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  • 1.
    Gerdle, Björn
    et al.
    Linköpings universitet, Institutionen för hälsa, medicin och vård, Avdelningen för prevention, rehabilitering och nära vård. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Wåhlén, Karin
    Linköpings universitet, Institutionen för hälsa, medicin och vård, Avdelningen för prevention, rehabilitering och nära vård. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Gordh, Torsten
    Uppsala Univ, Sweden.
    Bäckryd, Emmanuel
    Linköpings universitet, Institutionen för hälsa, medicin och vård, Avdelningen för prevention, rehabilitering och nära vård. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Carlsson, Anders
    Linköpings universitet, Institutionen för hälsa, medicin och vård, Avdelningen för prevention, rehabilitering och nära vård. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Ghafouri, Bijar
    Linköpings universitet, Institutionen för hälsa, medicin och vård, Avdelningen för prevention, rehabilitering och nära vård. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Plasma proteins from several components of the immune system differentiate chronic widespread pain patients from healthy controls - an exploratory case-control study combining targeted and non-targeted protein identification2022Ingår i: Medicine, ISSN 0025-7974, E-ISSN 1536-5964, Vol. 101, nr 46, artikel-id e31013Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Chronic widespread pain (CWP), including fibromyalgia (FM), is characterized by generalized musculoskeletal pain and hyperalgesia. Plasma proteins from proteomics (non-targeted) and from targeted inflammatory panels (cytokines/chemokines) differentiate CWP/FM from controls. The importance of proteins obtained from these two sources, the protein-protein association network, and the biological processes involved were investigated. Plasma proteins from women with CWP (n = 15) and CON (n = 23) were analyzed using two-dimensional gel electrophoresis analysis and a multiplex proximity extension assay for analysis of cytokines/chemokines. Associations between the proteins and group were multivarietly analyzed. The protein-protein association network and the biological processes according to the Gene Ontology were investigated. Proteins from both sources were important for group differentiation; the majority from the two-dimensional gel electrophoresis analysis. 58 proteins significantly differentiated the two groups (R-2 = 0.83). A significantly enriched network was found; biological processes were acute phase response, complement activation, and innate immune response. As with other studies, this study shows that plasma proteins can differentiate CWP from healthy subjects. Focusing on cytokines/chemokines is not sufficient to grasp the peripheral biological processes that maintain CWP/FM since our results show that other components of the immune and inflammation systems are also highly significant.

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  • 2.
    Jasim, Hajer
    et al.
    Karolinska Inst, Sweden.
    Ghafouri, Bijar
    Linköpings universitet, Institutionen för hälsa, medicin och vård, Avdelningen för prevention, rehabilitering och nära vård. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Carlsson, Anders
    Linköpings universitet, Institutionen för hälsa, medicin och vård, Avdelningen för prevention, rehabilitering och nära vård. Linköpings universitet, Medicinska fakulteten.
    Hedenberg-Magnusson, Britt
    Karolinska Inst, Sweden; Folktandvarden Stockholms Lan AB, Sweden.
    Ernberg, Malin
    Karolinska Inst, Sweden.
    Daytime changes of salivary biomarkers involved in pain2020Ingår i: Journal of Oral Rehabilitation, E-ISSN 1365-2842, Vol. 47, nr 7, s. 843-850Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The study aimed to investigate salivary levels of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), substance P (SP) and glutamate at five time points from morning to afternoon in a well-characterised healthy and pain-free individuals. Ten young adults were included. Unstimulated and stimulated whole saliva were collected from each participant repeatedly across the day. Blood samples were drawn in connection with the first and last saliva sample as reference standard. Levels of NGF and BDNF were determined using gel-free Western blot technology, glutamate levels were analysed using a colorimetric assay, and SP was determined using a commercially available ELISA. Salivary NGF and BDNF showed significant differences between the different collection times in both unstimulated (NGF; P = .006; BDNF; P = .026) and stimulated whole saliva (NGF; P = .006; BDNF; P = .019). The highest concentrations of the neuropeptides were expressed in the early morning, and they thereafter decreased across the day. In contrast, the expression of salivary glutamate and SP did not show any significant changes across the day. Plasma levels of NGF were higher in the evening sample (P = .028); otherwise, there were no significant differences for any of the other markers between morning and evening samples. NGF and BDNF in whole saliva showed a significant variation across the day. On the contrary, no variation in the levels of SP and glutamate was detected. These findings highlight the importance of consistency in the collection time and approach in biomarker studies using saliva.

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  • 3.
    Jasim, Hajer
    et al.
    Karolinska Inst, Sweden; Scandinavian Ctr Orofacial Neurosci SCON, Sweden.
    Ernberg, Malin
    Karolinska Inst, Sweden; Scandinavian Ctr Orofacial Neurosci SCON, Sweden.
    Carlsson, Anders
    Linköpings universitet, Institutionen för hälsa, medicin och vård, Avdelningen för prevention, rehabilitering och nära vård. Linköpings universitet, Medicinska fakulteten.
    Gerdle, Björn
    Linköpings universitet, Institutionen för hälsa, medicin och vård, Avdelningen för prevention, rehabilitering och nära vård. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Ghafouri, Bijar
    Linköpings universitet, Institutionen för hälsa, medicin och vård, Avdelningen för prevention, rehabilitering och nära vård. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Protein Signature in Saliva of Temporomandibular Disorders Myalgia2020Ingår i: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, Vol. 21, nr 7, artikel-id 2569Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In the last years, several attempts have been made to study specific biological markers of temporomandibular disorders (TMD). So far, no laboratory tests have been appropriately validated for the diagnosis and prognosis of these disorders. This study aimed to investigate the proteomic profile of the whole stimulated saliva of TMD myalgia patients in order to evaluate potential diagnostic and/or prognostic salivary candidate proteins which could be useful for the management of TMD. Twenty patients diagnosed with TMD myalgia according to the validated Diagnostic Criteria for TMD (DC/TMD) and 20 matched healthy pain-free controls were enrolled. Saliva samples were collected in the morning. Comparative proteomic analysis was performed with two-dimensional gel electrophoresis followed by identification with liquid chromatography-tandem mass spectrometry. Statistical analysis of the quantitative proteomics data revealed that 20 proteins were significantly altered in patients compared to controls. Among these proteins, 12 showed significantly increased levels, and 8 showed significantly decreased levels in patients with TMD myalgia compared to controls. The identified proteins are involved in metabolic processes, immune response, and stress response. This proteomic study shows that the salivary protein profile can discriminate patients with TMD myalgia from healthy subjects, but the protein signature has no correlation with the clinical features of TMD myalgia. Additional studies are needed to validate our observations in additional sample sets and to continue assessing the utility of saliva as a suitable sample for studying processes related to TMD myalgia.

  • 4.
    Jasim, Hajer
    et al.
    Karolinska Inst, Sweden.
    Carlsson, Anders K
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum. Linköpings universitet, Medicinska fakulteten.
    Gerdle, Björn
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum. Linköpings universitet, Medicinska fakulteten.
    Ernberg, Malin
    Karolinska Inst, Sweden.
    Ghafouri, Bijar
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum. Linköpings universitet, Medicinska fakulteten.
    Diurnal variation of inflammatory plasma proteins involved in pain2019Ingår i: Pain Reports, E-ISSN 2471-2531, Vol. 4, nr 5, artikel-id e776Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Introduction: Proteomics is a powerful approach for biochemical research because it directly studies the main functional components of biochemical systems. The understanding of the normal fluctuations of the proteome in health is essential to identify pain-specific biomarkers.

    Objective: To investigate fluctuations of the plasma proteome in healthy pain-free individuals.

    Methods: Blood samples were structurally collected in the early morning and evening from 10 clinically healthy individuals (26.3 ± 3.3 years). High abundant proteins were removed from plasma, and proteins were then analysed by nanoliquid chromatography combined with mass spectrometry. In addition, an assay of 71 cytokines/chemokines/growth factors was analysed.

    Results: Multivariate statistical analysis displayed that there were up to 64 proteins whose expression levels were significantly altered between the plasma samples collected during the morning and evening; no changes existed for the assay. The levels of 34 proteins were increased and 30 proteins were decreased during the evening compared with the morning sample. The increased proteins were involved in the biological processes such as protein activation cascade, complement activation, and stress response. The decreased proteins were involved in regulation of endopeptidase activity, inflammatory response, and protein metabolic processes.

    Conclusion: The circadian variations in the plasma proteome stress the need to collect blood samples of both patients and controls at a fixed time of the day. The results in this study might be useful for better understanding of the complexity of individual variation in the human plasma proteome over time and provide a baseline for improved pain biomarker discovery.

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  • 5.
    Jasim, Hajer
    et al.
    Karolinska Inst, Sweden; Karolinska Inst, Sweden; Folktandvarden Stockholm AB, Sweden.
    Carlsson, Anders
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Hedenberg-Magnusson, Britt
    Karolinska Inst, Sweden; Karolinska Inst, Sweden; Folktandvarden Stockholm AB, Sweden.
    Ghafouri, Bijar
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Ernberg, Malin
    Karolinska Inst, Sweden; Karolinska Inst, Sweden.
    Saliva as a medium to detect and measure biomarkers related to pain2018Ingår i: Scientific Reports, E-ISSN 2045-2322, Vol. 8, artikel-id 3220Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Saliva is often neglected as a body fluid of diagnostic or prognostic value, even though generally well accepted by the patients. This is due to lack of a standardized collection procedure. The aim of this study was to identify the ideal saliva collection technique and develop new sensitive methods to detect and analyse markers related to pain in healthy pain-free subjects. Plasma and five different saliva collection approached was evaluated during strictly controlled conditions. Levels of nerve growth factor (NGF), calcitonin gene-related peptide (CGRP) and brain derived neurotropic factor (BDNF) were determined using novel western blotting based technology. Glutamate and substance P (SP) was determined using commercial available methods. Several new isoforms were found for NGF, CGRP and BDNF in saliva. The isoform pattern showed significant variation in both expression and chemiluminescence levels between different collection methods. New sensitive methods to study pain related markers in saliva were developed in this study. Furthermore, we are first to demonstrate a correlation between the Glutamate concentration in stimulated whole saliva and blood. However, the fundamental conclusion drawn is the importance of consistency in the collection method.

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  • 6.
    Wåhlén, Karin
    et al.
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Olausson, Patrik
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Carlsson, Anders K
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Ghafouri, Nazdar
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Gerdle, Björn
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Ghafouri, Bijar
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Systemic alterations in plasma proteins from women with chronic widespread pain compared to healthy controls: a proteomic study2017Ingår i: Journal of Pain Research, E-ISSN 1178-7090, Vol. 10, s. 797-809Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Chronic widespread pain (CWP) is a complex pain condition that is difficult to treat. The prevalence of CWP approximates similar to 10% of the general population, with higher prevalence in women. Lack of understanding of molecular mechanisms has been a challenge for diagnosis and treatment of chronic pain. The aim of this study was to explore the systemic protein changes in CWP compared to those in healthy controls (CON). By applying 2-dimensional gel electrophoresis, we analyzed the protein pattern of plasma samples from women with CWP (n=16) and healthy women (n=23). The proteomic data were analyzed using multivariate statistical models, and altered proteins were identified using mass spectrometry. The proteome analysis was further validated by gel-free Western blot. Multivariate statistical data analysis of quantified proteins revealed 22 altered proteins in women with CWP, compared to CON group. Many of the identified proteins are previously known to be involved in different parts of the complement system and metabolic and inflammatory processes, e.g., complement factor B, vitamin D-binding protein, ceruloplasmin, transthyretin and alpha-2-HS-glycoprotein. These results indicate that important systemic protein differences exist between women with CWP and healthy women. Further, this study illustrates the potential use of proteomics to detect biomarkers that may provide new insights into the molecular mechanism(s) of chronic pain. However, further larger investigations are required in order to confirm these findings before it will be possible to identify proteins as potential pain biomarkers for clinical use.

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  • 7.
    Ghafouri, Bijar
    et al.
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Carlsson, Anders
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Holmberg, Sara
    Department Research and Dev, Sweden.
    Thelin, Anders
    Uppsala University, Sweden.
    Tagesson, Christer
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Hjärt- och Medicincentrum, Arbets- och miljömedicin.
    Biomarkers of lsystemic inflammation in farmers with musculoskeletal disorders; a plasma proteomic study2016Ingår i: BMC Musculoskeletal Disorders, E-ISSN 1471-2474, Vol. 17, nr 206Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Farmers have an increased risk for musculoskeletal disorders (MSD) such as osteoarthritis of the hip, low back pain, and neck and upper limb complaints. The underlying mechanisms are not fully understood. Workrelated exposures and inflammatory responses might be involved. Our objective was to identify plasma proteins that differentiated farmers with MSD from rural referents. Methods: Plasma samples from 13 farmers with MSD and rural referents were included in the investigation. Gel based proteomics was used for protein analysis and proteins that differed significantly between the groups were identified by mass spectrometry. Results: In total, 15 proteins differed significantly between the groups. The levels of leucine-rich alpha-2-glycoprotein, haptoglobin, complement factor B, serotransferrin, one isoform of kininogen, one isoform of alpha-1-antitrypsin, and two isoforms of hemopexin were higher in farmers with MSD than in referents. On the other hand, the levels of alpha-2-HS-glycoprotein, alpha-1B-glycoprotein, vitamin D-binding protein, apolipoprotein A1, antithrombin, one isoform of kininogen, and one isoform of alpha-1-antitrypsin were lower in farmers than in referents. Many of the identified proteins are known to be involved in inflammation. Conclusions: Farmers with MSD had altered plasma levels of protein biomarkers compared to the referents, indicating that farmers with MSD may be subject to a more systemic inflammation. It is possible that the identified differences of proteins may give clues to the biochemical changes occurring during the development and progression of MSD in farmers, and that one or several of these protein biomarkers might eventually be used to identify and prevent work-related MSD.

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  • 8.
    Hadrévi, Jenny
    et al.
    Umeå University, Sweden.
    Turkina, Maria V
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Carlsson, Anders
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Östergötlands Läns Landsting, Sinnescentrum, Smärt och rehabiliteringscentrum. Linköpings universitet, Medicinska fakulteten.
    Gerdle, Björn
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Linköpings universitet, Medicinska fakulteten. Östergötlands Läns Landsting, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Larsson, Britt
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Linköpings universitet, Medicinska fakulteten. Östergötlands Läns Landsting, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Hellström, Fredrik
    University of Gävle, Sweden.
    Ghafouri, Bijar
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Linköpings universitet, Medicinska fakulteten. Östergötlands Läns Landsting, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Myosin light chain and calcium regulating protein differences in chronic musculoskeletal neck and shoulder pain2016Ingår i: Journal of Integrated OMICS, ISSN 2182-0287, Vol. 6, nr 1, s. 1-8Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Proteomic screening analysis has detected myosin light chain (MLC) as a protein implied to be involved in chronic musculoskeletal neck and shoulder pain. Several analyses of MLC proteins have stated a difference in phosphorylation being the determining factor for protein activation hence altered contrability of the muscle in i.e. senescence. In continuation of a previous publication, this study is an attempt to analyze the different MLC isoforms by mass spectrometry and immune-analyses in myalgic and healthy trapezius muscle. In the present study no differences in phosphorylation level between the corresponding individual proteins were detected using LC-MSMS and immunoblotting; instead we assigned different isoforms of regulatory MLCs. To further elucidate the contrability: calcium (Ca2+) regulatory proteins, sarco(endo)plasmic reticulum Ca2+ ATPase 1 (SERCA-1) and calsequestrine (CSQ) were analyzed by western blot. The analysis revealed a significantly increased abundance of SERCA-1 protein in the myalgic muscle and a significantly increased abundance of CSQ in healthy muscle. Myalgic muscle contraction patterns have in previous studies shown to differ from healthy muscle which may be connected to the Ca2+ availability in the muscle. Here we present the proteomic characterization of differences in Ca2+ regulating proteins and particularly regulatory MLCs in trapezius muscle of women with chronic musculoskeletal neck and shoulder pain.

  • 9.
    Bäckryd, Emmanuel
    et al.
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Ghafouri, Bijar
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum. Region Östergötland, Hjärt- och Medicincentrum, Arbets- och miljömedicin.
    Carlsson, Anders K
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Olausson, Patrik
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Gerdle, Björn
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Multivariate proteomic analysis of the cerebrospinal fluid of patients with peripheral neuropathic pain and healthy controls: a hypothesis-generating pilot study2015Ingår i: Journal of Pain Research, E-ISSN 1178-7090, Vol. 8, s. 321-333Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Pain medicine lacks objective biomarkers to guide diagnosis and treatment. Combining two-dimensional gel proteomics with multivariate data analysis by projection, we exploratively analyzed the cerebrospinal fluid of eleven patients with severe peripheral neuropathic pain due to trauma and/or surgery refractory to conventional treatment and eleven healthy controls. Using orthogonal partial least squares discriminant analysis, we identified a panel of 36 proteins highly discriminating between the two groups. Due to a possible confounding effect of age, a new model with age as outcome variable was computed for patients (n=11), and four out of 36 protein spots were excluded due to a probable influence of age. Of the 32 remaining proteins, the following seven had the highest discriminatory power between the two groups: an isoform of angiotensinogen (upregulated in patients), two isoforms of alpha-1-antitrypsin (downregulated in patients), three isoforms of haptoglobin (upregulated in patients), and one isoform of pigment epithelium-derived factor (downregulated in patients). It has recently been hypothesized that the renin–angiotensin system may play a role in the pathophysiology of neuropathic pain, and a clinical trial of an angiotensin II receptor antagonist was recently published. It is noteworthy that when searching for neuropathic pain biomarkers with a purely explorative methodology, it was indeed a renin–angiotensin system protein that had the highest discriminatory power between patients and controls in the present study. The results from this hypothesis-generating pilot study have to be confirmed in larger, hypothesis-driven studies with age-matched controls, but the present study illustrates the fruitfulness of combining proteomics with multivariate data analysis in hypothesis-generating pain biomarker studies in humans.

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