A New Method of Myostatin Inhibition in Mice via Oral Administration of Lactobacillus casei Expressing Modified Myostatin Protein, BLS-M22.

Myostatin is a member of the transforming growth factor-beta superfamily and is an endogenous negative regulator of muscle growth. This study aimed to determine whether an oral administration of Lactobacillus casei expressing modified human myostatin (BLS-M22) could elicit sufficient levels of myostatin-specific antibody and improve the dystrophic features of an animal model of Duchenne muscular dystrophy (DMD; mdx mouse). BLS-M22 is a recombinant L. casei engineered to harbor the pKV vector and poly-gamma-glutamic acid gene linked to a modified human myostatin gene. Serological analysis showed that anti-myostatin IgG titers were significantly increased, and serum creatine kinase was significantly reduced in the BLS-M22-treated mdx mice compared to the control mice. In addition, treatment of BLS-M22 resulted in a significant increase in body weight and motor function (Rotarod behavior test). Histological analysis showed an improvement in the dystrophic features (fibrosis and muscle hypertrophy) of the mdx mice with the administration of BLS-M22. The circulating antibodies generated after BLS-M22 oral administration successfully lowered serum myostatin concentration. Myostatin blockade resulted in serological, histological, and functional improvements in mdx mice. Overall, the findings suggest the potential of BLS-M22 to treat DMD; however, further clinical trials are essential to ascertain its efficacy and safety in humans.

Intravitreal tanibirumab, a fully human monoclonal antibody against vascular endothelial growth factor receptor 2, partially suppresses and regresses laser-induced choroidal neovascularization in a rat model.

PURPOSE: The study investigated the effect of intravitreally administered tanibirumab, a fully human monoclonal antibody against vascular endothelial growth factor receptor 2, in a rat model of laser-induced choroidal neovascularization (CNV). METHODS: CNV was induced by laser photocoagulation on day 0 in the eyes of Brown Norway rats. Intravitreal injection of tanibirumab or phosphate-buffered saline (PBS) was done on day 0 (prevention arm) or day 7 (treatment arm). Seven days after injection, the eyes were enucleated and retinal pigment epithelium-choroid-sclera flat mounts were prepared. Areas of CNV were determined in the flat mounts using tetramethylrhodamine isothiocyanate Bandeiraea simplicifolia (BS) isolectin labeling and intravenously administered fluorescein isothiocyanate-dextran and quantified using an image analysis program. RESULTS: In the prevention arm, the mean area of CNV measured by BS isolectin labeling was reduced by 28.2% and 53.9% in tanibirumab-treated eyes (20 and 60 µg, respectively) compared with PBS-treated control eyes on day 7 (P=0.038 and P<0.001, respectively). In the treatment arm, the mean area of CNV measured by BS isolectin labeling was reduced by 28.7% and 46.0% in tanibirumab-treated eyes (20 and 60 µg, respectively) compared with PBS-treated control eyes on day 14 (P=0.048 and P<0.001, respectively). CONCLUSIONS: Intravitreally administered tanibirumab partially suppressed the formation of new CNV and partially regressed preformed laser-induced CNV in the rat model. Tanibirumab may be a feasible treatment for CNV associated with age-related macular degeneration or other causes.

Stanniocalcin-1 protects retinal ganglion cells by inhibiting apoptosis and oxidative damage.

Optic neuropathy including glaucoma is one of the leading causes of irreversible vision loss, and there are currently no effective therapies. The hallmark of pathophysiology of optic neuropathy is oxidative stress and apoptotic death of retinal ganglion cells (RGCs), a population of neurons in the central nervous system with their soma in the inner retina and axons in the optic nerve. We here tested that an anti-apoptotic protein stanniocalcin-1 (STC-1) can prevent loss of RGCs in the rat retina with optic nerve transection (ONT) and in cultures of RGC-5 cells with CoCl2 injury. We found that intravitreal injection of STC-1 increased the number of RGCs in the retina at days 7 and 14 after ONT, and decreased apoptosis and oxidative damage. In cultures, treatment with STC-1 dose-dependently increased cell viability, and decreased apoptosis and levels of reactive oxygen species in RGC-5 cells that were exposed to CoCl2. The expression of HIF-1α that was up-regulated by injury was significantly suppressed in the retina and in RGC-5 cells by STC-1 treatment. The results suggested that intravitreal injection of STC-1 might be a useful therapy for optic nerve diseases in which RGCs undergo apoptosis through oxidative stress.

Reactive oxygen species by isoflurane mediates inhibition of nuclear factor κB activation in lipopolysaccharide-induced acute inflammation of the lung.

BACKGROUND: Although anesthetic-induced inhibition of lipopolysaccharide (LPS)-induced lung injury has been recognized, the underlying mechanism is obscure. Some studies suggest that reactive oxygen species (ROS) by isoflurane play a crucial role for anesthetic-induced protective effects on the brain or the heart; however, it still remains controversial. In this study, we examined the role of isoflurane-derived ROS in isoflurane-induced inhibition of lung injury and nuclear factor κB (NFκB) activation in LPS-challenged rat lungs. METHODS: Male Sprague-Dawley rats were subjected to inhalation of 1.0 minimum alveolar concentration of isoflurane for 60 minutes, and intratracheal LPS 0.1 mg was administered 60 minutes later. In some cases, ROS scavenger, 2-mercaptopropinyl glycine or N-acetylcysteine was given 30 minutes before isoflurane. ROS generation was measured by fluorometer before LPS challenge and 4 hours after. Isoflurane's preconditioning effect was assessed by histologic examination, protein content, neutrophil recruitment, and determination of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 levels in bronchoalveolar lavage fluid and lung tissue. Western blotting measured phosphorylation of inhibitory κB α (ser 32/36), NFκB p65, and inducible nitric oxide synthase (iNOS). TNF-α and IL-6 mRNA expression and immunofluorescence staining for iNOS were also assessed. RESULTS: Isoflurane preconditioning reduced inflammatory lung injury and TNF-α, IL-1ß, and IL-6 release in the lung. Isoflurane upregulated ROS generation before LPS but inhibited a ROS burst after LPS challenge. ROS scavenger administration before isoflurane abolished the isoflurane preconditioning effect as well as isoflurane-induced inhibition of phosphorylation of inhibitory κBα, NFκB p65, iNOS activation, and mRNA expression of TNF-α and IL-6 in acute LPS-challenged lungs. CONCLUSIONS: This study suggests a crucial role of upregulated ROS generation by isoflurane for modification of inflammatory pathways by isoflurane preconditioning in acute inflammation of the lung.

Discovery of gliotoxin as a new small molecule targeting thioredoxin redox system.

Thioredoxin redox system has been implicated as an intracellular anti-oxidant defense system leading to reduction of cellular oxidative stresses utilizing electrons from NADPH. From high content screening of small molecules targeting the system, gliotoxin, a fungal metabolite, was identified as an active compound. Gliotoxin potently accelerates NADPH oxidation and reduces H(2)O(2). The compound reduces H(2)O(2) to H(2)O by replacing the function of peroxiredoxin in vitro and decreases intracellular level of H(2)O(2) in HeLa cells. The anti-oxidant activity of gliotoxin was further validated H(2)O(2)-mediated cellular phenotype of angiogenesis. The proliferation of endothelial cells was inhibited by the compound at nanomolar range. In addition, H(2)O(2)-induced tube formation and invasion of the cells were blocked by gliotoxin. Together, these results demonstrate that gliotoxin is a new small molecule targeting thioredoxin redox system.

Activity assay of mammalian 2-cys peroxiredoxins using yeast thioredoxin reductase system.

2-Cys peroxiredoxin (Prx) is a novel cellular peroxidase that reduces peroxides in the presence of thioredoxin, thioredoxin reductase, and nicotinamide adenine dinucleotide phosphate (NADPH) and that functions in H(2)O(2)-mediated signal transduction. Recent studies have shown that 2-cys Prx can be inactivated by cysteine overoxidation in conditions of oxidative stress. Therefore, peroxidase activity, rather than the protein level, of 2-cys Prx is the more important measure to predict its cellular function. Here, we introduce a modified activity assay method for mammalian 2-cys Prx based on yeast nonselenium thioredoxin reductase. Yeast thioredoxin reductase is expressed in Escherichia coli cells and purified at high yield (40 mg/L of culture broth) as an active flavoprotein by combined diethyl aminoethyl (DEAE) and phenyl hydrophobic chromatography. The optimal concentrations of yeast thioredoxin and thioredoxin reductase required to achieve maximum mammalian 2-cys Prx activity are 3.0 and 1.5 microM, respectively. This modified assay method is useful for measuring 2-cys Prx activity in cell lysates and can also be adapted for a 96-well plate reader for high-throughput screening of chemical compounds that target 2-cys Prx.

9-polylysine protein transduction domain: enhanced penetration efficiency of superoxide dismutase into mammalian cells and skin.

Antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), have been considered to have a beneficial effect against various diseases that are mediated by the reactive oxygen species (ROS). Although a variety of modified recombinant antioxidant enzymes have been generated to protect against oxidative stresses, the lack of their transduction ability into cells resulted in a limited ability to detoxify intracellular ROS. To render the SOD enzyme capable of detoxifying intracellular ROS when added extracellularly, cell-permeable recombinant SOD proteins were generated. A human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene was fused with a gene fragment that encodes the 9 amino acids Tat protein transduction domain (RKKRRQRRR) of HIV-1 and lysine rich peptide (KKKKKKKKK) in a bacterial expression vector in order to produce a genetic in-frame Tat-SOD and 9Lys-SOD fusion protein, respectively. The expressed and purified Tat-SOD and 9Lys-SOD fusion proteins can transduce into human fibroblast cells, and they were enzymatically active and stable for 24 h. The cell viability of the fibroblast cells that were treated with paraquat, an intracellular superoxide anion generator, was increased by the transduced Tat-SOD or 9Lys-SOD. The transduction efficacy of 9Lys-SOD was more efficient than that of Tat-SOD. We evaluated the ability of the SOD fusion pmteins to transduce into animal skin. This analysis showed that Tat-SOD and 9Lys-SOD fusion proteins efficiently penetrated into the epidermis as well as the dermis of the subcutaneous layer, when sprayed on mice skin (judged by the immunohistochemistry and specific enzyme activities). The enzymatic activity of the transduced 9Lys-SOD was higher than that of Tat-SOD, indicating that the penetration of 9Lys-SOD was more efficient when put into the skin. These results suggest Tat-SOD and 9Lys-SOD fusion proteins can be used as anti-aging cosmetics, or in protein therapy, for various disorders that are related to this antioxidant enzyme and ROS.