Thursday, April 10, 2025
4/10/2025
Thymosin Beta-4: A novel treatment for Acanthamoeba keratitis - PROJECT SUMMARY/ABSTRACT Acanthamoeba castellanii causes Acanthamoeba keratitis (AK), a serious eye infection that can lead to gradual vision loss, permanent blindness, and the need for keratoplasty. The parasite releases proteases that degrade host cell membranes. In response, host cells undergo apoptosis and secrete pro-inflammatory cytokines. A. castellanii has two life cycle stages, metabolically active amoebae and environmentally stable cysts. According to the CDC, both forms may be found in soil, fresh or sea water, sewage, swimming or medicinal therapy pools, dental treatment units, dialysis machines, heating/ventilation/air conditioning equipment and contact lens equipment and solutions. The majority of AK cases (~85%) occur in contact lens wearers. In immunocompromised hosts, A. castellanii can also cause opportunistic infections, such as granulomatous amoebic encephalitis (GAE), skin lesions, and nasopharyngeal, lung, and kidney infections. The importance of this pathogen is exemplified by an increasing number of reported cases, especially AK and GAE, both of which are challenging to diagnose and treat because of a lack of standardized treatment protocols and efficacious therapies. No single AK drug can eradicate both forms of the pathogen and also be non-toxic to the tissues of the eye. Thymosin β4 (Tβ4) is a small (4.9 kDa) actin-binding protein that is ubiquitously expressed in higher eukaryotes but may be absent in lower eukaryotes. In addition to actin-binding, the peptide is also secreted and can increase angiogenesis and cell proliferation, while inhibiting apoptosis and inflammation. Importantly, Tβ4 has been shown to promote corneal wound healing and reduce corneal inflammation. Topical Tβ4 has been used to treat an animal model of bacterial keratitis, was the subject of a completed US phase 3 clinical trial for the treatment of dry eye syndrome and is the subject of a current US phase 3 clinical trial for neurotrophic keratitis, a degenerative corneal disease. Given these advanced ophthalmic uses, we hypothesize that Tβ4 is a viable novel treatment for AK. We will test this hypothesis in two Aims. In the first aim, we will calculate IC50, minimum inhibitory constant (MIC) and onset of action in vitro using infected cultures of immortalized retinal (RPE-1) and corneal (HCE-S) cells. We will also determine the effect of Tβ4 on parasite virulence functions (e.g., stage conversion, adhesion, secretion). On the host side, we will measure apoptosis and cytokine release in the presence of parasite and peptide. In the second aim, we will employ two innovative ex vivo models of disease, a porcine corneal button model and a 3D human corneal tissue model. Tissues (3D human model or animal eye) will be infected with A. castellanii and subsequently treated with Tβ4. Histopathology and qPCR of Acanthamoeba 18S rRNA will be performed on both models to determine resolution of pathology and reduction of Acanthamoeba infection, respectively. At completion, we expect to have defined the efficacy of Tβ4 in the context of AK. The project will have beneficial impact because they will provide a strong mechanism-based proof-of-principle for the further development of topical Tβ4 as a novel treatment for AK.
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