Antimicrobial Effects of Adjunctive Thymosin beta-4 Therapy in Bacterial Keratitis

Purpose: Our lab has been focused on developing thymosin beta-4 (Tβ4 ) as a potential therapeutic for bacterial keratitis, an extremely debilitating ocular infection that is prevalent world wide. Tβ4 is a naturally occurring g-actin sequestering peptide, which we have previously shown significantly improves disease outcome in a P. aeruginosa (PA)-induced keratitis model when used adjunctively with ciprofloxacin (cipro). Following observations of decreased bacterial load in Tβ4 +cipro-treated corneas, this project aims to investigate the synergy between Tβ4 and cipro regarding bactericidal effects and antimicrobial peptide (AMP) regulation.Methods: Minimum inhibitory concentration (MIC) assays were used to quantify the bactericidal activity of ciprofloxacin, Tβ4, and a combination of cipro with Tβ4 to evaluate synergy. Micro serial dilutions were carried out using 96 well plates and concentrations of PA prepared for 105 and 106 CFU/mL inoculum. Additionally, we began to further elucidate the mechanism behind Tβ4 mediated effects by assessing the impact of Tβ4 on the activation of AMP pathways. Human corneal epithelial cells (HUCLs) were stimulated with LPS (25 μg/mL) for 6 and 24 hours. Experimental groups included: CTRL, Tβ4, cipro, and Tβ4 +cipro. Additionally, mRNA and protein levels of AMPs, including LL-37, S100A8, hBD-3, keratin6A, and TLR-4 were also assessed. Results: Though we expected to see a further decrease in MIC for adjunct Tβ4 therapy in comparison with cipro alone, the two groups exhibited similar results without a significant difference in MIC. Surprisingly however, the MIC assay showed very little bacterial growth with ciprofloxacin alone, even at significantly low concentrations. Unexpectedly, Tβ4 alone did not show significant bactericidal activity. Regarding AMP expression, RT-PCR results revealed upregulation of a number of AMPs in response to Tβ4 stimulation at 6 hrs, as well as further upregulation in combo groups at 24 hrs, indicating a possible synergistic effect. This trend has been similarly observed with select lipoxygenase enzymes and SPM receptors.Conclusion: We believe that the results from our experiments help provide evidence by which Tb4 influences the antimicrobial effects observed in the bacterial keratitis model. More importantly, suggestions of a synergistic effect between Tb4 and cipro at the gene level, as well as findings of an extremely low MIC for ciprofloxacin may allow for lower concentrations of antibiotic to be used in the clinical setting. This would not only decrease the risk for potential side effects/toxicity issues related to the use of antibiotics, but potentially reduce antimicrobial resistance

Thymosin-␤4 Inhibits Corneal Epithelial Cell Apoptosis after Ethanol Exposure In Vitro

PURPOSE. The purpose of this study was to determine the effect of thymosin beta 4 (T␤ 4 ) treatment on human corneal epithelial cells exposed to ethanol in vitro. The efficacy of T␤ 4 in preventing mitochondrial disruption and in inhibiting caspasemediated apoptosis was examined. METHODS. Nontransformed human corneal epithelial cells (HCECs) at passage 4 were untreated or treated with ethanol (20% for 20 seconds) or a combination of ethanol and T␤ 4 . The cells were allowed to recover from ethanol treatment for 24 hours. Mitochondrial membrane integrity and the release of cytochrome c to the cytoplasm were assessed using microscopy, Western blot, and ELISA. Bcl-2 expression and cell proliferation were measured using ELISA. Colorimetric activity assays were completed for caspase-2, -3, -8, and -9. RESULTS. T␤ 4 treatment decreased deleterious mitochondrial alterations, significantly decreased cytochrome c release from mitochondria, and increased Bcl-2 expression in ethanol-exposed human corneal epithelial cells. In ethanol-exposed corneal epithelium T␤ 4 treatment inhibited caspase-2, -3, -8, and -9 activity, with caspase-8 showing the most significant inhibition. T␤ 4 treatment resulted in no significant effect on the proliferation of human corneal epithelial cells after ethanol exposure. CONCLUSIONS. T␤ 4 plays an antiapoptotic role under conditions of epithelial cell challenge with an external stress such as exposure to ethanol. T␤ 4 may function as an antiapoptotic agent by inhibiting the release of cytochrome c from mitochondria and by suppressing the activation of caspases. (Invest Ophthalmol Vis Sci

Thymosin beta 4 and the eye: the journey from bench to bedside

<p><b>Introduction</b>: Thymosin beta 4 (Tβ4) has important applications in ocular repair and Phase 3 clinical trials using Tβ4 to treat dry eye and neurotrophic keratopathy are currently ongoing. These exciting clinical possibilities for Tβ4 in the eye are the result of seminal basic scientific discoveries and contributions from so many talented investigators.</p> <p><b>Areas covered</b>: My personal Tβ4 journey began at the NIH in 1998 and propelled my career as a clinician scientist. As a tribute to the amazing individuals who have guided and supported me along with my brilliant colleagues and students who have contributed and collaborated with me over the years, this review will tell the cumulative story of how Tβ4 became a major potential new therapy for corneal wound healing disorders. The journey has been marked by the thrilling exhilaration from fundamental breakthroughs in the laboratory and clinic, combined with the challenging and often harsh realities of submitting grants and obtaining funding.</p> <p><b>Expert opinion</b>: The electrifying possibility of Tβ4 as a revolutionary novel dry eye therapy is something that could have only been dreamed about just a few years ago. We believe that Tβ4 eyedrops will help many patients suffering from several ocular surface related disorders.</p

Angiogenic activity of human soluble intercellular adhesion molecule-1.

Serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) are elevated in a number of pathological conditions associated with angiogenesis, including tumor growth. Because the increased levels of sICAM-1 suggested that it may be angiogenic, we tested the ability of sICAM-1 to promote angiogenesis, Human recombinant sICAM-1 stimulates chemokinetic endothelial cell migration, endothelial cell tube formation on Matrigel,and sprouting of aortic rings, sICAM-1 also mediates angiogenesis in the chick chorioallantoic membrane assay. Additionally, we found a M-r 49,000 molecule that binds to sICAM-1 that may be the surface Ligand on endothelial cells, The evidence that sICAM-1 has angiogenic activity suggests a possible role linking inflammation and neovascularization, Furthermore, sICAM-1 may enhance tumor growth by promoting angiogenesis and escape from immunosurveillance.X11104sciescopu

Ocular dirofilariasis: Ophthalmic implication of climate change on vector-borne parasites

Purpose: To describe a geographically rare case of ophthalmic dirofilariasis. Observations: An 81-year-old male of good socioeconomic status living in the state of Michigan in the United States, presented to the eye clinic with a painful red left eye. He had not traveled outside of the state of Michigan in over three years. He was found to have a 7 cm long subconjunctival roundworm, which was ultimately extracted. Conclusions and importance: With increasing global temperatures, ocular dirofilariasis is being introduced in more northern climates and should be included in the differential diagnosis in areas previously isolated from these vector-borne parasites