Browsing by Subject "Serum"
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Item Burn Serum Stimulated Mitochondrial Fission Was Decreased with IL-6 Antibody Treatment(2017-01-17) Sehat, Alvand; Song, Juquan; Kulangara, Rohan; Maredia, Navin; Maxwell, Christian; Panwar, Kunal; Huebinger, Ryan M.; Carlson, Deborah L.; Zang, Qun S.; Wolf, Steven E.INTRODUCTION: Burn patients suffer muscle mass loss associated with hyperinflammation and hypercatabolism. We previously observed that burn serum resulted in cell death with elevated mitochondrial fragmentation in C2C12 myoblast. IL-6 as the key cytokine response to thermal injury has been showed to increase mitochondrial fragmentation. We thus posit that inhibition of IL-6 expression in burn serum will alleviate mitochondrial fragmentation. The aim of this study is to investigate the neutralization effect of IL-6 antibody in burn serum stimulated myoblasts. METHODS: Murine myoblasts C2C12 cells were cultured with recombinant IL-6 protein from 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, and 100 ng/ml. Cells were labeled with MitoTracker Green dye and live cell images were captured with Confocal microscopy. Next, C2C12 cells were exposed to medium containing 1) 10% serum from control rat, 2) 10% serum from burn rat, and 3) 10% serum from burn rat and 0.5 ug/ml of IL-6 antibody. All cells were labeled as the first experiment and live cell images were recorded. The caspase 3 activity was further examined from cell protein lysate. RESULTS: The 1 ng/ml dose of r-IL6 showed a fourfold increase in mitochondrial volume (μm3) at 24 hours post challenge. The intensity signal of Mitotracker was significantly increased in the 1 ng/ml IL-6 dose group at 48 hours. The 1 ng/ml dose of r-IL6 showed an increase in mitochondrial fragmentation. In cells cultured with 10% serum from control rats, mitochondrial morphology maintained the elongated linear shape during the 48 hours. In cells cultured with 10% serum from burned rats, a reduction in mitochondrial sizes was significant. In cells cultured with 10% serum and IL-6 antibody treatment this effect was reversed. Further, the intensity signals increased in response to the burn serum challenge. However, with addition of IL-6 antibody treatment the intensity signals decreased. In addition, burn serum increased the total volume of mitochondria about 1.4 fold, while with IL-6 antibody treatment the total volume was decreased. Consistently, a significant decrease in the expression of caspase 3 in the IL-6 antibody treatment group was observed. CONCLUSION: IL-6 stimulates an increase in mitochondria fragmentation in myoblasts, while IL-6 antibody treatment decreases mitochondrial fragmentation and cell death in burn serum stimulated myoblasts. This project has shown that targeting cytokine levels may be an effective treatment strategy in the management of burn patients.Item Burn Serum Stimulates Mitochondrial Fission in C2C12 Myoblasts(2021-04-22) Sehat, Alvand; Wolf, Steven; Carlson, Deborah; Huebinger, RyanBACKGROUND: Burn patients suffer muscle mass loss associated with a hypercatabolic status. Impairment of mitochondrial function has been observed in the muscle of burn patient's. OBJECTIVE: We hypothesize that muscle atrophy due to burn injur y is associated with an alteration in mitochondrial dynamics. This study was designed to investigate changes in mitochondrial fission and fusion in response to a burn serum challenge in myoblasts in vitro. METHODS: Cultured murine myoblasts, C2C12 cells, w ere exposed to 10% rat serum isolated either from 40% total body surface area (TBSA) scald burn rats or control rats. Cells were then labeled with MitoTracker Green dye and live cell images were recorded by confocal microscopy. The expression of mitochondr ial fission/fusion factors was examined by Western blots. RESULTS: In cells cultured with 10% serum from control rats, mitochondrial morphology maintained the elongated linear shape during the 48 hours we observed. However, 24 hours of culturing in 10% scaled rat serum resulted in a significant reduction in mitochondrial size. .Further, the number of total mitochondria increased, indicating a stimulation of mitochondrial fragmentation in response to the burn serum challenge. In addition, burn serum increases the total volume of mitochondria about 1.4 fold. Consistently, Western blot analysis showed a significant decrease in the expression of mitochondrial fusion protein Mfn1. CONCLUSION: Burn serum stimulates an increase in mitochondria fission in myoblasts.Item Mitochondrial Dynamics in Renal Cell with Burn Serum Stimulation: An Observational Study(2017-01-17) Maxwell, Christian T. W.; Huebinger, Ryan M.; Song, Juquan; Sehat, Alvand J.; Wolf, Steven E.BACKGROUND: Myoblasts and myocardiocytes have increased cell death with burn serum stimulation, which is associated with mitochondrial fission and function impairment. Acute kidney injury is a significant issue in burn patients; however, the role of the mitochondrial response in renal cells (RCs) has not been explored. The purpose of this summer project was to establish a method that observes mitochondrial dynamics in renal epithelial cells upon exposure to burn serum. HYPOTHESIS: The mitochondrial fission/fusion cycle in human RCs is disrupted following exposure to burn serum. METHODS: Human primary renal proximal tubule epithelial cells were cultured with Renal Epithelial Cell Growth Media in an incubator at 37°C with 5% CO2. Upon reaching 65-70% confluency, RCs were treated with growth media containing 10% rat serum (RS) either from control rats or 40% total body surface area scald burn rats. 3nM of MitoTracker Green FM dye was added to all cell treatments to stain the mitochondria (MT). Live cell images were taken under a Nikon Ti Eclipse Confocal microscope at 6, 24, 48, 72, and 96 hour time points; subsequently, images were analyzed with Nikon software to identify fluorescent intensity, mitochondrial volume and elongation. T-tests were then applied to analyze the significance with Bonferroni correction. Significance after correction was determined if p < .01 . OUTCOMES: RC morphology was cuboidal and refractile in the culture media. During serum stimulation experiments, the MT of RCs gradually increased in fluorescent intensity and maintained a rod-like shape in both groups. Specifically, the MT exposed to normal RS had significantly higher intensity at the 72 and 96 hour marks than those in burn RS. Despite these results, cell viability was determined to be 48 hours, since 60% or more of cells had detached at the 72 and 96 hour marks, indicating cell death. There were no significant differences in elongation and volume between burn and normal serum stimulation at each time point. CONCLUSIONS: The cell culture protocol for human primary renal epithelial cells was established this summer. Although rats and humans share evolutionarily conserved traits, the RS did not significantly influence the more evolved human RC.Item Mitochondrial Fission with Function Impairment in Burn Serum Treated C2C12 Cells(2016-01-19) Sehat, Alvand; Song, Juquan; Kumar, Puneet; Cai, Anthony; Huebinger, Ryan M.; Carlson, Deborah, L.; Zang, Qun S.; Wolf, Steven E.BACKGROUND: Burn patients suffer muscle mass loss associated with a hypercatabolic status. Mitochondria dynamics cycle is affected by metabolic status, and mitochondrial fission mediated high glucose induced cell death. Mitochondria function impairment associated with muscle mass loss has been observed in severe burn patients. We hypothesize that severe burn impaired muscle mass loss is associated with increased mitochondria fission with function impairment. The study was to investigate mitochondrial dynamics in response to burn serum stimulation. METHODS: Murine myoblast C2C12 cells were treated with DMEM media containing 10% rat serum isolated either from 40% TBSA scald burn rats, or control rats. Mitochondria was labeled with 3nM of MitoTracker Green FM dye, and live cell images were taken sequentially under a Nikon Ti Eclipse Confocal microscope. Cell lysates were collected for molecular biological analysis. Mitochondrial function was evaluated with Enzo Mito-ID membrane potential cytotoxicity kit. Target protein signals from cell lysate were detected by SDS-PAGE and western blot analysis. RESULTS: Mitochondrial morphology maintained the elongated linear shape in C2C12 cells when treated with 10% control rat serum. In contrast, when cells were treated with 10% burn serum, mitochondria reduced the elongated linear shape at 24 to 48 hours, and the florescent dye diffused at 72 hours. The cell florescent images showed an increase in circularity and fragmentation of mitochondria in C2C12 cells with burn serum stimulation. Meanwhile mitochondrial membrane potential decreased with 6hr post-burn serum stimulation. Western blot data showed that mitofusion-1 (Mfn1) significantly decreased in C2C12 cells with burn serum stimulation, confirming the observation of mitochondrial fission in response to burn serum. Cell death marker caspase 3 increased its expression in C2C12 cells with burn serum stimulation, suggesting a superfluous cell death in skeletal muscle after burn. CONCLUSION: Our results show an increase in the mitochondria fission/fusion ratio in C2C12 cells stimulated with burn serum isolated 6 hours after burn. The mechanism of mitochondrial fission with function impairment leading to muscle death is under investigation.Item The USPA2 Protein and Serum Resistance of Moraxella Catarrhalis(2006-05-15) Attia, Ahmed Sherif; Hansen, Eric J.Most isolates of Moraxella (Branhamella) catarrhalis are resistant to the bactericidal activity of normal human serum. Several M. catarrhalis gene products have been linked to the serum-resistant phenotype but none of them was shown to be directly involved in this phenotype. This study provides the first evidence for the direct involvement of the UspA2 protein of several serum-resistant M. catarrhalis strains in the serum-resistant phenotype. This was achieved by using transformation and allelic exchange to introduce hybrid uspA2 genes into M. catarrhalis, together with cloning and expression of different UspA2 proteins in Haemophilus influenzae. Using different types of human sera, it was concluded that serum-sensitive M. catarrhalis strains are killed via the classical complement pathway. Analysis of complement deposition on four different serum-resistant M. catarrhalis strains and their serum-sensitive uspA2 mutants showed similar amounts of early complement components binding to these cells, but a significant reduction occurred in the amount of polymerized C9 on the wild-type strains relative to that on the uspA2 mutants. The binding of the UspA2 proteins of these strains to the complement regulator vitronectin was shown to be responsible for the protection of these strains against complementmediated killing. This represents the first example of vitronectin-mediated serum resistance on a microbe. In contrast, binding of the complement regulator C4BP by the M. catarrhalis strains used in this study did not correlate with serum resistance. Finally, analysis of the untranslated region upstream of the uspA2 open reading frame showed that the presence of a heteropolymeric nucleotide repeat (AGAT) in this region is necessary for both normal expression of the UspA2 protein and serum resistance. Also, it was shown that changes in the number of AGAT repeats affected transcription of the uspA2 gene, with 15-18 AGAT repeats yielding maximal levels of transcription. These results indicate that these AGAT repeats play a regulatory role in the expression of the uspA2 gene.