Multiplex array proteomics detects increased MMP-8 in CSF after spinal cord injury

Light, Matthew; Minor, Kenneth H.; DeWitt, Peter; Jasper, Kyle H; Davies, Stephen J.

doi:10.1186/1742-2094-9-122

Cited by 26 publications

(17 citation statements)

References 33 publications

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“…As recent efforts at defining the CSF proteome have illustrated, there is a diverse array of proteins found in both healthy and diseased CSF [37, 62, 63]. WB remains an indispensable technique for the study of protein mass, modifications, and relative abundance in the CSF.…”

Section: Discussionmentioning

confidence: 99%

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Total protein is an effective loading control for cerebrospinal fluid western blots

Collins

Peller

et al. 2015

Journal of Neuroscience Methods

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Background Cerebrospinal fluid (CSF) has been used to identify biomarkers of neurological disease. CSF protein biomarkers identified by high-throughput methods, however, require further validation. While Western blotting (WB) is well-suited to this task, the lack of a validated loading control for CSF WB limits the method’s accuracy. New Method We investigated the use of total protein (TP) as a CSF WB loading control. Using iodine-based reversible membrane staining, we determined the linear range and consistency of the CSF TP signal. We then spiked green fluorescent protein (GFP) into CSF to create defined sample-to-sample differences in GFP levels that were measured by WB before and after TP loading correction. Levels of CSF complement C3 and cystatin C measured by WB with TP loading correction and ELISA in amyotrophic lateral sclerosis and healthy control CSF samples were then compared. Results CSF WB with the TP loading control accurately detected defined differences in GFP levels and corrected for simulated loading errors. Individual CSF sample Western blot and ELISA measurements of complement C3 and cystatin C were significantly correlated and the methods showed a comparable ability to detect between-groups differences. Comparison with Existing Method CSF TP staining has a greater linear dynamic range and sample-to-sample consistency than albumin, a commonly used CSF loading control. The method accurately corrects for simulated errors in loading and improves the sensitivity of CSF WB compared to using no loading control. Conclusions The TP staining loading control improves the sensitivity and accuracy of CSF WB results.

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Section: Discussionmentioning

confidence: 99%

“…Previous studies using PAGE/WB of CSF samples have used a variety of loading controls, including albumin [35], transthyretin [36], and transferrin [37]. Others have used no loading control or equal CSF volume loading [38, 39].…”

Section: Introductionmentioning

confidence: 99%

Total protein is an effective loading control for cerebrospinal fluid western blots

Collins

Peller

et al. 2015

Journal of Neuroscience Methods

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“…In septic conditions, MMP-8 was upregulated in microglia at inflammatory brain sites, and its inhibition reduced neuroinflammation. These results point toward microglia as cellular targets in the treatment of CNS disorders, such as MS, spinal cord injury, and traumatic brain injury, all of which involve upregulation of MMP-8 as a pathological feature (13,14,46). The in vivo effects of M8I and MMP-8 shRNA might not be confined to microglia.…”

Section: Discussionmentioning

confidence: 97%

“…In EAE, MMP-8 modulates Th1/Th2 polarization, causing inflammation and demyelination (13), and knockout of MMP-8 or administration of an MMP-8 inhibitor attenuates BBB breakdown and EAE progression. A recent study also demonstrated an increase in MMP-8 in cerebrospinal fluid (CSF) in rats with spinal cord injury, suggesting that MMP-8 is a biomarker for subacute spinal cord injury (14).…”

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confidence: 99%

Matrix Metalloproteinase-8 Plays a Pivotal Role in Neuroinflammation by Modulating TNF-α Activation

Lee

Han

Woo

et al. 2014

The Journal of Immunology

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Matrix metalloproteinases (MMPs) play important roles in normal brain development and synaptic plasticity, although aberrant expression of MMPs leads to brain damage, including blood–brain barrier disruption, inflammation, demyelination, and neuronal cell death. In this article, we report that MMP-8 is upregulated in LPS-stimulated BV2 microglial cells and primary cultured microglia, and treatment of MMP-8 inhibitor (M8I) or MMP-8 short hairpin RNA suppresses proinflammatory molecules, particularly TNF-α secretion. Subsequent experiments showed that MMP-8 exhibits TNF-α–converting enzyme (TACE) activity by cleaving the prodomain of TNF-α (A74/Q75, A76/V77 residues) and, furthermore, that M8I inhibits TACE activity more efficiently than TAPI-0, a general TACE inhibitor. Biochemical analysis of the underlying anti-inflammatory mechanisms of M8I revealed that it inhibits MAPK phosphorylation, NF-κB/AP-1 activity, and reactive oxygen species production. Further support for the proinflammatory role of microglial MMP-8 was obtained from an in vivo animal model of neuroinflammatory disorder. MMP-8 is upregulated in septic conditions, particularly in microglia. Administration of M8I or MMP-8 short hairpin RNA significantly inhibits microglial activation and expression/secretion of TNF-α in brain tissue, serum, and cerebrospinal fluid of LPS-induced septic mice. These results demonstrate that MMP-8 critically mediates microglial activation by modulating TNF-α activity, which may explain neuroinflammation in septic mouse brain.

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“…A second study employed multiplex analysis of inflammatory mediators in CSF and demonstrated significant increases in MMP-8 protein after traumatic injury to the spinal cord. The authors concluded that MMP-8 might represent a novel subacute-phase CSF biomarker for SCI[86].…”

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confidence: 99%

The Next Generation of Biomarker Research in Spinal Cord Injury

et al. 2016

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In traumatic spinal cord injury (SCI) patients, the assessment of the exact degree of lesion severity and neurological prognosis has proven to be extremely challenging. The current tools for predicting functional outcome in SCI patients such as clinical examination and magnetic resonance imaging are often inaccessible to unstable or polytraumatized patients, lack sensitivity, and are unreliable in the acute phase of the injury. Multiple candidate protein biomarkers known to be linked to the pathology have been studied for their potential to predict neurological outcome over time. This hypothesis-driven approach, however, has yielded only minimal success, and the reported individual markers lack sensitivity and correlation with specific outcome measures, which highlights the need for an unbiased high-throughput screening approach to identify novel candidate biomarkers. Antibodies were suggested to represent better biomarkers as their counterpart antigens, as they are highly specific and abundantly present in blood due to their inherent amplification and long half-life. Moreover, antibodies are easily accessible and are amenable to high-throughput screening. We therefore suggest an unbiased, high-throughput, and powerful antibody profiling procedure, named Serological antigen selection, based on complementary DNA (cDNA) phage display to combine the multiple benefits of stable and frequent antibodies and those of an unbiased method. The application of such an innovative and unbiased approach can complement the more traditional approaches to aid in the discovery of novel SCI-associated biomarkers.

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Multiplex array proteomics detects increased MMP-8 in CSF after spinal cord injury

Cited by 26 publications

References 33 publications

Total protein is an effective loading control for cerebrospinal fluid western blots

Total protein is an effective loading control for cerebrospinal fluid western blots

Matrix Metalloproteinase-8 Plays a Pivotal Role in Neuroinflammation by Modulating TNF-α Activation

The Next Generation of Biomarker Research in Spinal Cord Injury

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