Browsing by Subject "Interleukin-6"
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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 Quantitative Assessment of Synovitis in Legg-Calve-Perthes Disease Using Gadolinium Enhanced MRI(2016-04-01) Neal, David Charles; Kim, Harry K. W.; Barker, Blake; Green, GordonPURPOSE: Hip synovitis in LCPD is associated with pain, decreased motion, and poor outcome. However, a reliable quantitative method to assess hip synovitis, its progression, and response to therapy is not currently available. Gadolinium MRI (Gd-MRI) has been used to assess synovitis in other inflammatory conditions, but no study has been performed to quantify hip synovitis in LCPD. Purposes of the study are to develop a reliable quantitative method to measure hip synovitis using Gd-MRI in LCPD and to determine the temporal progression of synovitis. METHODS: 22 patients (22 hips) were enrolled based on the following criteria: first Gd-MRI obtained during active stage of LCPD (Waldenström Stage I to II) and serial MRI obtained using the same protocol (average number of MRIs 3±1, range 2-6). The areas of synovial enhancement in all slices of Gd-MRIs were measured using digital image analysis software. Individual areas were converted to volumes and the measurements were compared over time and Waldenström stage of disease. Intra-/ inter-observer reliabilities of measurements were assessed by 2 independent observers. Intraclass correlation coefficient (ICC) was analyzed. Total volume of synovitis was compared between the affected and unaffected sides using a t-test with statistical significance at p<0.05. RESULTS: Age at diagnosis ranged from 5 to 11 years (mean 7.4 ± 1.6). The first Gd-MRI was obtained at a mean duration of 1.0 ± 1.2 months after diagnosis (range 0 - 3 months). Synovial enhancement on initial Gd-MRI was significantly higher on the affected side (7031 ± 3109 mm3) vs. the unaffected side (367 ± 308 mm3, p<0.001). On final MRI, mean synovial enhancement was 4184 ± 3030 mm3 on the affected vs. 484 ± 415 mm3 (p<0.001) on the unaffected side (Figure 1). Mean synovial enhancement (synovitis) decreased with progression of Waldenström Staging from Stage I (avg. 7535 ± 3524 mm3) to Stage III (avg. 3800 ± 2413 mm3). The intraclass correlation coefficient was 0.98. The interobserver agreement was 0.94 CONCLUSION: Synovitis is highly prevalent in LCPD and most severe during the early stages of the disease. Overall, synovitis decreased within the first 20 months after diagnosis. This decrease correlated with the progression of the Waldenström stage. SIGNIFICANCE: A reliable quantitative method to assess synovitis in LCPD using Gd-MRI was developed. LCPD is associated with chronic synovitis which decreased with Waldenström stage.