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

INTRODUCTION: A variety of methods have been used to study inflammatory changes in the acutely injured spinal cord. Recently novel multiplex assays have been used in an attempt to overcome limitations in numbers of available targets studied in a single experiment. Other technical challenges in developing pre-clinical rodent models to investigate biomarkers in cerebrospinal fluid (CSF) include relatively small volumes of sample and low concentrations of target proteins. The primary objective of this study was to characterize the inflammatory profile present in CSF at a subacute time point in a clinically relevant rodent model of traumatic spinal cord injury (SCI). Our other aim was to test a microarray proteomics platform specifically for this application. METHODS: A 34 cytokine sandwich ELISA microarray was used to study inflammatory changes in CSF samples taken 12 days post-cervical SCI in adult rats. The difference between the median foreground signal and the median background signal was measured. Bonferroni and Benjamini-Hochburg multiple testing corrections were applied to limit the False Discovery Rate (FDR), and a linear mixed model was used to account for repeated measures in the array. RESULTS: We report a novel subacute SCI biomarker, elevated levels of matrix metalloproteinase-8 protein in CSF, and discuss application of statistical models designed for multiplex testing. CONCLUSIONS: Major advantages of this assay over conventional methods include high-throughput format, good sensitivity, and reduced sample consumption. This method can be useful for creating comprehensive inflammatory profiles, and biomarkers can be used in the clinic to assess injury severity and to objectively grade response to therapy.

Gelatinase B [matrix metalloproteinase (MMP)-9] and collagenases (MMP-8/-13) are upregulated in cerebrospinal fluid during aseptic and bacterial meningitis in children.

We investigated the protein expression of gelatinases [matrix metalloproteinase (MMP)-2 and -9] and collagenases (MMP-8 and -13) in cerebrospinal fluid (CSF) from patients with bacterial (BM, n = 17) and aseptic (AM, n = 14) meningitis. In both, MMP-8 and -9 were increased in 100% of patients, whereas MMP-13 was detectable in 53% and 82% respectively. Three patients with clinical signs of meningitis, without CSF pleocytosis, scored positive for all three MMPs. MMP-8 appeared in two isoforms, granulocyte-type [polymorphonuclear cell (PMN)] and fibroblast/macrophage (F/M) MMP-8. Analysis of kinetic changes from serial lumbar punctures showed that these MMPs are independently regulated, and correlate only partly with CSF cytosis or levels of the endogenous inhibitor, tissue inhibitor of matrix metalloproteinase-1. In vitro, T cells, peripheral blood mononuclear cells (PBMCs) and granulocytes (PMN) release MMP-8 and -9, whereas MMP-13 could be found only in the former two cell types. Using models of exogenous (n-formyl-Met-Leu-Phe, T cell receptor cross-linking) and host-derived stimuli (interleukin-2), the kinetics and the release of the MMP-8, -9 and -13 showed strong variation between these immune cells and suggest release from preformed stocks. In addition, MMP-9 is also synthesized de novo in PBMCs and T cells. In conclusion, invading immune cells contribute only partially to MMPs in CSF during meningitis, and parenchymal cells are an equally relevant source. In this context, in patients with clinical signs of meningitis, but without CSF pleocytosis, MMPs seem to be a highly sensitive marker for intrathecal inflammation. The present data support the concept that broad-spectrum enzyme inhibition targeting gelatinases and collagenases is a potential strategy for adjunctive therapy in infectious meningitis.

Expression of matrix metalloproteinases, sICAM-1 and IL-8 in CSF from children with meningitis.

The combined expression of the inflammatory mediators, matrix metalloproteinases (MMPs), soluble form of intracellular adhesion molecule ICAM-1 (sICAM-1) and interleukin (IL)-8, was evaluated in children infected with bacterial or viral meningitis. MMP-2 and IL-8 were detected in all CSF samples and were enhanced in both bacterial and viral infected samples, compared to those from control children. The expression of MMP-9 as well as sICAM-1 was not detected in control CSF while observed in viral infected and further elevated in bacterial infected samples. This pilot study supports a role for MMPs, IL-8 and sICAM in infectious meningitis and suggests further research to determine their possible use as biomarkers for various forms of meningeal infection as well as the use of their specific antagonists as potential therapeutic agents for central nervous system (CNS) inflammatory processes.

Matrix metalloproteinase (MMP)-8 and MMP-9 in cerebrospinal fluid during bacterial meningitis: association with blood-brain barrier damage and neurological sequelae.

To evaluate the spectrum and regulation of matrix metalloproteinases (MMPs) in bacterial meningitis (BM), concentrations of MMP-2, MMP-3, MMP-8, and MMP-9 and endogenous inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were measured in the cerebrospinal fluid (CSF) of 27 children with BM. MMP-8 and MMP-9 were detected in 91% and 97%, respectively, of CSF specimens from patients but were not detected in control patients. CSF levels of MMP-9 were higher (P<.05) in 5 patients who developed hearing impairment or secondary epilepsy than in those who recovered without neurological deficits. Levels of MMP-9 correlated with concentrations of TIMP-1 (P<.001) and tumor necrosis factor-alpha (P=.03). Repeated lumbar punctures showed that levels of MMP-8 and MMP-9 were regulated independently and did not correlate with the CSF cell count. Therefore, MMPs may derive not only from granulocytes infiltrating the CSF space but also from parenchymal cells of the meninges and brain. High concentrations of MMP-9 are a risk factor for the development of postmeningitidal neurological sequelae.