoided. Proteomics’ most recent application in uveitis is in gene therapy for specific diseases. The field of proteomics has enhanced our understanding of uveitis and holds potential for future drug development and the development of less intrusive methods of approaching uveitis as well as other ocular pathologies.Conflict of InterestThe authors declare that they have no conflict of interest. The authors declare that no financial support was received for authorship of this article.References1. Ahmad MT, Zhang P, Dufresne C, Ferrucci L, Semba RD. The Human Eye Proteome Project: Updates on an Emerging Proteome. Proteomics. 2018 Mar;18(5–6):e1700394. 2. Semba RD, Enghild JJ, Venkatraman V, Dyrlund TF, Van Eyk JE. The Human Eye Proteome Project: Perspectives on an emerging proteome. Vol. 13, Proteomics. 2013. p. 2500–11. 3. Bansal R, Gupta A. Protein Biomarkers in Uveitis. Front Immunol. 2020 Dec 3;11.

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