The effect of pre-analytical handling on the stability of fractalkine, monocyte chemoattractant protein 1 (MCP1), interleukin 6 and interleukin 8 in samples of human cerebrospinal fluid.

Cytokine networks in cerebrospinal fluid (CSF) are important to our understanding of several neuroinflammatory diseases. Knowledge about optimal handling of samples is limited but important to minimize bias and reduce costs in CSF biomarker studies. The aim of this study was to examine the effect of storage temperature and time delay from CSF sample collection until freezing on the concentration of 11 different cytokines thought to be associated with chronic pain. CSF samples from 21 individuals undergoing hip or knee arthroplasty under spinal anesthesia were divided between two tubes. One tube was stored and centrifuged (within 30 min) at room temperature, and one tube was stored in ice water and centrifuged (within 30 min) at 4 °C. Each tube was split into six vials that were frozen at -80 °C, 0.5, 1, 2, 3, 4, or 5 h after collection. Cytokines were analyzed using a multiplex panel. A random effect panel data regression was conducted for each biomarker including the variables of storage temperature until freezing and time delay. Four cytokines had detectable levels: Fractalkine, monocyte chemoattractant protein 1(MCP-1), interleukine 6 (IL-6), and interleukine 8 (IL-8). There was no significant effect of storage temperature and time delay on MCP-1, IL-6, or IL-8 concentrations. Fractalkine concentration showed no clear trend. No concentration differences were observed between samples kept in ice water and those at room temperature except at the 3-h time point, and there was no overall significant effect of time delay on fractalkine concentration. We found no clear effect of storage temperature and time delay up to five hours from sample collection until freezing on the CSF concentrations of fractalkine, MCP-1, IL-6, or IL-8.

Cerebrospinal Fluid and Blood CX3CL1 as a Potential Biomarker in Early Diagnosis and Prognosis of Dementia.

BACKGROUND: A growing body of evidence highlights the crucial role of neuroinflammation and chemokine involvement in cognitive impairment pathophysiology. Fractalkine (CX3CL1) appears to be a relevant causative factor in the development of dementia, particularly at the early stages of the disease. However, limited data are available on the levels of CX3CL1 in the cerebrospinal fluid (CSF) and blood. Additionally, to date, its utility as a biomarker for MCI or AD has not been studied. OBJECTIVE: The aim of the present study was to evaluate the clinical utility of CX3CL1 in the early diagnosis of cognitive impairment. We also compared the diagnostic usefulness of CX3CL1 with other biomarkers associated with neuroinflammation. METHODS: A total of 60 patients with cognitive impairment, including 42 patients with AD and 18 subjects with MCI, as well as 20 cognitively healthy controls were enrolled in the study. CSF and blood concentrations of CX3CL1, CCL-2, and YKL-40 were measured by ELISA. RESULTS: Significantly higher CSF and blood concentrations of CX3CL1 were observed in MCI and AD patients compared to older individuals without cognitive impairment. The increase in the levels of CX3CL1 and YKL-40 in non-demented subjects was associated with MCI. The area under the ROC curve for CX3CL1 in MCI subjects was larger in comparison to classical AD markers. CONCLUSION: Presented results indicate a crucial role of CX3CL1 in the pathology of cognitive impairment and the potential usefulness of this protein in the early diagnosis of MCI and AD.

Glial activation and inflammation along the Alzheimer's disease continuum.

BACKGROUND: Neuronal and glial cell interaction is essential for synaptic homeostasis and may be affected in Alzheimer's disease (AD). We measured cerebrospinal fluid (CSF) neuronal and glia markers along the AD continuum, to reveal putative protective or harmful stage-dependent patterns of activation. METHODS: We included healthy controls (n = 36) and Aß-positive (Aß+) cases (as defined by pathological CSF amyloid beta 1-42 (Aß42)) with either subjective cognitive decline (SCD, n = 19), mild cognitive impairment (MCI, n = 39), or AD dementia (n = 27). The following CSF markers were measured: a microglial activation marker-soluble triggering receptor expressed on myeloid cells 2 (sTREM2), a marker of microglial inflammatory reaction-monocyte chemoattractant protein-1 (MCP-1), two astroglial activation markers-chitinase-3-like protein 1 (YKL-40) and clusterin, a neuron-microglia communication marker-fractalkine, and the CSF AD biomarkers (Aß42, phosphorylated tau (P-tau), total tau (T-tau)). Using ANOVA with planned comparisons, or Kruskal-Wallis tests with Dunn's pairwise comparisons, CSF levels were compared between clinical groups and between stages of biomarker severity using CSF biomarkers for classification based on amyloid pathology (A), tau pathology (T), and neurodegeneration (N) giving rise to the A/T/N score. RESULTS: Compared to healthy controls, sTREM2 was increased in SCD (p < .01), MCI (p < .05), and AD dementia cases (p < .001) and increased in AD dementia compared to MCI cases (p < .05). MCP-1 was increased in MCI (p < .05) and AD dementia compared to both healthy controls (p < .001) and SCD cases (p < .01). YKL-40 was increased in dementia compared to healthy controls (p < .01) and MCI (p < .05). All of the CSF activation markers were increased in subjects with pathological CSF T-tau (A+T-N+ and A+T+N+), compared to subjects without neurodegeneration (A-T-N- and A+T-N-). DISCUSSION: Microglial activation as indicated by increased sTREM2 is present already at the preclinical SCD stage; increased MCP-1 and astroglial activation markers (YKL-40 and clusterin) were noted only at the MCI and AD dementia stages, respectively, and in Aß+ cases (A+) with pathological T-tau (N+). Possible different effects of early and later glial activation need to be explored.

Comparison of bead array and glass nanoreactor multi-analyte platforms for the evaluation of CNS and peripheral inflammatory markers during HIV infection.

While human immunodeficiency virus (HIV) infection has become a treatable disease with the development of combination antiretroviral therapy (cART), chronic inflammation that affects the central nervous system and other organs is still common. Reliable methods are needed to study HIV-associated inflammatory biomarkers. In this study involving both plasma and cerebrospinal fluid (CSF), we compared multiplex bead array (MBA) to a relatively new technology based on microfluidics and glass nanoreactor (GNR) technology for the measurement of three commonly studied markers from HIV-infected individuals. We found that results correlated between the two platforms for MCP-1 in both fluids as well as for plasma TNFα (all p < .005). However, results between the two platforms did not correlate for CSF TNFα or fractalkine from plasma or CSF. A statistically significant decrease in CSF TNFα over time (p < .0001) was only detectable with the MBA platform, and TNFα on the MBA was the only CSF biomarker to correlate with CSF HIV RNA (rho = 0.71, p < .0001). Meanwhile, the GNR platform was superior in terms of intra-assay fractalkine (FKN) variability and the detection of a significant FKN decrease over time. Additionally, the only significant correlation between blood biomarkers and plasma HIV RNA was with FKN on the GNR platform (rho = 0.38, p = .015). Given the variability in results between platforms, more research is needed on methods to quantitate HIV-associated inflammation.

The role of fractalkine (CX3CL1) in regulation of CD4(+) cell migration to the central nervous system in patients with relapsing-remitting multiple sclerosis.

Fractalkine (CX3CL1) levels are increased in the cerebrospinal fluid (CSF) of patients with clinically isolated syndrome (CIS), as well as in the CSF and serum samples from patients with relapsing-remitting multiple sclerosis (RRMS). A higher percentage of circulating CD4(+) T-cells expressed its surface receptor (CX3CR1) and intracellular adhesion molecule (ICAM-1) in RRMS patients in comparison to healthy controls (HCs). The CX3CR1(+)ICAM-1(+)CD4(+) T-cells are enriched in the CSF of the RRMS patients. In vitro migration studies revealed that CD4(+) T-cells, which migrated toward a CX3CL1 gradient, expressed higher levels of ICAM-1 than non-migrating cells. CX3CL1 significantly increased IFN-γ and TNF-α gene expression and IFN-γ secretion by CD4(+) T-cells derived from the RRMS patients. CX3CL1 upregulated ICAM-1 expression on the surface of RRMS patient-derived but not HC-derived CD4(+) T-cells. Thus, CX3CL1 induces recruitment of CX3CR1(+)ICAM-1(+)CD4(+) T-cells into the central nervous system (CNS) during the early inflammatory response in MS.

Altered expression of CX3CL1 in patients with epilepsy and in a rat model.

Chemokine C-X3-C motif ligand 1 (CX3CL1, alias fractalkine), is highly expressed in the central nervous system and participates in inflammatory responses. Recent studies indicated that inflammatory processes within the brain constitute a common and crucial mechanism in the pathophysiological characteristics of epilepsy. This study investigated the expression pattern of CX3CL1 in epilepsy and its relationship with neuronal loss. Double immunolabeling, IHC, and immunoblotting results showed that CX3CL1 expression was up-regulated in the temporal neocortex of patients with temporal lobe epilepsy. In a rat model of epilepsy, CX3CL1 up-regulation began 6 hours after epilepsy, with relatively high expression for 60 days. In addition, ELISA revealed that the concentrations of CX3CL1 in cerebrospinal fluid and serum were higher in epileptic patients than in patients with neurosis but lower than in patients with inflammatory neurological diseases. Moreover, H&E staining demonstrated significant neuronal loss in the brains of epileptic patients and in the rat model. Finally, the expression of tumor necrosis factor-related apoptosis-inducing ligand was significantly increased in both patients and the animal model, suggesting that tumor necrosis factor-related apoptosis-inducing ligand may play a role in CX3CL1-induced cell death. Thus, our results indicate that CX3CL1 may serve as a possible biomarker of brain inflammation in epileptic patients.

CX(3)CR1 drives cytotoxic CD4(+)CD28(-) T cells into the brain of multiple sclerosis patients.

Immunosenescence, or ageing of the immune system, contributes to the increased morbidity and mortality seen in the elderly population. Premature immunosenescence is shown to occur in a subgroup of patients with autoimmune diseases. One of the main characteristics of immunosenescence is the expansion of CD4(+)CD28(-) T cells in the blood. In this study, we investigate the potential contribution of these cells to disease processes in a subgroup of multiple sclerosis (MS) and rheumatoid arthritis (RA) patients. Characterization of CD4(+)CD28(-) T cells in patients and healthy controls reveals that they have an inflammation-seeking effector-memory T cell phenotype with cytotoxic properties, as they expel cytotoxic granules in response to a polyclonal stimulus or MS-related autoantigens. We identify CX(3)CR1, the fractalkine receptor, as a selective marker to discriminate CD4(+)CD28(-) T cells from their CD4(+)CD28(+) counterparts. CX(3)CR1 expression enables CD4(+)CD28(-) T cells to migrate towards a fractalkine gradient in vitro. In addition, we find increased levels of fractalkine in the cerebrospinal fluid and inflammatory lesions of MS patients. We demonstrate for the first time that CD4(+)CD28(-) T cells accumulate in MS lesions of a subgroup of patients. Moreover, we have indications that these cells are cytotoxic in the target tissue. Overall, our findings suggest that CD4(+)CD28(-) T cells migrate in response to a chemotactic gradient of fractalkine to sites of inflammation, where they contribute to the inflammatory processes in a subgroup of patients with MS and RA.

Cerebrospinal fluid biomarkers for Parkinson disease diagnosis and progression.

OBJECTIVE: There is a clear need to develop biomarkers for Parkinson disease (PD) diagnosis, differential diagnosis of Parkinsonian disorders, and monitoring disease progression. We and others have demonstrated that a decrease in DJ-1 and/or α-synuclein in the cerebrospinal fluid (CSF) is a potential index for Parkinson disease diagnosis, but not for PD severity. METHODS: Using highly sensitive and quantitative Luminex assays, we measured total tau, phosphorylated tau, amyloid beta peptide 1-42 (Aß(1-42)), Flt3 ligand, and fractalkine levels in CSF in a large cohort of PD patients at different stages as well as healthy and diseased controls. The utility of these 5 markers was evaluated for disease diagnosis and severity/progression correlation alone, as well as in combination with DJ-1 and α-synuclein. The major results were further validated in an independent cohort of cross-sectional PD patients as well as in PD cases with CSF samples collected longitudinally. RESULTS: The results demonstrated that combinations of these biomarkers could differentiate PD patients not only from normal controls but also from patients with Alzheimer disease (AD) and multiple system atrophy. Particularly, with CSF Flt3 ligand, PD could be clearly differentiated from multiple system atrophy, a disease that overlaps with PD clinically, with excellent sensitivity (99%) and specificity (95%). In addition, we identified CSF fractalkine/Aß(1-42) that positively correlated with PD severity in cross-sectional samples as well as with PD progression in longitudinal samples. INTERPRETATION: We have demonstrated that this panel of 7 CSF proteins could aid in Parkinson disease diagnosis, differential diagnosis, and correlation with disease severity and progression.