Anne-Li Lind

The effects of age and gender on plasma levels of 63 cytokines

Cytokines play important roles as regulators of cell functions, and over the last decades a number of cytokine assays have been developed. The aim of the present study was to investigate the effects of age and gender on a large number of cytokines. Plasma samples were collected from 33 healthy blood donors. The samples were analyzed using a multiplex proximity extension assay (PEA) allowing simultaneous measurement of 92 cytokines and four technical controls. Biomarkers with less than 80% quantitative results were excluded leaving 63 cytokines that were analyzed for the effects of gender and age. The plasma level of three of the investigated biomarkers (DNER, MCP-4 and MMP-10) were found to be significantly different for the two genders (adjusted p-value<0.05), and 15 of the biomarkers (CCL11, CCL25, CDCP1, CSF-1, CXCL11, CXCL9, FGF-23, Flt3L, HGF, IL-10RB, MCP-3, MCP-4, MMP-10, OPG, VEGF-A) were significantly associated with age. This study reveals the effects of age and gender on a large number of cytokine assays. CXCL5 and TNFB were significantly higher in females, while the other markers with significant gender-dependent differences were higher in males. For the markers that were significantly associated with age, only CXCL6 was found to decrease with age, while the other biomarkers increased with age.

Evidence of both systemic inflammation and neuroinflammation in fibromyalgia patients, as assessed by a multiplex protein panel applied to the cerebrospinal fluid and to plasma

In addition to central hyperexcitability and impaired top–down modulation, chronic inflammation probably plays a role in the pathophysiology of fibromyalgia (FM). Indeed, on the basis of both animal experiments and human studies involving the analysis of cytokines and other inflammation-related proteins in different body fluids, neuroinflammatory mechanisms are considered to be central to the pathophysiology of many chronic pain conditions. However, concerning FM, previous human plasma/serum and/or cerebrospinal fluid (CSF) cytokine studies have looked only at a few predetermined cytokine candidates. Instead of analyzing only a few substances at a time, we used a new multiplex protein panel enabling simultaneous analysis of 92 inflammation-related proteins. Hence, we investigated the CSF and plasma inflammatory profiles of 40 FM patients compared with CSF from healthy controls (n=10) and plasma from blood donor controls (n=46). Using multivariate data analysis by projection, we found evidence of both neuroinflammation (as assessed in CSF) and chronic systemic inflammation (as assessed in plasma). Two groups of proteins (one for CSF and one for plasma) highly discriminating between patients and controls are presented. Notably, we found high levels of CSF chemokine CX3CL1 (also known as fractalkine). In addition, previous findings concerning IL-8 in FM were replicated, in both CSF and plasma. This is the first time that such an extensive inflammatory profile has been described for FM patients. Hence, FM seems to be characterized by objective biochemical alterations, and the lingering characterization of its mechanisms as essentially idiopathic or even psychogenic should be seen as definitively outdated.

A Multiplex Protein Panel Applied to Cerebrospinal Fluid Reveals Three New Biomarker Candidates in ALS but None in Neuropathic Pain Patients

The objective of this study was to develop and apply a novel multiplex panel of solid-phase proximity ligation assays (SP-PLA) requiring only 20 μL of samples, as a tool for discovering protein biomarkers for neurological disease and treatment thereof in cerebrospinal fluid (CSF). We applied the SP-PLA to samples from two sets of patients with poorly understood nervous system pathologies amyotrophic lateral sclerosis (ALS) and neuropathic pain, where patients were treated with spinal cord stimulation (SCS). Forty-seven inflammatory and neurotrophic proteins were measured in samples from 20 ALS patients and 15 neuropathic pain patients, and compared to normal concentrations in CSF from control individuals. Nineteen of the 47 proteins were detectable in more than 95% of the 72 controls. None of the 21 proteins detectable in CSF from neuropathic pain patients were significantly altered by SCS. The levels of the three proteins, follistatin, interleukin-1 alpha, and kallikrein-5 were all significantly reduced in the ALS group compared to age-matched controls. These results demonstrate the utility of purpose designed multiplex SP-PLA panels in CSF biomarker research for understanding neuropathological and neurotherapeutic mechanisms. The protein changes found in the CSF of ALS patients may be of diagnostic interest.

Inflammatory Serum Protein Profiling of Patients with Lumbar Radicular Pain One Year after Disc Herniation

Earlier studies suggest that lumbar radicular pain following disc herniation may be associated with a local or systemic inflammatory process. In the present study, we investigated the serum inflammatory protein profile of such patients. All 45 patients were recruited from Oslo University Hospital, Ullevål, Norway, during the period 2007–2009. The new multiplex proximity extension assay (PEA) technology was used to analyze the levels of 92 proteins. Interestingly, the present data showed that patients with radicular pain 12 months after disc herniation may be different from other patients with regard to many measurable serum cytokines. Given a false discovery rate (FDR) of 0.10 and 0.05, we identified 41 and 13 proteins, respectively, which were significantly upregulated in the patients with severe pain one year after disc herniation. On the top of the list ranked by estimated increase we found C-X-C motif chemokine 5 (CXCM5; 217% increase), epidermal growth factor (EGF; 142% increase), and monocyte chemotactic protein 4 (MCP-4; 70% increase). Moreover, a clear overall difference in the serum cytokine profile between the chronic and the recovered patients was demonstrated. Thus, the present results may be important for future protein serum profiling of lumbar radicular pain patients with regard to prognosis and choice of treatment. We conclude that serum proteins may be measurable molecular markers of persistent pain after disc herniation.

Spinal Cord Stimulation Alters Protein Levels in the Cerebrospinal Fluid of Neuropathic Pain Patients : A Proteomic Mass Spectrometric Analysis

Electrical neuromodulation by spinal cord stimulation (SCS) is a well‐established method for treatment of neuropathic pain. However, the mechanism behind the pain relieving effect in patients remains largely unknown. In this study, we target the human cerebrospinal fluid (CSF) proteome, a little investigated aspect of SCS mechanism of action.

The body mass index (BMI) is significantly correlated with levels of cytokines and chemokines in cerebrospinal fluid

Cytokines and chemokines regulate many functions in the body including the brain. The interactions between adipose tissue and the central nervous system (CNS) are important for the regulation of energy balance. CNS function is also influenced by age. The aim of the present study was to investigate the effects of body mass index (BMI) and age on cytokine and chemokine levels in cerebrospinal fluid. Cerebrospinal fluid samples (n=89) were collected from patients undergoing routine surgical procedures. The samples were analyzed using the multiplex proximity extension assay (PEA) in which 92 different cytokines are measured simultaneously using minute sample volume. We found no significant correlations between age and cytokine levels for any of the studied markers. In contrast, at a false discovery rate of 10%, 19 markers were significantly associated with BMI (in decreasing significance: FGF-5, ADA, Beta-NGF, CD40, IL-10RB, CCL19, TGF-alpha, SIRT2, TWEAK, SCF, CSF-1, 4E-BP1, DNER, LIF-R, STAMPB, CXCL10, CXCL6, VEGF-A and CX3CL1). This study reveals a clear effect of BMI on cytokine and chemokine levels in cerebrospinal fluid.

Targeting Oxidative Injury and Cytokines' Activity in the Treatment with Anti‐Tumor Necrosis Factor‐α Antibody for Complex Regional Pain Syndrome 1

Cytokines and oxygen free radicals have been implicated in the potential pathogenic development of complex regional pain syndrome (CRPS). We aimed to analyze the relationship between clinical status, circulating levels of cytokines, and markers of oxidative damage during the treatment with anti‐TNFα antibodies. The patient chosen for treatment had not had improvement through a number of conventional therapies and fulfilled the current diagnostic criteria for CRPS‐1. We investigated the clinical variables before and after systemic administration of 1.4 mg/kg anti‐TNFα antibody (infliximab), repeated after 1 month in a dose of 3 mg/kg. Blood samples were collected before and after anti‐TNFα antibodies administration, and plasma was analyzed for 8‐isoprostane‐prostaglandin F2α (8‐iso‐PGF2α, a marker of oxidative injury) and cytokines (TNF‐α, IL‐4, IL‐6, IL‐7, IL‐8, IL‐10, IL‐17A). Plasma concentrations of 8‐iso‐PGF2α were measured with radioimmunoassay (RIA), and the kinetics of cytokines were detected in plasma by antibody‐based proximity ligation (PLA). Pathologically high levels of 8‐iso‐PGF2α were found in the patient. Immediately after each administration of infliximab, the levels of 8‐iso‐PGF2α decreased. Although the patient showed an improvement of the cutaneous dystrophic symptoms and diminished pain associated with these lesions, the levels of circulating TNFα increased after the administration of anti‐TNFα antibodies. In a patient with CRPS‐1 treated with anti‐TNFα antibodies, we report increased levels of circulating TNFα and a temporary mitigation of oxidative stress as measured by plasma F2‐isoprostane. This case report provides evidence 2 supporting the indication of monitoring the oxidative stress biomarkers during treatment with anti‐TNFα antibodies in CRPS 1.

Quantification of 10 elements in human cerebrospinal fluid from chronic pain patients with and without spinal cord stimulation.

Neuropathic pain affects 1-10% of the general population and is caused by a lesion or disease of the somatosensory nervous system. Spinal cord stimulation (SCS), a method where implanted electrodes stimulate the spinal cord, has been successfully used to treat drug-resistant neuropathic pain, but the mechanism of action is largely unknown. Studies show that SCS changes the protein levels in CSF (cerebrospinal fluid) of pain patients. Several neurological conditions have been shown to alter the elemental composition of CSF. Therefore changes in the levels of ions and trace elements in the CSF may correspond to SCS use. This study used ICP-MS (Inductively coupled plasma mass spectrometry) and ICP-AES (Inductively coupled plasma atomic emission spectroscopy) to quantify 10 elements in CSF from chronic neuropathic pain patients using SCS. The element concentrations in CSF from patients with SCS treatment on/off, were measured. No effect on the element concentrations in CSF from treatment with SCS could be detected. Also, the elemental concentrations in pooled CSF from patients without chronic neuropathic pain was determined and compared to the patients using SCS. The concentration of the elements Ca, Sr, Na, K, P, Mg and Ti were, significantly higher in patients compared to the CSF-control.

Research on genes predisposing for chronic pain: a challenge for pain researchers in Scandinavia

Abstract Finding predisposing factors or vulnerability genes for chronic pain development would provide opportunities to tailor treatment for each patient. Such knowledge also pinpoints crucial functions required for the pathophysiological process. Both these outcomes are necessary for further improving chronic pain prevention and treatment. Pain can be modulated by a myriad of processes including endogenous opioid production, inflammation and tissue repair, which can trigger synaptic plasticity both centrally and peripherally, affecting both excitatory and inhibitory signaling by neurons, as well as glial signaling contributing to these processes. The genetic foundation for this web of interactions should provide future drug targets for chronic pain prevention and treatment. As the body of data grows, with increased patient cohort sizes, and more standardized characterizations of the pain state, we can hope to identify many new gene candidates for treatment of chronic pain. We are convinced that pain researchers in the Nordic countries have excellent possibilities for networking and cooperation, to carry out successful projects in the field of the genetics of pain. The “New Scandinavian Association for the Study of Pain” (newSASP) may provide an important facilitating arena to achieve this goal.

High levels of cerebrospinal fluid chemokines point to the presence of neuroinflammation in peripheral neuropathic pain: a cross-sectional study of 2 cohorts of patients compared with healthy controls

Abstract Animal models suggest that chemokines are important mediators in the pathophysiology of neuropathic pain. Indeed, these substances have been called “gliotransmitters,” a term that illustrates the close interplay between glial cells and neurons in the context of neuroinflammation and pain. However, evidence in humans is scarce. The aim of the study was to determine a comprehensive cerebrospinal fluid (CSF) inflammatory profile of patients with neuropathic pain. Our hypothesis was that we would thereby find indications of a postulated on-going process of central neuroinflammation. Samples of CSF were collected from 2 cohorts of patients with neuropathic pain (n = 11 and n = 16, respectively) and healthy control subjects (n = 11). The samples were analyzed with a multiplex proximity extension assay in which 92 inflammation-related proteins were measured simultaneously (Proseek Multiplex Inflammation I; Olink Bioscience, Uppsala, Sweden). Univariate testing with control of false discovery rate, as well as orthogonal partial least squares discriminant analysis, were used for statistical analyses. Levels of chemokines CXCL6, CXCL10, CCL8, CCL11, CCL23 in CSF, as well as protein LAPTGF-beta-1, were significantly higher in both neuropathic pain cohorts compared with healthy controls, pointing to neuroinflammation in patients. These 6 proteins were also major results in a recent similar study in patients with fibromyalgia. The findings need to be confirmed in larger cohorts, and the question of causality remains to be settled. Because it has been suggested that prevalent comorbidities to chronic pain (eg, depression, anxiety, poor sleep, and tiredness) also are associated with neuroinflammation, it will be important to determine whether neuroinflammation is a common mediator.