Neurofilament light chain and total tau in the differential diagnosis and prognostic evaluation of acute and chronic inflammatory polyneuropathies.

BACKGROUND AND PURPOSE: To investigate the diagnostic and prognostic value of axonal injury biomarkers in patients with inflammatory polyneuropathies. METHODS: Neurofilament light chain (NfL) and total tau (T-tau) were measured in the cerebrospinal fluid (CSF) and plasma in 41 patients with Guillain-Barré syndrome (GBS), 32 patients with chronic inflammatory demyelinating polyneuropathy (CIDP), 10 with paraproteinemia-related demyelinating polyneuropathy (PDN), and 8 with multifocal motor neuropathy (MMN), in comparison with 39 disease-free controls and 59 other controls. Outcome was measured with the GBS-disability score (GBS-ds) or Inflammatory Neuropathy Cause and Treatment (INCAT) disability score. RESULTS: Neurofilament light chain levels in CSF and plasma were higher in GBS, CIDP, and PDN vs. disease-free controls. Patients with MMN had higher NfL levels in plasma vs. disease-free controls, but lower levels in CSF and plasma vs. patients with amyotrophic lateral sclerosis (ALS). T-tau levels in plasma were higher in GBS, CIDP, PDN, and MMN vs. all control groups. Neurofilament light chain levels in CSF and plasma in patients with GBS correlated with GBS-ds, as higher levels were associated with inability to run after 6 and 12 months. NfL levels in CSF and plasma in CIDP did not correlate significantly with outcome. CONCLUSIONS: Acute and chronic inflammatory neuropathies are associated with an increase in levels of NfL in CSF and plasma, but NfL is validated as a prognostic biomarker only in GBS. NfL could be used in differentiating patients with MMN from ALS. T-tau in plasma is a novel biomarker that could be used in a diagnostic assessment of patients with acute and chronic inflammatory polyneuropathies.

Characterization of neuroinflammation and periphery-to-CNS inflammatory cross-talk in patients with disc herniation and degenerative disc disease.

The aim of the study was to identify inflammatory cytokines/chemokines associated with neuroinflammation and periphery-to-CNS inflammatory cross-talk in degenerative disc disease (DDD) and lumbar disc herniation (LDH), common causes of low back pain (LBP). A secondary aim was to investigate the associations between cytokines and symptom severity. METHODS: In total, 40 DDD and 40 LDH patients were recruited from a surgical waiting list, as well as 39 healthy controls (HC) and 40 cerebrospinal fluid (CSF) controls. The subjects completed questionnaires and pressure algometry was performed at the lumbar spine and forearm. The CSF, serum and disc tissues were collected during surgery. Inflammatory mediators TNF, INFg, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13 and MCP1 were analysed by immunoassay (Meso Scale Discovery) and quantitative real-time polymerase chain reaction (qPCR) was used for analysis of IL-6, IL-8, MCP1 and TSPO expression in intervertebral discs (IVDs). RESULTS: In the LDH group, we found elevated IL-8 concentrations in CSF indicating neuroinflammation, while IL-8 and MCP1 concentrations in serum were lower compared to HC. The IVD expression of IL-6, IL-8 and TSPO was lower in LDH patients compared to DDD. LDH patients had a positive correlation between IL-8 concentrations in CSF and serum and IL-8 in CSF was associated with higher pain intensity and increased spinal pressure pain sensitivity. The MCP1 concentration in serum was associated with higher global pain ratings and increased spinal pressure pain sensitivity, while IL-6 serum concentration correlated with the intensity of the neuropathic pain component (leg pain) in LDH patients. IVD expression of TSPO in LDH patients was associated with increased intensity of back pain. No differences were found in cytokine CSF concentrations between DDD patients and CSF controls, but DDD patients had lower IL-8 and MCP1 serum concentrations than HC. In female DDD patients, IL-8 and MCP1 concentrations in serum were associated with increased intensity of back pain. CONCLUSION: Our results suggest that neuroinflammation mediated by elevated IL-8 concentrations in CSF and IL-8 mediated periphery-to-CNS inflammatory cross-talk contributes to pain in LDH patients and suggest a link between TSPO expression in discs and low back pain.

Autoantibodies to citrullinated proteins induce joint pain independent of inflammation via a chemokine-dependent mechanism.

OBJECTIVE: An interesting and so far unexplained feature of chronic pain in autoimmune disease is the frequent disconnect between pain and inflammation. This is illustrated well in rheumatoid arthritis (RA) where pain in joints (arthralgia) may precede joint inflammation and persist even after successful anti-inflammatory treatment. In the present study, we have addressed the possibility that autoantibodies against citrullinated proteins (ACPA), present in RA, may be directly responsible for the induction of pain, independent of inflammation. METHODS: Antibodies purified from human patients with RA, healthy donors and murinised monoclonal ACPA were injected into mice. Pain-like behaviour was monitored for up to 28 days, and tissues were analysed for signs of pathology. Mouse osteoclasts were cultured and stimulated with antibodies, and supernatants analysed for release of factors. Mice were treated with CXCR1/2 (interleukin (IL) 8 receptor) antagonist reparixin. RESULTS: Mice injected with either human or murinised ACPA developed long-lasting pronounced pain-like behaviour in the absence of inflammation, while non-ACPA IgG from patients with RA or control monoclonal IgG were without pronociceptive effect. This effect was coupled to ACPA-mediated activation of osteoclasts and release of the nociceptive chemokine CXCL1 (analogue to human IL-8). ACPA-induced pain-like behaviour was reversed with reparixin. CONCLUSIONS: The data suggest that CXCL1/IL-8, released from osteoclasts in an autoantibody-dependent manner, produces pain by activating sensory neurons. The identification of this new pain pathway may open new avenues for pain treatment in RA and also in other painful diseases associated with autoantibody production and/or osteoclast activation.

Increased levels of IL-6 in the cerebrospinal fluid of patients with chronic schizophrenia--significance for activation of the kynurenine pathway.

BACKGROUND: Accumulating evidence indicates that schizophrenia is associated with brain immune activation. While a number of reports suggest increased cytokine levels in patients with schizophrenia, many of these studies have been limited by their focus on peripheral cytokines or confounded by various antipsychotic treatments. Here, well-characterized patients with schizophrenia, all receiving olanzapine treatment, and healthy volunteers were analyzed with regard to cerebrospinal fluid (CSF) levels of cytokines. We correlated the CSF cytokine levels to previously analyzed metabolites of the kynurenine (KYN) pathway. METHODS: We analyzed the CSF from patients and controls using electrochemiluminescence detection with regard to cytokines. Cell culture media from human cortical astrocytes were analyzed for KYN and kynurenic acid (KYNA) using high-pressure liquid chromatography or liquid chromatography/mass spectrometry. RESULTS: We included 23 patients and 37 controls in our study. Patients with schizophrenia had increased CSF levels of interleukin (IL)-6 compared with healthy volunteers. In patients, we also observed a positive correlation between IL-6 and the tryptophan:KYNA ratio, indicating that IL-6 activates the KYN pathway. In line with this, application of IL-6 to cultured human astrocytes increased cell medium concentration of KYNA. LIMITATIONS: The CSF samples had been frozen and thawed twice before analysis of cytokines. Median age differed between patients and controls. When appropriate, all present analyses were adjusted for age. CONCLUSION: We have shown that IL-6, KYN and KYNA are elevated in patients with chronic schizophrenia, strengthening the idea of brain immune activation in patients with this disease. Our concurrent cell culture and clinical findings suggest that IL-6 induces the KYN pathway, leading to increased production of the N-methyl-D-aspartate receptor antagonist KYNA in patients with schizophrenia.

Influence of model and matrix on cytokine profile in rat and human.

OBJECTIVE: Emerging evidence indicates that low-grade inflammation is part of the clinical picture of OA and that there is a need to identify soluble biomarkers of ongoing inflammation in the joint from a translational aspect. The aim of this study was to compare levels of pro-inflammatory biomarkers in SF, serum and/or EDTA plasma. METHODS: SF and blood from rats subjected to Freund's complete adjuvant (FCA; n = 48) or monoiodoacetate (MIA; n = 88) monoarthritis and from control rats were collected over time. SF, EDTA plasma and serum were obtained from six individuals with OA of the knee and healthy controls. Levels of IL-6, KC/GRO, IL-8, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 3α (MIP-3α), IL-1ß, TNF and l(+)-lactate were assessed either by immune assay or by a colorimetric method. RESULTS: Elevated levels of biomarkers were shown in monoarthritic animals in SF compared with the control groups, although with considerably lower magnitude in the MIA groups, which also indicated a biphasic pattern. Levels of KC/GRO and MIP-3α in serum from the FCA model and IL-6 in the MIA model followed the pattern of SF. In serum samples from OA individuals, MIP-3α correlated significantly with levels in SF. CONCLUSION: While we found increased levels of markers in joint fluid and blood, no single systemic biochemical biomarkers that were a common denominator between the animal models and the patient material could be identified. Our data indicate that it is critical to delineate the temporal profile of multiple local and systemic factors in order to pinpoint soluble biomarkers for OA.

Neurodegenerative biomarkers outperform neuroinflammatory biomarkers in amyotrophic lateral sclerosis.

OBJECTIVE: To describe the diagnostic and prognostic performance, and longitudinal trajectories, of potential biomarkers of neuroaxonal degeneration and neuroinflammation in amyotrophic lateral sclerosis (ALS). METHODS: This case-control study included 192 incident ALS patients, 42 ALS mimics, 114 neurological controls, and 117 healthy controls from Stockholm, Sweden. Forty-four ALS patients provided repeated measurements. We assessed biomarkers of (1)neuroaxonal degeneration: neurofilament light (NfL) and phosphorylated neurofilament heavy (pNfH) in cerebrospinal fluid (CSF) and NfL in serum, and (2)neuroinflammation: chitotriosidase-1 (CHIT1) and monocyte chemoattractant protein 1 (MCP-1) in CSF. To evaluate diagnostic performance, we calculated the area under the curve (AUC). To estimate prognostic performance, we applied quantile regression and Cox regression. We used linear regression models with robust standard errors to assess temporal changes over time. RESULTS: Neurofilaments performed better at differentiating ALS patients from mimics (AUC: pNfH 0.92, CSF NfL 0.86, serum NfL 0.91) than neuroinflammatory biomarkers (AUC: CHIT1 0.71, MCP-1 0.56). Combining biomarkers did not improve diagnostic performance. Similarly, neurofilaments performed better than neuroinflammatory biomarkers at predicting functional decline and survival. The stratified analysis revealed differences according to the site of onset: in bulbar patients, neurofilaments and CHIT1 performed worse at predicting survival and correlations were lower between biomarkers. Finally, in bulbar patients, neurofilaments and CHIT1 increased longitudinally but were stable in spinal patients. CONCLUSIONS: Biomarkers of neuroaxonal degeneration displayed better diagnostic and prognostic value compared with neuroinflammatory biomarkers. However, in contrast to spinal patients, in bulbar patients neurofilaments and CHIT1 performed worse at predicting survival and seemed to increase over time.

Cardiac troponin T is elevated and increases longitudinally in ALS patients.

Objective: To test whether high-sensitivity cardiac troponin T (hs-cTnT) could act as a diagnostic or prognostic biomarker in ALS, comparing hs-cTnT to neurofilament light (NfL). Methods: We performed a case-control study, including 150 ALS patients, 28 ALS mimics, and 108 healthy controls, and a follow-up study of the ALS patients, during 2014-2020 in Stockholm, Sweden. We compared concentrations of hs-cTnT in plasma and NfL in the cerebrospinal fluid between cases and controls. To evaluate the diagnostic performance, we calculated the area under the curve (AUC). Hazard ratios (HRs) were estimated from Cox models to assess associations between hs-cTnT and NfL at ALS diagnosis and risk of death. The longitudinal analysis measured changes of hs-cTnT and NfL since ALS diagnosis. Results: We noted higher levels of hs-cTnT in ALS patients (median: 16.5 ng/L) than in ALS mimics (11 ng/L) and healthy controls (6 ng/L). Both hs-cTnT and NfL could distinguish ALS patients from ALS mimics, with higher AUC noted for NfL (AUC 0.88; 95%CI 0.79-0.97). Disease progression correlated weakly with hs-cTnT (Pearson's r = 0.18, p = 0.04) and moderately with NfL (Pearson's r = 0.41, p < 0.001). Shorter survival was associated with higher levels of NfL at diagnosis (HR 1.08, 95%CI 1.04-1.11), but not hs-cTnT. hs-cTnT increased (12.61 ng/L per year, 95%CI 7.14-18.06) whereas NfL decreased longitudinally since ALS diagnosis. Conclusions: NfL is a stronger diagnostic and prognostic biomarker than hs-cTnT for ALS. However, hs-cTnT might constitute a disease progression biomarker as it increases longitudinally. The underlying causes for this increase need to be investigated.

Can L(+)-lactate be used as a marker of experimentally induced inflammation in rats?

OBJECTIVE: The purpose of this study was to investigate if L(+)-lactate (lactate) can be used as a marker of progression of joint inflammation in comparison with a reference marker, prostaglandin E2 (PGE(2)), and to analyse implications for drug treatments. MATERIALS AND METHODS: The assessment of the inflammation time course and the treatment efficacy studies were performed on two occasions. At specific time points, synovial fluid was extracted from Sprague-Dawley rats (n = 87) challenged with either carrageenan (Cg) or Freund's complete adjuvant (FCA) or from six non-inflamed rats. Naproxen (7.5 or 30 micromol/kg) or rofecoxib (30 micromol/kg) was administered per os 2 h post Cg or at 48 h post FCA. Levels of PGE(2) and lactate were assessed either by immuno-assay or by colorimetric assay. RESULTS: Increased levels of both markers were detected following Cg or FCA injection. Pharmacological treatments resulted in lower concentrations of PGE(2) whereas levels of lactate remained unaffected compared to the vehicle-treated group. CONCLUSION: Our results suggest that lactate may be useful as an additional biomarker of inflammatory processes, especially for monitoring the non-cox-inhibitor sensitive cascade.

Monosodium iodoacetate-induced monoarthritis develops differently in knee versus ankle joint in rats.

OBJECTIVE: Disability and movement-related pain are major symptoms of joint disease, motivating the development of methods to quantify motor behaviour in rodent joint pain models. We compared effects on behaviour, assessed the levels of biochemical mediators and made a detailed histopathological evaluation after induction of rat monoiodoacetate (MIA) monoarthritis into the ankle or knee joint. DESIGN: Twenty-seven male Lewis rats were used. Before and up to 28 days after induction, they were tested for weight bearing during walking (dynamic), and standing (static), and for mechanical sensitivity. At termination synovial fluid was taken from ankle and/or knee joints for analysis of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), macrophage inflammatory protein 3 alpha (MIP-3α), keratinocyte chemoattractant (KC)/human growth-regulated oncogene (GRO) and L(+)-lactate, and from separate rats joints were collected for histopathological assessment. RESULTS: MIA ankle joint injection gave a marked reduction of dynamic weight bearing during the first days, not seen in rats with knee joint injection. At three weeks, it was decreased in the group with knee injection, but not in those with ankle injection. However, the different injection sites caused similar reductions in static weight bearing during the early phase, which was normalized in the group with ankle injection but continued and was strengthened with time in the knee injected group. Histopathological assessment, biochemical mediators and joint swelling confirmed the disparate profiles. CONCLUSIONS: This work shows that ankle versus knee joint injection of MIA resulted in different profiles in rats, which may mirror what has been found in human patients with osteoarthritis.

No evidence for altered plasma NGF and BDNF levels in fibromyalgia patients.

There has been a surging interest in the putative role of peripheral growth factors in the pathophysiology of fibromyalgia, specifically in the peripheral sensitization that occurs in chronic pain disorders. This cross-sectional study set out to assess and compare brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in plasma samples from fibromyalgia patients and healthy controls. Plasma BDNF and NGF were measured in 89 fibromyalgia patients and 36 pain-free controls, and compared using ANCOVA controlling for potential confounders, as well as Bayesian methods for parameter estimation and model evaluation. BDNF and NGF levels in fibromyalgia patients did not differ from those in pain-free controls. Statistical methods were consistent, with both frequentist and Bayesian approaches leading to the same conclusions. Our study fails to replicate the finding that peripheral BDNF is altered in fibromyalgia, and instead our findings suggest that plasma levels of growth factor appear normative in fibromyalgia.

Methodological aspects of rat beta-endorphin analysis-influence of diurnal variation.

Beta-endorphin radioimmunoassays (RIAs) are widely performed following physical, emotional and environmental challenges in the rat. In the literature, a wide range of techniques have been described, but in the present study, we have focused on methodological aspects of beta-endorphin RIAs, investigating various characteristics of human and rat specific antibodies. Initial studies verified that the RIA outcome was not appropriate when using non-species compatible components. Novel rat beta-endorphin antibodies, r 4114 and r 4268, were raised in rabbits and characterised in terms of specificity, avidity and titer. Both of the new antisera showed 68.1% cross-reactivity with human beta-endorphin. The ED50 was 50+/-8 pmol/l, and the mean ED80 was 17 pmol/l for r 4268 but three-fold higher for r 4114. The intra-assay coefficient of variation (CV) was 7% at 100 pmol/l and the inter-assay CV was 10% at the same level for r 4268 and similar for r 4114. Using this novel rat beta-endorphin RIA for analyses of diurnal influence and removal from the Animal House cage, no significant changes were observed in either the hypothalamus or peri-aqueductal grey regions. These results suggest that rat beta-endorphin concentrations in these brain areas are not affected by order of removal or diurnal variation.

Using gait analysis to assess weight bearing in rats with Freund׳s complete adjuvant-induced monoarthritis to improve predictivity: Interfering with the cyclooxygenase and nerve growth factor pathways.

Lack of predictive power for drug effects has been a major criticism against animal pain models. It is therefore important to define the utility and limitations of different models. The aim of this study was to extend previous work on gait analysis as a tool to investigate pharmacological effects in monoarthritic rats, specifically to test the hypothesis that monoarthritis induced by Freund׳s complete adjuvant (FCA) provides a better estimate of overall analgesic efficacy of established, and novel, clinically effective and ineffective therapeutic approaches. Male rats injected intra-articularly into one ankle joint with FCA (1.0mg/ml) were treated with the monoclonal antibody to nerve growth factor (NGF), MEDI-578, the inhibitors of tropomyosin receptor kinases A, B and C (pan-Trk) AZ6623 or AZ7092, the transient receptor potential vanilloid 1 (TRPV1) inhibitor AZD1386, or the cyclooxygenase (COX) inhibitors naproxen, ibuprofen, valdecoxib or rofecoxib. Effects on weight bearing during locomotion were tested using video capture of print images. The apparent efficacy in this model was Trk inhibitors≥anti-NGF antibody>COX inhibitors. The TRPV1 inhibitor was ineffective. Together with previous data, the results support using gait-related parameters in the monoarthritis model. FCA as induction agent seems to provide a good overall prediction of analgesic efficacy in disorders with inflammatory joint pain.

Delayed Imatinib Treatment for Acute Spinal Cord Injury: Functional Recovery and Serum Biomarkers.

With no currently available drug treatment for spinal cord injury, there is a need for additional therapeutic candidates. We took the approach of repositioning existing pharmacological agents to serve as acute treatments for spinal cord injury and previously found imatinib to have positive effects on locomotor and bladder function in experimental spinal cord injury when administered immediately after the injury. However, for imatinib to have translational value, it needs to have sustained beneficial effects with delayed initiation of treatment, as well. Here, we show that imatinib improves hind limb locomotion and bladder recovery when initiation of treatment was delayed until 4 h after injury and that bladder function was improved with a delay of up to 24 h. The treatment did not induce hypersensitivity. Instead, imatinib-treated animals were generally less hypersensitive to either thermal or mechanical stimuli, compared with controls. In an effort to provide potential biomarkers, we found serum levels of three cytokines/chemokines--monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-3α, and keratinocyte chemoattractant/growth-regulated oncogene (interleukin 8)--to increase over time with imatinib treatment and to be significantly higher in injured imatinib-treated animals than in controls during the early treatment period. This correlated to macrophage activation and autofluorescence in lymphoid organs. At the site of injury in the spinal cord, macrophage activation was instead reduced by imatinib treatment. Our data strengthen the case for clinical trials of imatinib by showing that initiation of treatment can be delayed and by identifying serum cytokines that may serve as candidate markers of effective imatinib doses.

Evidence of different mediators of central inflammation in dysfunctional and inflammatory pain--interleukin-8 in fibromyalgia and interleukin-1 ß in rheumatoid arthritis.

The purpose of this study was to relate central inflammation to autonomic activity (heart rate variability (HRV)) in patients with rheumatoid arthritis (RA) and fibromyalgia (FM). RA patients had reduced parasympathetic activity and FM patients had increased sympathetic activity compared to healthy controls. Comparisons between RA and FM showed higher cerebrospinal fluid (CSF) interleukin (IL)-1ß inversely correlated to parasympathetic activity in RA. The FM patients had higher concentrations of CSF IL-8, IL-1Ra, IL-4 and IL-10, but none of these cytokines correlated with HRV. In conclusion, we found different profiles of central cytokines, i.e., elevated IL-1ß in inflammatory pain (RA) and elevated IL-8 in dysfunctional pain (FM).

NGF modulates CGRP synthesis in human B-lymphocytes: a possible anti-inflammatory action of NGF?

We investigated whether the sensory neuropeptide, calcitonin gene-related peptide (CGRP), could be synthesised by human lymphocytes. Our results indicate that in activated B-cells, there is a strong expression of CGRP gene transcripts, which is almost absent in resting cells. Since B-cells autocrinally produce NGF, the neutralisation of endogenous NGF by anti-NGF antibodies resulted in a marked reduction in CGRP expression in both resting and activated B-cells. Thus, NGF appears to directly affect the synthesis of CGRP in B-cells as in sensory neurons. By regulating CGRP synthesis in lymphocytes and neuronal cells, NGF can influence the intensity and duration of the immune response.

Bone and joint neuropathy in rats with type-2 diabetes.

We have previously demonstrated that Goto-Kakizaki (GK) rats with spontaneous type-2 diabetes and peripheral neuropathy exhibit regional osteopathic changes. In the present study on 18 GK rats and 21 control Wistar rats, the occurrence of the sensory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP), and the autonomic neuropeptide Y (NPY) was analysed in bone and joints, dorsal root ganglia and lumbar spinal cord by immunohistochemistry and radioimmunoassay (RIA). Immunohistochemistry disclosed a predominance of immunoreactivities in vessel-related nerve fibers, although some were also seen in free terminals. While SP, CGRP and NPY in periosteum, cortical bone and synovium was confined to neuronal tissue, the bone marrow in addition exhibited an abundance of NPY-positive megakaryocytes. Apart from this cellular source of NPY, the observations suggest that the three neuropeptides analysed in bone and joints are of neuronal origin. Quantification by RIA showed a significant decrease of NPY in cortical bone (-36%), bone marrow (-66%) and ankle (-29%) of GK rats. CGRP was decreased in the spinal cord (-19%) and dorsal root ganglia (-26%) but was unchanged in bone and joints, as with SP. Given the suggested anabolic role of NPY and CGRP on bone, neuropeptidergic deficit in diabetes may prove to be an important factor underlying the development of regional osteopenia.