Synthesis and characterization of the N-terminal acetylated 17-23 fragment of thymosin beta 4 identified in TB-500, a product suspected to possess doping potential

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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

Significance of Thymosin β4 and Implication of PINCH-1-ILK-α-Parvin (PIP) Complex in Human Dilated Cardiomyopathy

Myocardial remodeling is a major contributor in the development of heart failure (HF) after myocardial infarction (MI). Integrin-linked kinase (ILK), LIM-only adaptor PINCH-1, and α-parvin are essential components of focal adhesions (FAs), which are highly expressed in the heart. ILK binds tightly to PINCH-1 and α-parvin, which regulates FA assembly and promotes cell survival via the activation of the kinase Akt. Mice lacking ILK, PINCH or α-parvin have been shown to develop severe defects in the heart, suggesting that these proteins play a critical role in heart function. Utilizing failing human heart tissues (dilated cardiomyopathy, DCM), we found a 2.27-fold (p<0.001) enhanced expression of PINCH, 4 fold for α-parvin, and 10.5 fold (p<0.001) for ILK as compared to non-failing (NF) counterparts. No significant enhancements were found for the PINCH isoform PINCH-2 and parvin isoform β-parvin. Using a co-immunoprecipitation method, we also found that the PINCH-1-ILK-α-parvin (PIP) complex and Akt activation were significantly up-regulated. These observations were further corroborated with the mouse myocardial infarction (MI) and transaortic constriction (TAC) model. Thymosin beta4 (Tβ4), an effective cell penetrating peptide for treating MI, was found to further enhance the level of PIP components and Akt activation, while substantially suppressing NF-κB activation and collagen expression—the hallmarks of cardiac fibrosis. In the presence of an Akt inhibitor, wortmannin, we show that Tβ4 had a decreased effect in protecting the heart from MI. These data suggest that the PIP complex and activation of Akt play critical roles in HF development. Tβ4 treatment likely improves cardiac function by enhancing PIP mediated Akt activation and suppressing NF-κB activation and collagen-mediated fibrosis. These data provide significant insight into the role of the PIP-Akt pathway and its regulation by Tβ4 treatment in post-MI

β-thymosins and interstitial lung disease: study of a scleroderma cohort with a one-year follow-up

Background: beta-thymosins play roles in cytoskeleton rearrangement, angiogenesis, fibrosis and reparative process, thus suggesting a possible involvement in the pathogenesis of systemic sclerosis. The aim of the study was to investigate the presence of thymosins beta(4), beta(4) sulfoxide, and beta(10) in bronchoalveolar lavage fluid of scleroderma patients with interstitial lung disease and the relation of these factors with pulmonary functional and radiological parameters. Methods: beta-thymosins concentrations were determined by Reverse Phase-High Performance Liquid Chromatography-Electrospray-Mass Spectrometry in the bronchoalveolar lavage fluid of 46 scleroderma patients with lung involvement and of 15 controls. Results: Thymosin beta(4), beta(4) sulfoxide, and beta(10) were detectable in bronchoalveolar lavage fluid of patients and controls. Thymosin beta(4) levels were significantly higher in scleroderma patients than in controls. In addition, analyzing the progression of scleroderma lung disease at one-year follow-up, we have found that higher thymosin beta(4) levels seem to have a protective role against lung tissue damage. Thymosin beta(4) sulfoxide levels were higher in the smokers and in the scleroderma patients with alveolitis. Conclusions: We describe for the first time beta-thymosins in bronchoalveolar lavage fluid and their possible involvement in the pathogenesis of scleroderma lung disease. Thymosin beta(4) seems to have a protective role against lung tissue damage, while its oxidation product mirrors an alveolar inflammatory statu

Thymosin Beta 4 Prevents Oxidative Stress by Targeting Antioxidant and Anti-Apoptotic Genes in Cardiac Fibroblasts

Thymosin beta-4 (Tβ4) is a ubiquitous protein with diverse functions relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory responses. The effecter molecules targeted by Tβ4 for cardiac protection remains unknown. The purpose of this study is to determine the molecules targeted by Tβ4 that mediate cardio-protection under oxidative stress.Rat neonatal fibroblasts cells were exposed to hydrogen peroxide (H(2)O(2)) in presence and absence of Tβ4 and expression of antioxidant, apoptotic and pro-fibrotic genes was evaluated by quantitative real-time PCR and western blotting. Reactive oxygen species (ROS) levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant and antiapoptotic genes were silenced by siRNA transfections in cardiac fibroblasts and the effect of Tβ4 on H(2)O(2)-induced profibrotic events was evaluated.Pre-treatment with Tβ4 resulted in reduction of the intracellular ROS levels induced by H(2)O(2) in the cardiac fibroblasts. This was associated with an increased expression of antioxidant enzymes Cu/Zn superoxide dismutase (SOD) and catalase and reduction of Bax/Bcl(2) ratio. Tβ4 treatment reduced the expression of pro-fibrotic genes [connective tissue growth factor (CTGF), collagen type-1 (Col-I) and collagen type-3 (Col-III)] in the cardiac fibroblasts. Silencing of Cu/Zn-SOD and catalase gene triggered apoptotic cell death in the cardiac fibroblasts, which was prevented by treatment with Tβ4.This is the first report that exhibits the targeted molecules modulated by Tβ4 under oxidative stress utilizing the cardiac fibroblasts. Tβ4 treatment prevented the profibrotic gene expression in the in vitro settings. Our findings indicate that Tβ4 selectively targets and upregulates catalase, Cu/Zn-SOD and Bcl(2), thereby, preventing H(2)O(2)-induced profibrotic changes in the myocardium. Further studies are warranted to elucidate the signaling pathways involved in the cardio-protection afforded by Tβ4

Coenzyme Q10 Reduces Ethanol-Induced Apoptosis in Corneal Fibroblasts

Dilute ethanol (EtOH) is a widely used agent to remove the corneal epithelium during the modern refractive surgery. The application of EtOH may cause the underlying corneal fibroblasts to undergo apoptosis. This study was designed to investigate the protective effect and potential mechanism of the respiratory chain coenzyme Q10 (CoQ10), an electron transporter of the mitochondrial respiratory chain and a ubiquitous free radical scavenger, against EtOH-induced apoptosis of corneal fibroblasts. Corneal fibroblasts were pretreated with CoQ10 (10 µM) for 2 h, followed by exposure to different concentrations of EtOH (0.4, 2, 4, and 20%) for 20 s. After indicated incubation period (2–12 h), MTT assay was used to examine cell viability. Treated cells were further assessed by flow cytometry to identify apoptosis. Reactive oxygen species (ROS) and the change in mitochondrial membrane potential were assessed using dichlorodihydrofluorescein diacetate/2′,7′-dichlorofluorescein (DCFH-DA/DCF) assays and flow-cytometric analysis of JC-1 staining, respectively. The activity and expression of caspases 2, 3, 8, and 9 were evaluated with a colorimetric assay and western blot analysis. We found that EtOH treatment significantly decreased the viability of corneal fibroblasts characterized by a higher percentage of apoptotic cells. CoQ10 could antagonize the apoptosis inducing effect of EtOH. The inhibition of cell apoptosis by CoQ10 was significant at 8 and 12 h after EtOH exposure. In EtOH-exposed corneal fibroblasts, CoQ10 pretreatment significantly reduced mitochondrial depolarization and ROS production at 30, 60, 90, and 120 min and inhibited the activation and expression of caspases 2 and 3 at 2 h after EtOH exposure. In summary, pretreatment with CoQ10 can inhibit mitochondrial depolarization, caspase activation, and cell apoptosis. These findings support the proposition that CoQ10 plays an antiapoptotic role in corneal fibroblasts after ethanol exposure

Loss of endogenous thymosin β4 accelerates glomerular disease

Glomerular disease is characterized by morphologic changes in podocyte cells accompanied by inflammation and fibrosis. Thymosin $\beta_4$ regulates cell morphology, inflammation, and fibrosis in several organs and administration of exogenous thymosin $\beta_4$ improves animal models of unilateral ureteral obstruction and diabetic nephropathy. However, the role of endogenous thymosin $\beta_4$ in the kidney is unknown. We demonstrate that thymosin β4 is expressed prominently in podocytes of developing and adult mouse glomeruli. Global loss of thymosin $\beta_4$ did not affect healthy glomeruli, but accelerated the severity of immune-mediated nephrotoxic nephritis with worse renal function, periglomerular inflammation, and fibrosis. Lack of thymosin $\beta_4$ in nephrotoxic nephritis led to the redistribution of podocytes from the glomerular tuft toward the Bowman capsule suggesting a role for thymosin $\beta_4$ in the migration of these cells. Thymosin $\beta_4$ knockdown in cultured podocytes also increased migration in a wound-healing assay, accompanied by F-actin rearrangement and increased RhoA activity. We propose that endogenous thymosin $\beta_4$ is a modifier of glomerular injury, likely having a protective role acting as a brake to slow disease progression