High mobility group box 1 and a network of other biomolecules influence fatigue in patients with Crohn's disease.

BACKGROUND: Fatigue is common in patients with chronic inflammatory and autoimmune diseases, often with a severe impact on the patient's daily life. From a biological point of view, fatigue can be regarded as an element of the sickness behavior response, a coordinated set of responses induced by pathogens to enhance survival during an infection and immunological danger. The mechanisms are not fully understood but involve activation of the innate immune system, with pro-inflammatory cytokines, in particular interleukin (IL)-1ß, acting on cerebral neurons. These mechanisms are also active during chronic inflammatory conditions. High mobility group box 1 (HMGB1) protein has interleukin-1 like properties and is a strong inducer of innate immune responses. Its role in generation of fatigue is not clarified. Emerging evidence indicates that also other biomolecules may influence sickness behavior. We aimed to elucidate how HMGB1 influences fatigue in patients with Crohn's disease, and how the protein interacts with other candidate biomarkers of fatigue. METHODS: In 56 patients with newly diagnosed Crohn's disease, fatigue was evaluated using three different fatigue instruments: the fatigue visual analog scale (fVAS), Fatigue Severity Scale (FSS), and the vitality subscale of Medical Outcomes Study Short-Form Health Survey (SF-36vs). The biochemical markers IL-1 receptor antagonist (RA), soluble IL-1 receptor type 2 (sIL-RII), heat shock protein 90 alpha (HSP90α), HMGB1, anti-fully reduced (fr)HMGB1 antibodies (abs), hemopexin (HPX), and pigment epithelium-derived factor (PEDF) were measured in plasma. Multivariable regression and principal component analyses (PCA) were applied. RESULTS: Multivariable regression analyses revealed significant contributions to fatigue severity for HMGB1 in the FSS model, HSP90α in the fVAS model and IL-1RA in the SF-36vs model. Depression and pain scores contributed to all three models. In PCA, two components described 53.3% of the variation. The "inflammation and cellular stress dimension" was dominated by IL-1RA, sIL-1RII, HSP90α, HPX, and PEDF scores, where the "HMGB1 dimension" was dominated by HMGB1, anti-frHMGB1 abs, and fVAS scores. CONCLUSION: This study supports the hypothesis that HMGB1 and a network of other biomolecules influence fatigue severity in chronic inflammatory conditions. The well-known association with depression and pain is also acknowledged.

Neurofilament light in plasma is a potential biomarker of central nervous system involvement in systemic lupus erythematosus.

BACKGROUND: Neuropsychiatric manifestations (NP) are common in systemic lupus erythematosus (SLE). However, the pathophysiological mechanisms are not completely understood. Neurofilament light protein (NfL) is part of the neuronal cytoskeleton. Increased NfL concentrations, reflecting neurodegeneration, is observed in cerebrospinal fluid (CSF) in several neurodegenerative and neuroinflammatory conditions. We aimed to explore if plasma NfL could serve as a biomarker for central nervous system (CNS) involvement in SLE. METHODS: Sixty-seven patients with SLE underwent neurological examination; 52 underwent lumbar puncture, while 62 underwent cerebral magnetic resonance imaging (MRI). We measured selected auto-antibodies and other laboratory variables postulated to have roles in NP pathophysiology in the blood and/or CSF. We used SPM12 software for MRI voxel-based morphometry. RESULTS: Age-adjusted linear regression analyses revealed increased plasma NfL concentrations with increasing creatinine (ß = 0.01, p < 0.001) and Q-albumin (ß = 0.07, p = 0.008). We observed higher plasma NfL concentrations in patients with a history of seizures (ß = 0.57, p = 0.014), impaired motor function (ß = 0.36, p = 0.008), increasing disease activity (ß = 0.04, p = 0.008), and organ damage (ß = 0.10, p = 0.002). Voxel-based morphometry suggested an association between increasing plasma NfL concentrations and the loss of cerebral white matter in the corpus callosum and hippocampal gray matter. CONCLUSION: Increased plasma NfL concentrations were associated with some abnormal neurological, cognitive, and neuroimaging findings. However, plasma NfL was also influenced by other factors, such as damage accrual, creatinine, and Q-albumin, thereby obscuring the interpretation of how plasma NfL reflects CNS involvement. Taken together, NfL in CSF seems a better marker of neuronal injury than plasma NfL in patients with SLE.

Sample Preparation Strategies for Antibody-Free Quantitative Analysis of High Mobility Group Box 1 Protein.

Sickness behavior and fatigue are induced by cerebral mechanisms involving inflammatory cytokines. High mobility group box 1 (HMGB1) is an alarmin, and a potential key player in this process. Reliable quantification methods for total HMGB1 and its redox variants must be established in order to clearly understand how it functions. Current methods pose significant challenges due to interference from other plasma proteins and autoantibodies. We aimed to develop an antibody-free sample preparation method followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to measure HMGB1 in human plasma. Different methods were applied for the removal of interfering proteins and the enrichment of HMGB1 from spiked human plasma samples. A comparison of methods showed an overall low extraction recovery (<40%), probably due to the stickiness of HMGB1. Reversed-phase liquid chromatography separation of intact proteins in diluted plasma yielded the most promising results. The method produced an even higher degree of HMGB1 purification than that observed with immunoaffinity extraction. Detection sensitivity needs to be further improved for the measurement of HMGB1 in patient samples. Nevertheless, it has been demonstrated that a versatile and fully antibody-free sample preparation method is possible, which could be of great use in further investigations.

Anti-HMGB1 auto-Abs influence fatigue in patients with Crohn's disease.

Fatigue is common in all chronic inflammatory and autoimmune diseases. A conceptual model for understanding the biological basis of fatigue describes it as being a part of the sickness behaviour response generated by pro-inflammatory cytokines and other mediators. We hypothesised that the pro-inflammatory high mobility group box 1 (HMGB1) protein is a fatigue-inducing molecule and that auto-Abs against HMGB1 reduce fatigue. We measured Abs against disulphide (ds) HMGB1 and fully reduced (fr) HMGB1 in plasma from 57 patients with Crohn's disease. Fatigue was rated using the fatigue visual analogue scale (fVAS) and disease activity with faecal calprotectin, C-reactive protein and the Simple Endoscopic Score for Crohn's disease. Multivariable regression models identified anti-dsHMGB1 and anti-frHMGB1 Abs as the strongest contributing factors for fVAS scores (B = -29.10 (P = 0.01), R2 = 0.17, and B = -17.77 (P = 0.01), R2 = 0.17, respectively). Results indicate that anti-HMGB1 auto-Abs alleviate fatigue possibly by down-regulating HMGB1-induced sickness behaviour.

Neurofilament light is a biomarker of brain involvement in lupus and primary Sjögren's syndrome.

BACKGROUND: To test the hypothesis that neurofilament light (NfL) in CSF is a biomarker of CNS involvement in patients with systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS), we measured NfL in CSF from 52 patients with lupus and 54 with pSS and explored associations with clinical, structural, immunological and biochemical abnormalities. METHODS: In CSF, we measured NfL, anti-P antibodies, protein S100B and TWEAK by ELISA and anti-NR2 antibodies by electrochemiluminescence. Anti-phospholipid antibodies and routine immunological tests were performed in blood. IgG and albumin were measured in CSF and serum for assessment of the blood-brain barrier function (Q-albumin) and intrathecal IgG production (IgG index). Cerebral MRI and neuropsychological testing were performed. RESULTS: A multivariable regression model showed that increasing CSF anti-NR2 antibody levels were associated with increasing NfL levels in patients with SLE (B 1.27, 95% CI 0.88-1.65, p < 0.001). Age contributed significantly in the model (B 0.04, 95% CI 0.03-0.05, p < 0.001). Similar findings were observed in the pSS group. Adjusted for age and sex, no associations were found between NfL levels and any MRI data. In SLE patients, higher NfL concentrations were associated with impairments in psychomotor speed and motor function, and in pSS with motor dysfunction. These associations remained in multivariable regression models. CONCLUSIONS: Increased concentration of NfL in CSF is a marker of cerebral involvement in patients with SLE and pSS, is strongly associated with the presence of anti-NR2 antibodies, and correlates with cognitive impairment in several domains.

Fatigue in patients with plaque-type psoriasis: lack of an association with plasma cytokines.

BACKGROUND: Fatigue is common in patients with psoriasis, and cytokines have been postulated to influence fatigue. OBJECTIVES: This case-control study explored the plasma levels of selected cytokines in patients with psoriasis and compared them with fatigue and other clinical factors. MATERIALS AND METHODS: Eighty-four patients with chronic plaque-type psoriasis and 84 age- and gender-matched healthy subjects were enrolled. Psoriasis severity was measured using the Psoriasis Area and Severity Index (PASI), and skin-related quality of life using the Dermatology Life Quality Index (DLQI). Fatigue was rated with the fatigue Visual Analogue Scale (fVAS). Plasma levels of interleukin (IL)-1ß, IL-1Rα, IL-1RII, IL-6, and IL-10 were measured by electrochemiluminescence sandwich immunoassay and ELISA. RESULTS: IL-1Rα and IL-6 median concentrations were significantly higher in patients than healthy subjects: 203 pg/mL (interquartile range: 150-274) versus 166 pg/mL (128-212), p=0.008 for IL-Rα, and 0.82 pg/mL (0.25-1.40) versus 0.50 pg/mL (0.25-0.91), p=0.009 for IL-6. IL-1ß, IL-1RII, and IL-10 concentrations did not differ between patients and healthy subjects. Higher levels of IL-1Rα and IL-6 were associated with increased body mass index (BMI), but not with disease activity. Cytokine concentrations were not associated with fatigue. CONCLUSION: These findings do not support an association between fatigue and blood concentrations of selected pro- and anti-inflammatory cytokines. The increased IL-1Rα and IL-6 levels associated with increased BMI are probably caused by release of adipokines from adipose tissue; of these, leptin, in particular, is known to be a strong inflammatory stimulator.

Considerably Lower Levels of Hypocretin-1 in Cerebrospinal Fluid Is Revealed by a Novel Mass Spectrometry Method Compared with Standard Radioimmunoassay.

Low levels of hypocretin-1 (Hcrt1) in cerebrospinal fluid (CSF) are associated with narcolepsy type 1 (NT1). Although immunoassays are prone to antibody batch differences, detection methods and variation between laboratories, the standard method for Hcrt1 measurement is a radioimmunoassay (RIA). Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is an antibody- and radioactive free alternative for precise measurement of Hcrt1. We developed an LC-MS/MS method for measurement of Hcrt1 in CSF with automated sample preparation by solid-phase extraction (SPE). The LC-MS/MS method was compared with the RIA method for Hcrt1 detection. CSF samples from healthy subjects and NT1 patients was obtained by lumbar puncture. NT1 patients were diagnosed according to the minimal criteria by the International Classification of Sleep Disorders (ICSD). The LC-MS/MS method showed linearity across the range of calibrators and had a limit of detection (LOD) of 2.5 pg/mL and a limit of quantitation (LOQ) of 3.6 pg/mL. Comparison of the LC-MS/MS method with RIA revealed a 19 times lower level in healthy controls and 22 times lower level in NT1 patients with the LC-MS/MS method than with RIA. Bland-Altman analysis demonstrated agreement between the methods. These results question what is detected by RIA and strongly suggest that the physiological concentrations of the peptide are much lower than previously believed. LC-MS/MS proves to be an alternative for detection of Hcrt1 for diagnosis of narcolepsy.

Interleukin-1-related activity and hypocretin-1 in cerebrospinal fluid contribute to fatigue in primary Sjögren's syndrome.

BACKGROUND: Fatigue is a common and sometimes debilitating phenomenon in primary Sjögren's syndrome (pSS) and other chronic inflammatory diseases. We aimed to investigate how IL-1 ß-related molecules and the neuropeptide hypocretin-1 (Hcrt1), a regulator of wakefulness, influence fatigue. METHODS: Hcrt1 was measured by radioimmunoassay (RIA) in cerebrospinal fluid (CSF) from 49 patients with pSS. Interleukin-1 receptor antagonist (IL-1Ra), IL-1 receptor type 2 (IL-1RII), IL-6, and S100B protein were measured by enzyme-linked immunosorbent assay (ELISA). Fatigue was rated by the fatigue visual analog scale (fVAS). RESULTS: Simple univariate regression and multiple regression analyses with fatigue as a dependent variable revealed that depression, pain, and the biochemical variable IL-1Ra had a significant association with fatigue. In PCA, two significant components were revealed. The first component (PC1) was dominated by variables related to IL-1ß activity (IL-1Ra, IL-1RII, and S100B). PC2 showed a negative association between IL-6 and Hcrt1. fVAS was then introduced as an additional variable. This new model demonstrated that fatigue had a higher association with the IL-1ß-related PC1 than to PC2. Additionally, a third component (PC3) became significant between low Hcrt1 concentrations and fVAS scores. CONCLUSIONS: The main findings of this study indicate a functional network in which several IL-1ß-related molecules in CSF influence fatigue in addition to the classical clinical factors of depression and pain. The neuropeptide Hcrt1 seems to participate in fatigue generation, but likely not through the IL-1 pathway.

Heat shock protein 90 and inflammatory activity in newly onset Crohn's disease.

OBJECTIVE: The aim of the study was to investigate whether heat shock protein (HSP)90α plasma concentrations were associated with disease activity in patients with Crohn's disease. MATERIALS AND METHODS: This cross-sectional study included 53 patients who were newly diagnosed with Crohn's disease. Demographic data and disease distribution were recorded, and disease activity was rated using the Simple Endoscopic Score for Crohn's Disease (SES-CD) and the Harvey Bradshaw Index (HBI). Faecal calprotectin and plasma concentrations of CRP and HSP90α were measured. RESULTS: The median SES-CD was 7, and the median HSP90α level was 17.2 ng/mL. The HSP90α level was significantly correlated with SES-CD, CRP, and faecal calprotectin, but not with HBI. Linear regression analysis revealed that HSP90α was significantly associated with SES-CD (r2 = 0.27, p < .001) and with CRP (r2 = 0.18, p = .002). HSP90α concentrations were significantly higher in the 10 patients with the highest SES-CD scores compared to the 10 patients with the lowest SES-CD scores. CONCLUSIONS: Objective measures of disease activity and inflammation in Crohn's disease - SES-CD and CRP - were closely associated with HSP90α concentrations in plasma, suggesting that HSP90α may be a biomarker of Crohn's disease.

Association of hippocampal atrophy with cerebrospinal fluid antibodies against the NR2 subtype of the N-methyl-D-aspartate receptor in patients with systemic lupus erythematosus and patients with primary Sjögren's syndrome.

OBJECTIVE: Cognitive dysfunction is common in both systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (SS). Antibodies against the NR2 subtype of the N-methyl-D-aspartate receptor (anti-NR2 antibodies) cause hippocampal atrophy and cognitive impairment in mice and have been associated with memory impairment in both patients with SLE and patients with primary SS. In addition, a reduced volume of hippocampal gray matter has been demonstrated in both SLE and primary SS. This study was undertaken to investigate whether there is a connection between the presence of anti-NR2 antibodies and hippocampal atrophy in human diseases. METHODS: Fifty patients with SLE and 50 patients with primary SS underwent clinical examination and cerebral magnetic resonance imaging. Anti-NR2 antibodies in cerebrospinal fluid (CSF) were measured, and hippocampal gray matter volumes were compared between patients who were positive for and those who were negative for anti-NR2 antibodies. RESULTS: Patients with anti-NR2 antibodies in CSF had less hippocampal gray matter than patients without these antibodies. No other differences regarding gray matter volumes in other parts of the brain were identified. CONCLUSION: The present findings indicate that anti-NR2 antibodies in patients with SLE and primary SS cause neuronal death manifested as reduced hippocampal gray matter, as has been previously demonstrated in mice with autoimmune disease.