antibodies

2025-05-30 | Fusarium spp. and Aspergillus flavus infection induces pathogen-specific and pathogen-independent host immune response in patients with fungal keratitis

Fungal keratitis, caused primarily by Fusarium spp. and Aspergillus flavus, is a significant cause of corneal blindness, particularly in tropical regions. Current antifungal agents like natamycin and voriconazole have limited efficacy, underscoring the need for a deeper understanding of host immune responses. This study employed high-throughput RNA sequencing to investigate differential gene expression in human corneal tissues from patients with Fusarium spp. and A. flavus keratitis and compared them to control cadaver corneal samples. RNA was extracted from infected and control samples, followed by sequencing and differential expression analysis. Further confirmation of differential expression of selected genes were carried out by Real-Time quantitative PCR (RT-qPCR). Data analysis identified common and Fusarium spp. and A. flavus-specific differentially expressed genes (DEGs). Pathway enrichment analysis using common genes identified pathways enriched in both infections, such as interleukin 17 (IL-17), tumor necrosis factor (TNF), and chemokine signalling. Expression of hub genes, including S100 calcium binding protein A7 (S100A7), S100 calcium binding protein A8 (S100A8), S100 calcium binding protein A9 (S100A9) and C-X-C motif chemokine ligand 8 (CXCL8), identified in interleukin 17 (IL-17) signalling, was confirmed by RT-qPCR analysis. Fusarium spp.-specific DEGs, including complement C3 (C3), interleukin 6 (IL-6), interleukin 19 (IL-19) and leucine rich alpha-2-glycoprotein 1 (LRG1), are enriched in pathways such as positive regulation of immune responses, acute inflammatory responses, leukocyte cell-cell adhesion, and the regulation of cell-cell adhesion. A. flavus-specific DEGs, such as triggering receptor expressed on myeloid cells 2 (TREM2) and apolipoprotein E (APOE), are predominantly enriched in adaptive immune response, negative regulation of immune system process, negative regulation of immune response, cell migration and motility pathways. RT-qPCR confirmed the key pathogen-specific DEGs, highlighting their potential as biomarkers for pathogen-specific immune responses. These findings provide insights into the distinct immune pathways triggered by Fusarium spp. and A. flavus, offering new therapeutic targets for improving fungal keratitis treatment.

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2024-07-31 | Anti-Inflammatory Efficacy of Nanobody Specific to β-Glucan on a Fungal Cell Wall in a Murine Model of Fungal Keratitis

Purpose: to explore the anti-inflammatory effects of a nanobody (Nb) specific to β-glucan on fungal keratitis (FK). Methods: in order to verify the therapeutic and anti-inflammatory efficacy of Nb in FK, the severity of inflammation was assessed with inflammatory scores, hematoxylin–eosin (HE) staining, and myeloperoxidase (MPO) assays. In corneas of mice of FK model and human corneal epithelial cells stimulated by fungal hyphae, real-time reverse transcriptase polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were used to detect the expression levels of inflammatory cytokines and pattern recognition receptors (PRRs). In vivo, macrophages and neutrophils infiltration in the cornea stroma was detected by immunofluorescence (IFS) staining. Results: In murine models infected with Aspergillus fumigatus (A. fumigatus), Nb treatment could reduce the inflammatory scores. HE staining and MPO results showed Nb significantly alleviated corneal edema and reduced inflammatory cell infiltration 3 days post-infection. In addition, the expression levels of LOX-1 and Dectin-1 were significantly decreased in the Nb group in vivo. The expression of chemokines CCL2 and CXCL2 also decreased in the Nb group. Compared with the PBS group, the number of macrophages and neutrophils in the Nb group was significantly decreased, which was shown in IFS results. Moreover, Nb attenuated the expression of Dectin-1, LOX-1, and inflammatory mediators, including IL-6 and IL-8 in vitro. Conclusion: our study showed that Nb could alleviate FK by downregulating the expression of PRRs and inflammatory factors as well as reducing the infiltration of macrophages and neutrophils.

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2021-09-06 | Anterior Segment Optical Coherence Tomography Angiography Assessment of Corneal Vascularisation After Combined Fine-Needle Diathermy with Subconjunctival Ranibizumab: A Pilot Study.

To describe anterior segment optical coherence tomography angiography (AS-OCTA) imaging to monitor corneal vascularisation (CoNV) and scar reduction after combined fine-needle diathermy (FND) with subconjunctival ranibizumab. Prospective clinical study of six eyes from six subjects with corneal scar and CoNV which underwent combined FND with subconjunctival ranibizumab. All eyes were imaged using slit-lamp photography (SLP) and AS-OCTA (Optovue, Inc., Fremont, CA, wavelength: 840 nm) before and after the operation, with two independent masked assessors analysing all images. Main outcome measures were changes in median corneal scar area and vessel density (AS-OCTA) comparing pre- and postoperative imaging up to month 3 and 6. The mean age of the subjects was 60 ± 23 years, with three males and three females. CoNV and corneal scarring involving the visual axis were present in all eyes, secondary to previous infective keratitis (n = 3), severe blepharokeratoconjunctivitis (n = 2), or chemical injury (n = 1). Follow-up time frame ranged from 2 to 6 months postoperation. There was a reduction in median corneal scar area from 30.2 mm2 (IQR 18.7-38.5) before surgery to 14.8 mm2 (IQR 7.1-19.6) after surgery, with a median reduction of 37.1% (IQR = - 3.1-86.9, p = 0.046). There was also a reduction in median cornea vessel density (AS-OCTA) from 20.8% (IQR 16.1-20.8) before surgery to 17.6% (IQR 14.0-17.6) after surgery, with a median reduction of 15.1% (IQR 13.2-15.1, p < 0.001). Combined imaging of SLP and AS-OCTA is useful for monitoring treatment response of corneal scarring and CoNV after combined FND with subconjunctival Ranibizumab.

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1988-01-01 | Protection against Herpetic Ocular Disease by Immunotherapy with Monoclonal Antibodies to Herpes simplex Virus Glycoproteins

In this paper we describe the ability of monoclonal antibodies to prevent herpetic stromal or interstitial keratitis following corneal infection in an outbred mouse model. Monoclonal antibodies recognizing antigenic determinants on glycoproteins B, C, D, and E of herpes simplex virus type 1 were injected intraperitoneally into CF-1 outbred mice 24 or 48 h following inoculation of the cornea with the RE strain of herpes simplex virus type 1. Passive, postexposure immunization with monoclonal antibodies had little effect on the severity of the initial corneal infection or the frequency of latent viral infections in the trigeminal ganglia, except for virus-neutralizing antibodies specific for glycoproteins B and D. A significant correlation was found between the severity of epithelial keratitis and the frequency of latent ganglionic infections. However, immunization with monoclonal antibodies protected the mice against encephalitis and prevented the development of necrotizing stromal keratitis that leads to permanent corneal scarring and blindness. This form of herpetic ocular disease does not respond to antiviral chemotherapy. Since nonneutralizing monoclonal antibodies were just as effective in prevention of encephalitis and stromal keratitis as ones that neutralized the virus in vitro, and antibodies were not administered until 24 or 48 h after corneal inoculation, we suggest that inactivation of infectious virus is not the only protective mechanism in this model.

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small molecules

2026-08-05 | Targeted Deletion of the exoU Gene Attenuates Biofilm Formation and Virulence of Pseudomonas aeruginosa Isolated from Contact Lens-Related Corneal Infection in a Murine Model

Purpose: Pseudomonas aeruginosa is the leading cause of contact lens (CL)-related microbial keratitis (MK), a sight-threatening corneal infection, accounting for 60-70% of culture-positive cases. Cytotoxic P. aeruginosa strains, which carry the exoU gene, are frequently associated with severe CL-related MK. Beyond enhanced cytotoxicity, exoU strains frequently display increased resistance to fluoroquinolones and aminoglycosides antibiotics. While resistance genes contribute to this phenotype, biofilm formation is a key driver of antibiotic tolerance and persistence. Although exoU isolates are often strong biofilm producers, the direct role of exoU in biofilm formation remains undefined. This study investigated the contribution of the exoU gene of P. aeruginosa to in vitro biofilm formation and in vivo virulence in a murine keratitis model. Methods: Nine exoU strains isolated from CL related keratitis were screened for biofilm production using the crystal violet assay. The highest biofilm-producing strain (PA169) was selected for deletion of the exoU gene via homologous recombination using the recombineering plasmid pCasPA. Deletion was confirmed by PCR. Biofilm biomass and architecture were compared between the wild-type PA169 and mutant (exoU gene deleted) using the crystal violet assay and confocal laser scanning microscopy (CLSM). Cytotoxicity of wild-type and mutant strains toward human corneal epithelial cells (HCECs) was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. For in vivo virulence assessment wild-type exoU and the exoU mutant were evaluated in a BALB/c murine keratitis model. Disease clinical severity scoring, corneal bacterial burden (log CFU), and myeloperoxidase (MPO) activity of polymorphonuclear neutrophil were compared among groups using Kruskal-Wallis and one-way Anova tests. Results: Biofilm formation was significantly reduced in the exoU mutant (OD570/OD660 nm=1.4±0.08) compared to its wild-type PA169 (OD570/OD660 nm=2.2±0.15, p<0.01). CLSM confirmed a decrease in biofilm thickness in the mutant strain relative to the parent strain (8.33±0.58 µm vs 11.67±0.58 µm, respectively, p<0.01). Additionally, the mutant produced lower cytotoxicity than the wild-type (OD570 8.2±0.23 vs 2.1±0.25, p<0.01) in HCECs. In vivo, at 48 hours post infection, wild-type exoU strain resulted in significantly higher disease severity (median 3, IQR 3-4) compared with the mutant (median 2, IQR 2-3; p<0.01). Corneal bacterial burden was higher in the wild-type exoU group (6.1±0.05 log CFU), significantly exceeding the mutant (4.3±0.04 log CFU; p<0.0001). MPO activity was markedly elevated in exoU-infected corneas (3.7±0.40 OD) compared with the mutant (1.2±0.16 OD; p<0.0001), indicating enhanced neutrophil-mediated inflammation. Conclusion : The exoU gene contributed to biofilm formation in the PA169 strain and directly promotes bacterial persistence and severe corneal inflammation. The gene exoU encodes a phospholipase, therefore, targeting this phospholipase with, for example phospholipase inhibitors, may represent a promising therapeutic strategy for preventing sight-threatening CL–associated MK.

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2026-07-28 | Mechanisms Underlying Increased Susceptibility and Severity of Bacterial Keratitis in Diabetic Mice 2257255

Abstract Introduction Diabetic patients have a higher incidence of infections, with increased severity and a higher prevalence of multidrug resistance. This study was to investigate the role of programmed cell death (PCD) pathways as an underlying mechanism for increased susceptibility and severity of bacterial keratitis in diabetic C57BL/6 mice. Methods Streptozotocin-induced diabetic (DM) C57BL/6 mice were inoculated with Pseudomonas aeruginosa (Pa), and the PCD pathways in the control, normoglycemic mice and STZ-treated mice were determined by immunohistochemistry using antibodies that detect the active forms of Caspase-8 (intrinsic apoptosis) and RIPK3 (necroptosis). The role of PCD pathways was determined by Casp8 and RIPK3 inhibitors. The therapeutic effects of targeting necroptosis were assessed using RIPK3 and HMGB1 inhibitors as adjunctive treatments, concomitantly applied with antibiotics to eliminate the invading bacterial pathogen. Results At 1-day post-infection (dpi), hyperglycemia increased neutrophil but suppressed macrophage infiltration. While in the control corneas, cleaved casp8 was stained strongly in NL, phosphor-RIPK3 was mostly detected in DM epithelial layer and stromal infiltrates. Inhibition of caspase activity worsens the outcome of keratitis, whereas blocking RIOPK3 prevents Pa infection in NL and greatly reduces the severity of keratitis in DM corneas. At 16 h post-infection, concomitant application of RIPK3 inhibitor or cell impermeable inhibitor HMGB1, a prototype of alarmin released from lytic PCD pathways, reduced lingering inflammation in Pa-infected corneas at 3 dpi. Conclusion Hyperglycemia skews apoptosis towards necroptosis, leading to exacerbated bacterial keratitis in B6 mice. Preventing or reversing this PCD shift may serve as an adjunct therapy for treating microbial keratitis in diabetic patients. Funding Source NIH grants R01 EY035785, EY010869, and RPB Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

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2026-06-15 | Design, Synthesis, Biological Evaluation of Novel Substituted Benzoxazole-Schiff Base Derivatives as Antibacterial Agents

Abstract A series of benzoxazole-Schiff base hybrids were synthesized and evaluated for broad-spectrum anti-infective activity. Two compounds emerged as potent antibacterial agents, inhibiting S. aureus [including methicillin-resistant Staphylococcus aureus (MRSA)], B. subtilis at 2–8 µg/mL, and E. coli at 16 µg.mL. One lead analogue combined the highest antibacterial activity with negligible mammalian cytotoxicity and demonstrated strong synergism with clinically relevant antibiotics. At elevated concentrations, this compound eradicated MRSA, disrupted pre-formed biofilms, and significantly delayed resistance development compared with norfloxacin. In-silico ADME profiling predicted that several of the synthesized compounds comply with Lipinski’s rules and exhibit favorable oral bioavailability and intestinal permeability. Mechanistic investigations attributed bacterial lethality to rapid membrane depolarization and ultrastructural disintegration. In a murine MRSA keratitis model, topically applied this lead analogue achieved dose-dependent bacterial clearance from infected corneas, underscoring its therapeutic potential for treating ocular and potentially systemic infections.

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2026-06-11 | Exploring the Antifungal, Antibiofilm, and Wound Healing In Vitro Properties of N-(4-Methoxycinnamoyl)-Anthranilic Acid as a Supportive Strategy for Ocular Fungal Infections

Background: Fungal ocular infections, including keratitis and endophthalmitis, remain difficult to treat due to limited antifungal efficacy, poor tissue penetration, and biofilm-mediated resistance. This study evaluated the antifungal and host-protective potential of N-(4-methoxycinnamoyl)-anthranilic acid (NMCA) against Candida albicans and the multidrug-resistant Candidozyma auris. Methods: The antifungal activity of NMCA was assessed by analyzing fungal viability over time, ergosterol levels, and its interaction with fluconazole. Its antibiofilm activity was evaluated through biomass and metabolic activity measurements, together with the expression of genes involved in adhesion (ALS3, ALS5, HWP1) and membrane homeostasis (ERG11, OLE1). In addition, infected epithelial models were used to investigate epithelial damage, intracellular fungal burden, oxidative stress, and wound closure. Results: NMCA showed promising antifungal activity (MIC80 75 μg mL−1 against C. albicans and 100 µg mL−1 against C. auris), inducing a time-dependent reduction in fungal viability of about 4-log10 after 24 h. The compound also reduced ergosterol levels and showed synergistic interaction with fluconazole, as indicated by FICI values of 0.203 for C. albicans and 0.375 for C. auris. Moreover, NMCA markedly inhibited biofilm formation by reducing both biomass and metabolic activity up to approximately 80%, while modulating the expression of key adhesion- and membrane-related genes. Beyond its direct antifungal effects, NMCA reduced epithelial damage and intracellular fungal burden, attenuated oxidative stress, and significantly improved wound closure (reaching 76.26% and 90.46% closure in C. albicans- and C. auris-infected cells, respectively) in infected epithelial models. Conclusions: Although limited by the use of in vitro systems, these findings highlight the multifunctional profile of NMCA, which combines antifungal, antibiofilm, and tissue-protective activities. By simultaneously targeting pathogen viability, biofilm formation, and host cell integrity, NMCA appears to be a promising adjunctive candidate for the treatment of ocular fungal infections, where both pathogen eradication and tissue preservation are crucial for clinical outcomes.

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2026-05-19 | Microbiological profile, antibiotic susceptibility pattern and treatment outcome of infectious keratitis at a tertiary care centre in Central India

Aim: Analytical cross-sectional study of causative organism, antibiotic susceptibility patterns and treatment outcome of infectious keratitis in a tertiary care hospital in Central India.Materials and Method: A hospital based, observational study was carried out at the Ophthalmology department of a tertiary care center in Central India. Demography, clinicopathological and microbiological profile, antibiotic susceptibility pattern and clinical outcomes were analysed.Results: During the period of two years which were analyzed, 180 patients of keratitis were identified out of which 50 cases were of infective keratitis. In our study, infective keratitis included all non-viral causes of the disease. Maximum incidence of microbial keratitis was seen in the age group 41 years and above (64%). Males were more commonly affected than females (66% versus 34%). Out of fifty cases, 28% was due to bacterial causes, 32% were due to fungal causes and 2% was due to acanthamoeba. Staphylococcus (78.6%) and Pseudomonas (14.3%) were the commonest isolates in bacterial keratitis. Aspergillus species was isolated in 90.9% cases with fungal keratitis.Conclusion: In our study, both bacterial and fungal causes were equally observed. The microbial flora varied based on geographical area and local antimicrobial practices. Wide -spread use of antibiotics has led to resistance in some case. Sensitivity to older antibiotics, such as Chloramphenicol, is seen to be reemerging. Therefore, in cases of resistance, Chloramphenicol can be considered as an alternative. Prudent and cautious use of antibiotics is essential to prevent the development of resistance.

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proteins

2026-04-15 | Neuroprotectin D1 regulating the inflammation and nerve restoration in Aspergillus fumigatus keratitis

AIM: To investigate the anti-inflammatory and neurorestorative effects of neuroprotectin D1 (NPD1) in Aspergillus fumigatus (A. fumigatus) keratitis in C57BL/6 mice. METHODS: The left corneas of C57BL/6 mice were infected with A. fumigatus. Each mouse was injected intraperitoneally with 2 μg NPD1 at 1, 3, and 5d post infection (p.i.) and injected subconjunctivally with 5 μL NPD1 once a day. The severity of keratitis was observed by slit lamp and classified by clinical score at 1, 3, and 5d p.i. Hematoxylin and eosin (HE) staining was used to evaluate the histological changes and inflammatory cell infiltration of corneas at 3 and 5d p.i. Polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and Western blot were performed to detect the expression of IL-1β, MIP-2, CGRP, p-p38/p38 MAPK, Nrf-2, and HO-1. Immunofluorescence staining was utilized to assess the neutrophil infiltration and neural innervation. Flow cytometry assay was performed to test the number of total, M1 and M2 macrophages. RESULTS: In A. fumigatus infected C57BL/6 mice, NPD1 treatment ameliorated the severity of keratitis. Compared with the phosphate-buffered saline (PBS) control group, NPD1 treatment inhibited the infiltration of neutrophils and macrophages, increased the ratio of CD206+/CD86+ macrophages, reduced the phosphorylation level of p38 MAPK, and enhanced the expression of Nrf-2/HO-1 at 3d p.i. The number of corneal nerves and the mechanical sensitivity threshold of the NPD1 treatment group were significantly higher than the PBS group at 3 and 5d p.i. Meanwhile, the expression of IL-1β and MIP-2 in the NPD1 treatment group was significantly lower at 3 and 5d p.i. CONCLUSION: NPD1 improves the prognosis of A. fumigatus keratitis in mice by inhibiting the recruitment of neutrophils and macrophages, promoting M2 macrophage polarization and corneal nerve regeneration.

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2025-10-09 | Collagen Analogs Promote Tissue Regeneration in HSV-1-Infected Corneas in Animal Models

Herpes simplex virus type 1 (HSV-1) is a leading cause of infectious corneal blindness worldwide. Human donor corneal transplantation remains the primary treatment for scarred corneas resulting from herpes simplex keratitis (HSK), a severe inflammatory corneal disease caused by HSV-1 infection, despite a high risk of re-infection or immune rejection of the allografts. As possible alternatives to donor grafting for HSK, we developed cell-free, regeneration-stimulating corneal implants designed to work even under adverse inflammatory situations such as severe infections. The implants comprised short, fully synthetic collagen-like peptides conjugated to polyethylene glycol (CLP-PEG) and crosslinked using carbodiimide chemistry. Being cell-free, they lacked the cellular targets that an already activated immune system would encounter in these inflamed corneas. We tested the performance of these implants in guinea pig and rabbit models of HSK. Three different HSV-1 strains were used to create experimental HSK in rabbits and guinea pigs. There were no overall statistically significant species differences or species-strain differences in virus-induced mortality. At three months post-operation, all treated corneas showed tissue regeneration, but with haze or neovascularization. The initially cell-free CLP-PEG implants allowed for repopulation by ingrowing cells to regenerate neocorneal tissue, despite the inflammation. However, they did not prevent HSV-1 reactivation nor re-infection, as neovascularization and disorganization were observed within the neocorneas. A detailed histopathological examination revealed viral strain differences, but only KOS infection showed interspecies neovascularization differences. A more detailed examination with larger numbers of animals is merited to fully elucidate the effects of the different viral strains on rabbits versus guinea pigs.

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2025-09-26 | Scedosporium-induced keratitis: insights from a case study

Abstract Introduction Pseudallescheria boydii with its asexual form, Scedosporium apiospermum , is now recognized as an important emerging opportunistic pheoid-hypomycosis. Usually results in invasive infections in immunocompromised patients but rarely can infect the eyes. Herein, we report a challenging case of Pseudallescheria boydii keratitis in a lady with traumatic injury. Case Report A 38-year-old female presented with alleged history of penetrating injury to right eye with stick by her spouse for which she was started on antibiotic eye drops from nearby local hospital and became symptomatically better. Later she had alleged history of ashes thrown to her right eye 2 weeks ago. She was referred here from a nearby hospital, with complaints of pain, itching, watering of eye and blurring of vision. On examination, Limbal -limbal ulcer with peripherally thinned out cornea and mucoid discharge was present. Corneal scrapings revealed P boydii . She was managed with antibiotics such as moxifloxacin, tobramycin and antifungals such as Natamycin, fluconazole. She did not improve symptomatically. She was referred to a higher center for keratoplasty and the patient was thus discharged for the same. Conclusion Pseudallescheria boydii is a soil saprophytic, uncommon fungi entering body via respiratory or penetrating injury. It is often mistaken as Aspergillosis, This fungi shows resistance to amphotericin B, flucytosine and susceptible to triazoles. Surgical resection of lesion shows better outcome. The response of disseminated infection to combination therapy with interferon gamma and antifungal agents is encouraging.

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2025-09-19 | SA-XV, a 15-amino acid fragment of host defense peptide S100A12, targets mitochondria and is protective against fungal infections

Fungal infections are a huge emerging crisis, with more than 2 million people infected worldwide annually. Corneal infections caused by fungus are the major cause of vision loss and often warrant corneal transplantation. Both Fusarium spp. and Candida spp. are critical etiological agents of fungal keratitis and also common causes for invasive fungal infections with high mortality rates. In the previous work, we described growth inhibition of Fusarium spp. by S100A12, a host antimicrobial peptide. Here, to optimize a potential therapeutic, we have studied a 15-amino acid fragment of S100A12, SA-XV. Interestingly, SA-XV demonstrated remarkable antifungal activities, similar to the parent peptide, against both Fusarium spp. and Candida spp. SA-XV is a cell-penetrating peptide, and once internalized, it binds to fungal DNA, halts the cell cycle, and disrupts mitochondria, leading to the generation of reactive oxygen species and cell damage. The atomistic structure of the peptide determined by NMR reveals that SA-XV associates with the fungal membrane. The structural changes in SA-XV from α-helical to random coil conformation were observed in all-atom simulations. In addition, SA-XV aids in wound healing of corneal epithelial cells and attenuates the fungal burden in a murine model of fungal keratitis. Our results clearly demonstrate SA-XV as a promising antifungal candidate that targets both filamentous and nonfilamentous fungi for alternative therapeutic interventions.

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2025-05-02 | Herpes simplex virus type-1 infection and spread in a novel porcine corneal explant model is restricted to the epithelium

Herpes Keratitis (HK) is a debilitating infection of the cornea that remains the leading cause of infectious blindness in developed countries. Caused primarily by herpes simplex virus type 1 (HSV-1), it is associated with recurrent inflammation, leading to corneal scarring. This study investigated the initial events during acute HSV-1 infection in the cornea by adapting our human anogenital mucosal explant model to a HSV-1 infected porcine corneal explant model, We infected these corneas topically via high-density microarray patches (HD-MAPs) dipped in GFP-labelled HSV-1. Virus infection and spread was detected by both GFP protein and RNAscope, adapted for HSV-1 DNA. The punctures were consistent, usually in the epithelium but some extended into the underlying stroma. However, HSV-1 was restricted to the corneal epithelium, without spread through the anterior limiting membrane (ALM) or Bowman’s layer into the stroma nor to the uppermost epithelial layer. This layer expressed SPRR1A similarly to the stratum granulosum of skin which is refractory to HSV-1 infection. In corneas where infected epithelial cells extended to the ALM, SPRR1A was also observed in this layer, suggesting it may contribute to its barrier function. Such studies of HSV-1 infection and spread will help improve therapy for HK and vaccine design to prevent it.

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cell therapies

2025-03-01 | Successful Management of a Refractory Scedosporium (Pseudallescheria) boydii Keratitis With Sclerokeratoplasty.

Here, we present the successful sclerokeratoplasty surgery in resistant Scedosporium (Pseudallescheria) boydii keratitis. A 47-year-old male patient presented with complaints of 4-mm corneal infiltration and 1-mm hypopyon after an eye injury with organic material. Topical treatments with vancomycin (50 mg/mL), ceftazidime (50 mg/mL), voriconazole (10 mg/mL), and amphotericin B (0.5 mg/mL) were started hourly. Because of the significant progression despite the treatment, emergency therapeutic penetrating keratoplasty was performed. In this patient, in whom Scedosporium boydii growth was detected in mycological examination, although the infection appeared under control after keratoplasty, recurrence occurred on day 30. Hence, because of the rapid progression of the infection to the corneoscleral junction despite intensive treatment, emergency sclerocorneal transplant was performed. Globe integrity was preserved, and the patient had no recurrence in the 5-month follow-up. In severe S. boydii keratitis that causes infiltration up to the limbus border, removal of infected corneal tissue by wide excision and sclerocorneal transplant along with medical treatment may offer a successful treatment option. To the best of our knowledge, this is the first case in which sclerokeratoplasty was performed to successfully treat S. boydii keratitis.

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2025-02-12 | Outcomes of a Tenon Patch Graft for Corneal Perforations in Resolving Infective Keratitis.

To report the outcomes of a Tenon patch graft (TPG) in sealing corneal perforations in cases with resolving infective keratitis. This retrospective interventional study was conducted at a tertiary eye care center. All patients who underwent a TPG for corneal perforations because of clinically or/and microbiologically diagnosed infective keratitis between 2021 and 2023 were included. The surgical technique was consistent with that used in noninfective etiology. Out of 100 cases undergoing a TPG during the same period, 15 eyes of 14 patients had corneal perforations secondary to infective keratitis. The etiological profile included bacterial keratitis in 8, viral and fungal keratitis in 3, and Pythium keratitis in 1 eye of 15 eyes. One patient had bilateral infective keratitis after refractive surgery. The perforations were central in 7 and paracentral in 8 eyes. Successful restoration of tectonic integrity at 1 month was achieved in 14/15 eyes (93%). One patient had a repeat perforation at 3 weeks postoperatively, which was managed with a cyanoacrylate glue application. Infection control was achieved in all cases with adjunctive topical antimicrobial therapy. A TPG is a viable option for restoring globe integrity in corneal perforations encountered in resolving infective keratitis.

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2024-12-13 | Exploring interspecies differences in ex vivo models of Pseudomonas aeruginosa keratitis: a comparative study of human, pig and sheep corneas

Introduction. Interspecies differences in human, pig and sheep corneal thickness may affect the Pseudomonas aeruginosa colonization. Currently, there is no research investigating the impact of these differences, along with variable storage and culture conditions on infection in ex vivo cornea models. These factors could significantly influence utilizing ex vivo models for drug testing research.Aim. In this study, we aim to compare the relevance of sheep and pig cornea infection models to human.Methodology. The corneas were stored in McCarey-Kaufman medium or Eagle's Minimum Essential Medium or Dulbecco's Modified Eagle's Medium/Mixture F-12 Ham medium (incubator) and then infected after varying storage durations. The effect of added foetal bovine serum (FBS) to media and continuous shaking mimicking rinsing with tears on infection was also investigated. The infection outcome was evaluated by comparing c.f.u. between conditions.Results. The study found that storage conditions, culture media, FBS and continuous rinsing of corneas with media had no significant effect on infection progression in ex vivo keratitis models across selected species.Conclusions. Pig and sheep models yield results comparable to human corneas. These findings support the interchangeability of ex vivo human, pig and sheep keratitis models for P. aeruginosa infection studies, emphasizing their relevance and reliability in research contexts. This interchangeability is particularly useful for research groups where one particular animal model may not be available. The media in this ex vivo keratitis model can be free of animal components by the removal of FBS, which reduces the reliance on animal-derived products, aligning with ethical considerations and promoting more sustainable and humane scientific practices. This study advances the understanding of ex vivo keratitis models, demonstrating their robustness and potential for broader application in ophthalmic research and drug testing.

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2024-07-18 | Maintained transparency of preserved cornea after penetrating keratoplasty for fungal keratitis: A case report

This report presents a case of fungal keratitis treated with penetrating keratoplasty using a cryopreserved cornea, highlighting the successful maintenance of corneal transparency post infection resolution. A 57-year-old man complaining of pain in the right eye was referred to our hospital. Although diagnosed with fungal keratitis, his corneal scraping indicated the presence of Fusarium sp, and he was unresponsive to voriconazole, micafungin, and pimaricin treatments. His right visual acuity was hand motion. Therefore, we performed penetrating keratoplasty using a cryopreserved cornea. A cornea frozen and stored at -80 °C was used, with an 8.5-mm-diameter graft covering the infected area. Fungal keratitis subsided after corneal transplantation, and approximately 2 months later, corneal thickness gradually decreased, leading to improved corneal transparency. Therefore, a secondary optical corneal transplantation was deemed unnecessary. The final best-corrected visual acuity after additional cataract surgery was 20/40. Transparency was subsequently maintained for over a year. Cryopreserved corneas in penetrating keratoplasty may be viable for maintaining corneal transparency. Additionally, waiting several months for secondary optical corneal transplantation after therapeutic keratoplasty may be a good approach.

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2023-09-29 | OUTCOME OF THERAPEUTIC PENETRATING KERATOPLASTY IN A TERTIARY EYE HOSPITAL IN NEPAL

Background: Therapeutic penetrating keratoplasty is the most common variety of keratoplasty performed in the developing countries. The objective of this study was to assess the outcome of therapeutic penetrating keratoplasty in infective keratitis. Methods: This was a hospital based retrospective study where medical records of patients who underwent therapeutic penetrating keratoplasty between July 2021 and December 2022 were analyzed. Data collected included demographic parameters, indications for surgery, microbiology of the ulcers, donor details. Success was evaluated in terms of eradication of the disease, anatomical stability, graft clarity and visual acuity. SPSS software version 20.0 was used for statistical analysis. The mean, standard deviation, frequency and percentage were calculated Results: Sixty-seven patients of therapeutic penetrating keratoplasty who completed 2 months of follow-up after surgery were included in the study. Corneal scrapings and corneal buttons were positive for organisms in 19 cases (90.47%) and 20 cases (29.85%) respectively. In all,out of 67 eyes, 30 (44.77%) were positive for organisms in smear or culture or both, of which 27 cases showed pure fungus and 3 cases showed pure bacteria. Aspergillus (8, 29.62%) was the commonest fungus isolated. Eradication of infection after the primary procedure was seen in 48 cases (71.64%). Anatomical success was seen in 60 eyes (89.50%), clear grafts were seen in 23 eyes (34.32%). Visual acuity of ≥6/60 was seen in 14 eyes (20.89%). Conclusions: Therapeutic penetrating keratoplasty provided good success in terms of maintaining anatomical integrity and helps in eradication of infection while giving satisfactory functional outcome.

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other

2026-06-01 | Vaccination with an HSV-1 virus containing CD-137L reduces herpetic corneal disease when used both prophylactically and therapeutically

Corneal infections with herpes simplex type 1 (HSV-1) are a leading cause of blindness in the world. At present the standard of care for corneal disease (herpetic stromal keratitis, HSK) is anti-virals and topical steroids. With the development of anti-viral resistant strains of the virus, it is important to discover new therapies for this disease. We have focused on the development of vaccines that would select for an immune response that controls both virus and corneal disease. As present most vaccines that have been studied tend to be efficacious when used in a prophylactic regimen. However, these vaccines are not effective when treating latently infected individuals when their disease is reactivated resulting in recurrent HSK. We decided to test the concept that if we were to include the co-stimulatory molecule CD137L in our vaccine construct that this would lead to success when used in a therapeutic paradigm. We present data indicating that this vaccine construct is effective in ameliorating both primary and recurrent forms of HSK. At this time, we cannot attribute this observation to specific mechanisms that may be mediating its protective effect, but that is something we wish to address in the future.

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2026-01-28 | Corneal Neovascularization: Pathogenesis, Current Insights and Future Strategies.

The cornea is an avascular, immune-privileged tissue critical to maintaining transparency, optimal light refraction, and protection from microbial and immunogenic insults. Corneal neovascularization (CoNV) is a pathological sequela of multiple anterior segment diseases and presents a major cause for reduced visual acuity and overall quality of life. Various aetiologies, including infection (e.g., herpes simplex), inflammation (e.g., infective keratitis), hypoxia (e.g., contact lens overuse), degeneration (e.g., chemical burns), and trauma, disrupt the homeostatic avascular microenvironment, triggering an overactive compensatory response. This response is governed by a complex interplay of pro- and anti-angiogenic factors. This review investigates the potential for these mediators to serve as therapeutic targets. Current therapeutic strategies for CoNV encompass topical corticosteroids, anti-VEGF injections, fine-needle diathermy, and laser modalities including argon, photodynamic therapy and Nd:YAG. Emerging therapies involve steroid-sparing immunosuppressants (including cyclosporine and rapamycin), anti-fibrotic agents and advanced drug delivery systems, including ocular nanosystems and viral vectors, to enhance drug bioavailability. Adjunctive therapy to attenuate the protective corneal epithelium prior to target neovascular plexi are further explored. Gene-based approaches, such as Aganirsen (antisense oligonucleotides) and CRISPR/Cas9-mediated VEGF-A editing, have shown promise in preclinical studies for CoNV regression and remission. Given the multifactorial pathophysiology of CoNV, combination therapies targeting multiple molecular pathways may offer improved visual outcomes. Case studies of CoNV highlight the need for multifaceted approaches tailored to patient demographics and underlying ocular diseases. Future research and clinical trials are essential to elucidate optimal therapeutic strategies and explore combination therapies to ensure better management, improved treatment outcomes, and long-term remission of this visually disabling condition.

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2024-09-09 | A novel GFP-based strategy to quantitate cellular spatial associations in HSV-1 viral pathogenesis

ABSTRACT Periodic reactivation of herpes simplex virus type 1 (HSV-1) triggers immune responses that result in corneal scarring (CS), known as herpes stromal keratitis (HSK). Despite considerable research, fully understanding HSK and eliminating it remains challenging due to a lack of comprehensive analysis of HSV-1-infected immune cells in both corneas and trigeminal ganglia (TG). We engineered a recombinant HSV-1 expressing green fluorescent protein (GFP) in the virulent McKrae virus strain that does not require corneal scarification for efficient virus replication (GFP-McKrae). Next-generation sequencing (NGS) analysis, along with in vitro and in vivo assays, showed that GFP-McKrae virus was similar to WT-McKrae virus. Furthermore, corneal cells infected with GFP-McKrae were quantitatively analyzed using image mass cytometry (IMC). The single-cell reconstruction data generated cellular maps of corneas based on the expression of 25 immune cell markers in GFP-McKrae-infected mice. Corneas from mock control mice showed the presence of T cells and macrophages, whereas corneas from GFP-McKrae-infected mice on days 3 and 5 post-infection (PI) exhibited increased immune cells. Notably, on day 3 PI, increased GFP expression was observed in closely situated clusters of DCs, macrophages, and epithelial cells. By day 5 PI, macrophages and T cells became prominent. Finally, immunostaining methods detected HSV-1 or GFP and gD proteins in latently infected TG. This study presents a valuable strategy for identifying cellular spatial associations in viral pathogenesis and holds promise for future therapeutic applications. IMPORTANCE The goal of this study was to establish quantitative approaches to analyze immune cell markers in HSV-1-infected intact corneas and trigeminal ganglia from primary and latently infected mice. This allowed us to define spatial and temporal interactions between specific immune cells and their potential roles in virus replication and latency. To accomplish this important goal, we took advantage of the utility of GFP-McKrae virus as a valuable research tool while also highlighting its potential to uncover previously unrecognized cell types that play pivotal roles in HSV-1 replication and latency. Such insights will pave the way for developing targeted therapeutic approaches to tackle HSV-1 infections more effectively.

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2024-08-14 | CRISPR-Cas9-mediated genome editing delivered by a single AAV9 vector inhibits HSV-1 reactivation in a latent rabbit keratitis model

Herpes simples virus 1 (HSV-1) keratitis is a major cause of blindness globally. During primary infection, HSV-1 travels to the trigeminal ganglia and establishes lifelong latency. Although some treatments can reduce symptom severity and recurrence, there is no cure for HSV-1 keratitis. We used CRISPR-Cas9 to co-target gene sequences encoding two essential HSV-1 proteins, ICP0 and ICP27, as a potential therapy for HSV-1 keratitis. In HSV-1-infected Vero cells, the HSV-1 viral load and titer were significantly reduced by plasmid transfection or AAV2 vector transduction expressing Cas9 nuclease from Staphylococcus aureus (SaCas9) and paired guide RNAs (gRNAs). Off-target assessment showed minimal off-target editing activity from the selected gRNAs. We then tested our CRISPR-Cas9 gene editing approach in a latent rabbit model of HSV-1 keratitis. Corneal scarification with all-in-one AAV8(Y733F)-SaCas9 or AAV9-SaCas9 vector reduced viral shedding by over 50%. Interestingly, intravenous administration of the same AAV9-SaCas9 vector eliminated viral shedding in 92% of treated eyes. In addition, treated trigeminal ganglia showed a reduction in HSV-1 DNA and RNA expression. Our results support the utility of single-dose AAV9 all-in-one CRISPR-Cas9 gene editing as a safe and effective strategy for treating HSV-1 keratitis.Graphical abstract

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2012-01-13 | Improved Corneal Wound Healing through Modulation of Gap Junction Communication Using Connexin43-Specific Antisense Oligodeoxynucleotides

Purpose.: Gap junctions play a major role in corneal wound healing. This study used reproducible models of corneal wound healing to evaluate the effect of a gap junction channel modulator, connexin43 (Cx43) antisense oligodeoxynucleotides (AsODN), on corneal healing dynamics. Methods.: A mechanical scrape wound model was used to evaluate Cx43 AsODN penetration and initial wound reepithelialization 12 hours postsurgery. Thereafter, detailed analyses of corneal edema, inflammation, and healing were performed in an excimer laser surface ablation model. In vivo confocal microscopy determined clinical parameters (edema, haze) and cellular changes (stromal hypercellularity, reepithelialization), whereas histology and immunohistochemistry were used to quantify stromal edema, inflammation, and reepithelialization. Results.: Cx43 AsODN penetrated through the hydrophilic stroma where the epithelium had been removed and accumulated in the basal epithelium close to the wound edge. Twelve hours after scrape wounding, Cx43 AsODN–treated eyes showed a significant reduction in wound area compared with the vehicle alone (1.59 ± 0.37 and 2.29 ± 0.58 mm2, respectively, P < 0.01). After excimer laser ablation, stromal edema and inflammation were reduced, with endothelial structures being clearly visible, and reepithelialization rates were again increased in Cx43 AsODN–treated eyes. Histologic analysis confirmed reduced edema in the central wound site and at the periphery of treated corneas (P < 0.05), whereas immunohistochemistry showed lower Cx43 levels (P < 0.05), reduced myofibroblast activation, and improved epithelial basal lamina deposition in antisense-treated wounds (P < 0.01). Conclusions.: Application of Cx43 AsODN to the cornea reduces stromal edema and inflammation, promoting faster wound closure and a more uniform repair of the epithelial basal lamina after laser ablation.

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