Equine recurrent uveitis (ERU) is a spontaneous, painful, and vision threatening disease affecting up to 25% of equine populations worldwide. Current treatments of ERU are non-specific and have many side effects which limits them to short-term use. In order to develop an effective therapy for ERU, we investigated the use of adeno-associated virus (AAV) gene therapy, exploiting a natural immune tolerance mechanism induced by equine interleukin-10 (Equine-IL10). The purpose of this study was to evaluate the therapeutic efficacy of a single intravitreal (IVT) dose of AAV8-Equine-IL10 gene therapy for inhibition of experimental autoimmune uveitis (EAU) in rats. Each rat was dosed intravitreally (IVT) in both eyes with either balanced salt solution (BSS) (control; n = 4), AAV8-Equine-IL10 at a low dose (2.4x10⁹vg; n = 5) or high dose (2.4x10¹⁰vg; n = 5). EAU was induced in all groups of rats 7 days after IVT injections and euthanized 21 days post-injection. Ophthalmic examination and aqueous humor (AH) cell counts were recorded with the observer blinded to the treatment groups. Histopathology and qPCR were performed on selected ocular tissues. Data presented herein demonstrate that AAV8-Equine-IL10 treated rats exhibited a significant decrease in clinical inflammatory scores and AH cell counts compared to BSS-treated EAU eyes on days 10, 12 and 14 post EAU induction at both administered vector doses. Mean cellular histologic infiltrative scores were also significantly less in AAV8-Equine-IL10 dosed rats compared to the BSS group. Intravitreal injection of AAV8-Equine-IL10 resulted in Equine-IL10 cDNA expression in the ciliary body, retina, cornea, and optic nerve in a dose-dependent manner. A single IVT injection of AAV8-Equine-IL10 appeared to be well-tolerated and inhibited EAU even at the lowest administered dose. These results demonstrate safety and efficacy of AAV8-Equine-IL10 to prevent EAU and support continued exploration of AAV gene therapy for the treatment of equine and perhaps human recurrent uveitis.

Introduction. Aspergillus flavus and Fusarium keratoplasticum are common causative pathogens of fungal keratitis (FK), a severe corneal disease associated with significant morbidity and vision loss. Escalating incidence of antifungal resistance to available antifungal drugs poses a major challenge to FK treatment. Cold atmospheric plasma (CAP) is a pioneering nonpharmacologic antimicrobial intervention that has demonstrated potential as a broad-spectrum antifungal treatment. Gap statement. Previous research highlights biofilm-associated resistance as a critical barrier to effective FK treatment. Although CAP has shown promise against various fungal infections, its efficacy against biofilm and conidial forms of FK pathogens remains inadequately explored. Aim. This study aims to investigate the antifungal efficacy of CAP against clinical fungal keratitis isolates of A. flavus and F. keratoplasticum in vitro. Methodology. Power parameters (22–27 kV_pp, 300–400 Hz and 20–80 mA) of a dielectric barrier discharge CAP device were optimized for inactivation of A. flavus biofilms. Optimal applied voltage and total current were applied to F. keratoplasticum biofilms and conidial suspensions of A. flavus and F. keratoplasticum. The antifungal effect of CAP treatment was investigated by evaluating fungal viability through means of metabolic activity, c.f.u. enumeration (c.f.u. ml⁻¹) and biofilm formation. Results. For both fungal species, CAP exhibited strong time-dependent inactivation, achieving greater than 80 % reduction in metabolic activity and c.f.u. ml⁻¹ within 300 s or less, and complete inhibition after 600 s of treatment. Conclusion. Our findings indicate that CAP is a promising broad-spectrum antifungal intervention. CAP treatment effectively reduces fungal viability in both biofilm and conidial suspension cultures of A. flavus and F. keratoplasticum, suggesting its potential as an alternative treatment strategy for fungal keratitis.

Fungal keratitis (FK) is an invasive infection of the cornea primarily associated with Aspergillus and Fusarium species. FK is treated empirically with a limited selection of topical antifungals with varying levels of success. Though clinical infections are typically characterized by a dense network of mature mycelium, traditional models used to test antifungal susceptibility of FK isolates exclusively evaluate susceptibility in fungal cultures derived from asexual spores known as conidia. The purpose of this study was to characterize differences in fungal response when topical antifungal treatment is initiated at progressive phases of fungal development. We compared the efficacy of voriconazole and luliconazole against in vitro cultures of A. flavus and F. keratoplasticum at 0, 24, and 48 h of fungal development. A porcine cadaver corneal model was used to compare antifungal efficacy of voriconazole and luliconazole in ex vivo tissue cultures of A. flavus and F. keratoplasticum at 0, 24, and 48 h of fungal development. Our results demonstrate phase-dependent susceptibility of both A. flavus and F. keratoplasticum to both azoles in vitro as well as ex vivo. We conclude that traditional antifungal susceptibility testing with conidial suspensions does not correlate with fungal susceptibility in cultures of a more advanced developmental phase. A revised method of antifungal susceptibility testing that evaluates hyphal susceptibility may better predict fungal response in the clinical setting where treatment is often delayed until days after the initial insult.

The purpose of this study is to evaluate sublethal cold atmospheric plasma (CAP) treatment of filamentous fungal pathogen susceptibility to commonly used antifungal drugs in vitro. Response to CAP in combination with voriconazole, fluconazole, amphotericin B, and caspofungin was evaluated in Aspergillus flavus and Fusarium keratoplasticum conidia and mycelium; conidial response to fluconazole was also assessed in three strains of F. falciforme. Conidial susceptibility to antifungal drugs alone or in combination with CAP was assessed using a modified CLSI broth microdilution assay with MIC determination and colony-forming unit (CFU) enumeration. Mycelial viability and biofilm thickness changes in response to antifungal drugs alone or in combination with CAP were assessed over 24 hours post treatment. CAP enhanced antifungal drug efficacy against all fungal species, though effects differed by drug and growth form. CAP enhanced antifungal drug susceptibility in conidia, with the strongest effect observed for F. keratoplasticum conidia, where caspofungin MIC decreased fourfold (from 16 to 4−8 μg/mL) and sensitivity to fluconazole, which exerted no effect in absence of CAP, was restored when combined with sublethal CAP treatment. In A. flavus, CAP lowered the MIC of voriconazole (from 0.25 to 0.06–0.125 μg/mL) but increased the MIC of amphotericin B (from 4 to >4 μg/mL), despite reductions in viable cell counts. Differential responses to CAP and fluconazole were observed across three strains of F. falciforme, suggesting variability in CAP response. In treated biofilms, CAP alone initially reduced mycelial viability and biofilm thickness, but partial recovery of the fungus was observed over time in most cases. When combined with antifungal drugs, CAP significantly enhanced reduction of mycelial viability and thickness beyond antifungal treatment alone. In F. keratoplasticum biofilms, the combination of CAP and antifungal drugs produced sustained reductions in mycelial viability and biofilm thickness, whereas A. flavus biofilms were more resistant to CAP treatment and exhibited consistent recovery after 24 hours.

Fungal keratitis (FK) is an invasive infection of the cornea primarily associated with Aspergillus and Fusarium species. FK is treated empirically with a limited selection of topical antifungals with varying levels of success. Though clinical infections are typically characterized by a dense network of mature mycelium, traditional models used to test antifungal susceptibility of FK isolates exclusively evaluate susceptibility in fungal cultures derived from asexual spores known as conidia. The purpose of this study was to characterize differences in fungal response when topical antifungal treatment is initiated at progressive phases of fungal development. We compared efficacy of voriconazole and luliconazole against in vitro cultures of A. flavus and F. keratoplasticum at 0, 24, and 48 h of fungal development. A porcine cadaver corneal model was used to compare antifungal efficacy of voriconazole and luliconazole in ex vivo tissue cultures of A. flavus and F. keratoplasticum at 0, 24, and 48 h of fungal development. Our results demonstrate phase-dependent susceptibility of both A. flavus and F. keratoplasticum to both azoles in vitro as well as ex vivo. We conclude that traditional antifungal susceptibility testing with conidial suspensions does not correlate with fungal susceptibility in cultures of a more advanced developmental phase. A revised method of antifungal susceptibility testing that evaluates hyphal susceptibility may better predict fungal response in the clinical setting where treatment is often delayed until days after the initial insult.