Hsa-miR-605 regulates the proinflammatory chemokine CXCL5 in complex regional pain syndrome.

Complex regional pain syndrome (CRPS) is a chronic pain condition characterized by inflammation and debilitating pain. CRPS patients with pain refractory to more conventional analgesics can be treated with subanesthetic doses of ketamine. Our previous studies found that poor responders to ketamine had a 22-fold downregulation of the miRNA hsa-miR-605 in blood prior to ketamine treatment. Hence, we sought to investigate the functional significance of miR-605 downregulation and its impact on target gene expression, as investigating target mRNAs of differentially expressed miRNAs can provide important insights on aberrant gene expression that may contribute to disease etiology. Using a bioinformatics prediction, we identified that miR-605 can target the proinflammatory chemokine CXCL5, which plays a role in leukocyte recruitment and activation. We hypothesized that downregulation of miR-605 in poor responders to ketamine could increase CXCL5 expression and thereby contribute to inflammation in these patients. We confirmed that miR-605 regulates CXCL5 by using a miRNA mimic and inhibitor in human primary endothelial cells. Inhibition of miR-605 increased CXCL5 secretion and migration of human monocytic cells, thereby demonstrating a functional impact of miR-605 on chemotaxis. Additionally, CXCL5 mRNA was upregulated in whole blood from poor responders to ketamine, and CXCL5 protein was increased in plasma from CRPS patients. Thus, our studies suggest that miR-605 regulation of CXCL5 can regulate inflammation.

Serum Soluble Interleukin-2 Receptor Does Not Differentiate Complex Regional Pain Syndrome from Other Pain Conditions in a Tertiary Referral Setting.

Previously, we showed that serum soluble interleukin-2 receptor (sIL-2R) levels, a marker for T-cell activation, were higher in complex regional pain syndrome (CRPS) patients than in healthy controls, suggesting pathogenic T-cell activation in CRPS. Additionally, sIL-2R levels discriminated well between CRPS and healthy controls with a high sensitivity (90%) and specificity (89.5%), suggesting a possible role for sIL-2R in the diagnosis of CRPS. In order to further validate this marker in the diagnostic workup of CRPS, we conducted this prospective cohort study in which we determined sIL-2R levels in patients that were referred to our tertiary referral center with a suspicion of CRPS in a limb, and subsequently compared sIL-2R levels between the patients that were diagnosed with CRPS (CRPS group) and those who were not (no CRPS group). A group of anonymous blood bank donors were used as a healthy control group. Furthermore, we explored the relationship between sIL-2R and CRPS disease severity using the CRPS severity score. Median sIL-2R levels of both the CRPS group (2809.0 pg/ml; Q3-Q1: 3913.0-1589.0) and no CRPS group (3654.0 pg/ml; Q3-Q1: 4429.0-2095.5) were significantly higher than that of the control group (1515.0 pg/ml; Q3-Q1: 1880.0-1150.0): CRPS vs. controls, p < .001; no CRPS vs. controls, p < 0.001. Serum sIL-2R levels did not differ significantly between the CRPS and no CRPS group. A statistically significant negative correlation was observed between sIL-2R levels and the CRPS severity score (r s = -0.468, p = 0.024). Our results confirm our previous findings of higher sIL-2R levels in CRPS patients than in healthy controls. We further showed that serum sIL-2R cannot differentiate between CRPS and other pain conditions of a limb in a tertiary referral setting. Interestingly, a negative correlation was found between sIL-2R and CRPS disease severity; this finding warrants further research into the relationship between sIL-2R and CRPS disease severity.

Novel immune biomarkers in complex regional pain syndrome.

We investigated serum levels of 29 cytokines and immune-activated kynurenine and tetrahydrobiopterin pathway metabolites in 15 complex regional pain syndrome (CRPS) subjects and 14 healthy controls. Significant reductions in interleukin-37 and tryptophan were found in CRPS subjects, along with positive correlations between kynurenine/tryptophan ratio and TNF-α levels with kinesiophobia, tetrahydrobiopterin levels with McGill pain score, sRAGE, and xanthurenic acid and neopterin levels with depression, anxiety and stress scores. Using machine learning, we identified a set of binary variables, including IL-37 and GM-CSF, capable of distinguishing controls from established CRPS subjects. These results suggest possible involvement of various inflammatory markers in CRPS pathogenesis.

Commonalities and differences in abnormal peripheral metabolites between patients with fibromyalgia and complex regional pain syndrome.

Background: Fibromyalgia (FM) and complex regional pain syndrome (CRPS) share many pathological mechanisms related to chronic pain that could contribute to multifactorial pathological mechanisms.Methods: We investigated peripheral metabolites in FM and CRPS patients compared to healthy controls based on cross-sectional study.Results: Mean corpuscular hemoglobin (p < 0.001), mean corpuscular volume (p = 0.014), and total bilirubin levels (p = 0.017) were lower in FM patients than in healthy controls. On the other hand, CRPS patients showed lower levels of total bilirubin than healthy controls (p = 0.037). Creatinine level was lower in FM patients (p = 0.057) compared to healthy controls, particularly when comparing the low-hemoglobin subgroup among FM patients (p = 0.035) with the low-hemoglobin subgroup among healthy controls. Red blood cell count (r = -0.620, p = 0.031), hematocrit (r = -0.593, p = 0.042), and creatinine level (r = -0.598, p = 0.040) showed negative correlations with McGill Pain Questionnaire-Affective (MPQ-A) scores in FM patients. A negative correlation was observed between MCV and McGill Pain Questionnaire-Sensory scores (r = -0.680, p = 0.015) in CRPS patients.Conclusion: We found specific peripheral metabolites that may exhibit different tendency between FM and CRPS patients as well as some common metabolites, which may be associated with peripheral pathology in the patients. Considering this study had a few limitations such as a small sample sizes and using a liberal threshold of significance in the correlation analysis, future studies with larger sample sizes may be needed to generalize these findings.

Transfer of complex regional pain syndrome to mice via human autoantibodies is mediated by interleukin-1-induced mechanisms.

Neuroimmune interactions may contribute to severe pain and regional inflammatory and autonomic signs in complex regional pain syndrome (CRPS), a posttraumatic pain disorder. Here, we investigated peripheral and central immune mechanisms in a translational passive transfer trauma mouse model of CRPS. Small plantar skin-muscle incision was performed in female C57BL/6 mice treated daily with purified serum immunoglobulin G (IgG) from patients with longstanding CRPS or healthy volunteers followed by assessment of paw edema, hyperalgesia, inflammation, and central glial activation. CRPS IgG significantly increased and prolonged swelling and induced stable hyperalgesia of the incised paw compared with IgG from healthy controls. After a short-lasting paw inflammatory response in all groups, CRPS IgG-injected mice displayed sustained, profound microglia and astrocyte activation in the dorsal horn of the spinal cord and pain-related brain regions, indicating central sensitization. Genetic deletion of interleukin-1 (IL-1) using IL-1αß knockout (KO) mice and perioperative IL-1 receptor type 1 (IL-1R1) blockade with the drug anakinra, but not treatment with the glucocorticoid prednisolone, prevented these changes. Anakinra treatment also reversed the established sensitization phenotype when initiated 8 days after incision. Furthermore, with the generation of an IL-1ß floxed(fl/fl) mouse line, we demonstrated that CRPS IgG-induced changes are in part mediated by microglia-derived IL-1ß, suggesting that both peripheral and central inflammatory mechanisms contribute to the transferred disease phenotype. These results indicate that persistent CRPS is often contributed to by autoantibodies and highlight a potential therapeutic use for clinically licensed antagonists, such as anakinra, to prevent or treat CRPS via blocking IL-1 actions.

Unilateral L4-dorsal root ganglion stimulation evokes pain relief in chronic neuropathic postsurgical knee pain and changes of inflammatory markers: part II whole transcriptome profiling.

BACKGROUND: In our recent clinical trial, increased peripheral concentrations of pro-inflammatory molecular mediators were determined in complex regional pain syndrome (CRPS) patients. After 3 months adjunctive unilateral, selective L4 dorsal root ganglion stimulation (L4-DRGSTIM), significantly decreased serum IL-10 and increased saliva oxytocin levels were assessed along with an improved pain and functional state. The current study extended molecular profiling towards gene expression analysis of genes known to be involved in the gonadotropin releasing hormone receptor and neuroinflammatory (cytokines/chemokines) signaling pathways. METHODS: Blood samples were collected from 12 CRPS patients for whole-transcriptome profiling in order to assay 18,845 inflammation-associated genes from frozen blood at baseline and after 3 months L4-DRGSTIM using PANTHER™ pathway enrichment analysis tool. RESULTS: Pathway enrichment analyses tools (GOrilla™ and PANTHER™) showed predominant involvement of inflammation mediated by chemokines/cytokines and gonadotropin releasing hormone receptor pathways. Further, screening of differentially regulated genes showed changes in innate immune response related genes. Transcriptomic analysis showed that 21 genes (predominantly immunoinflammatory) were significantly changed after L4-DRGSTIM. Seven genes including TLR1, FFAR2, IL1RAP, ILRN, C5, PKB and IL18 were down regulated and fourteen genes including CXCL2, CCL11, IL36G, CRP, SCGB1A1, IL-17F, TNFRSF4, PLA2G2A, CREB3L3, ADAMTS12, IL1F10, NOX1, CHIA and BDKRB1 were upregulated. CONCLUSIONS: In our sub-group analysis of L4-DRGSTIM treated CRPS patients, we found either upregulated or downregulated genes involved in immunoinflammatory circuits relevant for the pathophysiology of CRPS indicating a possible relation. However, large biobank-based approaches are recommended to establish genetic phenotyping as a quantitative outcome measure in CRPS patients. Trial registration The study protocol was registered at the 15.11.2016 on German Register for Clinical Trials (DRKS ID 00011267). https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00011267.

What is normal trauma healing and what is complex regional pain syndrome I? An analysis of clinical and experimental biomarkers.

Complex regional pain syndrome (CRPS) typically develops after fracture or trauma. Many of the studies so far have analyzed clinical and molecular markers of CRPS in comparison with healthy or pain controls. This approach, however, neglects mechanisms occurring during physiological trauma recovery. Therefore, we compared the clinical phenotype, sensory profiles, patient-reported outcomes, and exosomal immunobarrier microRNAs (miRs) regulating barrier function and immune response between CRPS and fracture controls (FCs) not fulfilling the CRPS diagnostic criteria. We included upper-extremity FCs, acute CRPS I patients within 1 year after trauma, a second disease control group (painful diabetic polyneuropathy), and healthy controls. Fracture controls were not symptoms-free, but reported some pain, disability, anxiety, and cold pain hyperalgesia in quantitative sensory testing. Patients with CRPS had higher scores for pain, disability, and all patient-reported outcomes. In quantitative sensory testing, ipsilateral and contralateral sides differed significantly. However, on the affected side, patients with CRPS were more sensitive in only 3 parameters (pinprick pain and blunt pressure) when compared to FCs. Two principal components were identified in the cohort: pain and psychological parameters distinguishing FC and CPRS. Furthermore, the immunobarrier-protective hsa-miR-223-5p was increased in plasma exosomes in FCs with normal healing, but not in CRPS and healthy controls. Low hsa-miR-223-5p was particularly observed in subjects with edema pointing towards barrier breakdown. In summary, normal trauma healing includes some CRPS signs and symptoms. It is the combination of different factors that distinguish CRPS and FC. Fracture control as a control group can assist to discover resolution factors after trauma.

Exosome microRNA signatures in patients with complex regional pain syndrome undergoing plasma exchange.

BACKGROUND: Therapeutic plasma exchange (PE) or plasmapheresis is an extracorporeal procedure employed to treat immunological disorders. Exosomes, nanosized vesicles of endosomal origin, mediate intercellular communication by transferring cargo proteins and nucleic acids and regulate many pathophysiological processes. Exosomal miRNAs are potential biomarkers due to their stability and dysregulation in diseases including complex regional pain syndrome (CRPS), a chronic pain disorder with persistent inflammation. A previous study showed that a subset of CRPS patients responded to PE. METHODS: As a proof-of-concept, we investigated the PE-induced exosomal miRNA changes in six CRPS patients. Plasma cytokine levels were measured by HPLC and correlated with miRNA expression. Luciferase assay following co-transfection of HEK293 cells with target 3'UTR constructs and miRNA mimics was used to evaluate miRNA mediated gene regulation of target mRNA. Transient transfection of THP-1 cells with miRNA mimics followed by estimation of target gene and protein expression was used to validate the findings. RESULTS: Comparison of miRNAs in exosomes from the serum of three responders and three poor-responders showed that 17 miRNAs differed significantly before and after therapy. Of these, poor responders had lower exosomal hsa-miR-338-5p. We show that miR-338-5p can bind to the interleukin 6 (IL-6) 3' untranslated region and can regulate IL-6 mRNA and protein levels in vitro. PE resulted in a significant reduction of IL-6 in CRPS patients. CONCLUSIONS: We propose that lower pretreatment levels of miR-338-5p in poor responders are linked to IL-6 levels and inflammation in CRPS. Our data suggests the feasibility of exploring exosomal miRNAs as a strategy in patient stratification for maximizing therapeutic outcome of PE.

The serum protease network-one key to understand complex regional pain syndrome pathophysiology.

Complex regional pain syndrome (CRPS) develops after fracture. The acute CRPS phenotype resembles exaggerated inflammation, which is explained by local and systemic activation of a proinflammatory network including peptides and cytokines. Epidemiologic data suggest that inactivation of the peptidase angiotensin-converting enzyme in patients treated for hypertension increases the odds to develop CRPS. This hint leads us to investigate the serum protease network activity in patients with CRPS vs respective controls. For this purpose, we developed a dabsyl-bradykinin (DBK)-based assay and used it to investigate patients with CRPS, as well as healthy and pain (painful diabetic neuropathy [dPNP]) controls. The major result is that the degradation of DBK to fragments 1-8 and 1-5 in healthy control and dPNP is shifted to higher values for DBK1-8 and lower values for DBK1-5 at 1 hour of incubation in patients with CRPS. Using this novel reporter peptide assay, we have been able to show that the resolving protease network for mediators such as BK might be different in patients with CRPS; having a look at the clinical signs, which resemble inflammation, this resolving protease network is probably less effective in CRPS.

Altered regulation of the T-cell system in patients with CRPS.

The aim of this study was to investigate T-cell subsets and immunomodulatory factors in patients with complex regional pain syndrome (CRPS). We found decreased numbers of pro-inflammatory Th17 cells in patients with CRPS as compared to healthy volunteers. The expression of Th17 related RORγT mRNA was also significantly decreased. Patients with CRPS showed an increased proportion of CD39+ Tregs. CD39 is a known inhibitor of Th17 cell differentiation. Systemic cytokine levels were almost unchanged in patients with CRPS. These findings suggest that the decrease in Th17 cells in CRPS is regulated by an increase in CD39+ Tregs and that this anti-inflammatory T-cell shift may be a mechanism to control inflammation in CRPS. GERMAN CLINICAL TRIAL REGISTER: Registration Trial DRKS00005954.

Elevated Plasma Levels of sIL-2R in Complex Regional Pain Syndrome: A Pathogenic Role for T-Lymphocytes?

The immune system has long been thought to be involved in the pathophysiology of complex regional pain syndrome (CRPS). However, not much is known about the role of the immune system and specifically T-cells in the onset and maintenance of this disease. In this study, we aimed to evaluate T-cell activity in CRPS by comparing blood soluble interleukin-2 receptor (sIL-2R) levels between CRPS patients and healthy controls. CRPS patients had statistically significant elevated levels of sIL-2R as compared to healthy controls (median sIL-2R levels: 4151 pg/ml (Q3 - Q1 = 5731 pg/ml - 3546 pg/ml) versus 1907 pg/ml (Q3 - Q1: 2206 pg/ml - 1374 pg/ml), p < 0.001, resp.). Furthermore, sIL-2R level seems to be a good discriminator between CRPS patients and healthy controls with a high sensitivity (90%) and specificity (89.5%). Our finding indicates increased T-cell activity in patients with CRPS. This finding is of considerable relevance as it could point towards a T-cell-mediated inflammatory process in this disease. This could pave the way for new anti-inflammatory therapies in the treatment of CRPS. Furthermore, sIL-2R could be a promising new marker for determining inflammatory disease activity in CRPS.

Identification of KRT16 as a target of an autoantibody response in complex regional pain syndrome.

OBJECTIVE: Using a mouse model of complex regional pain syndrome (CRPS), our goal was to identify autoantigens in the skin of the affected limb. METHODS: A CRPS-like state was induced using the tibia fracture/cast immobilization model. Three weeks after fracture, hindpaw skin was homogenized, run on 2-d gels, and probed by sera from fracture and control mice. Spots of interest were analyzed by liquid chromatography-mass spectroscopy (LC-MS) and the list of targets validated by examining their abundance and subcellular localization. In order to measure the autoantigenicity of selected protein targets, we quantified the binding of IgM in control and fracture mice sera, as well as in control and CRPS human sera, to the recombinant protein. RESULTS: We show unique binding between fracture skin extracts and fracture sera, suggesting the presence of auto-antigens. LC-MS analysis provided us a list of potential targets, some of which were upregulated after fracture (KRT16, eEF1a1, and PRPH), while others showed subcellular-redistribution and increased membrane localization (ANXA2 and ENO3). No changes in protein citrullination or carbamylation were observed. In addition to increased abundance, KRT16 demonstrated autoantigenicity, since sera from both fracture mice and CRPS patients showed increased autoantibody binding to recombinant kRT16 protein. CONCLUSIONS: Pursuing autoimmune contributions to CRPS provides a novel approach to understanding the condition and may allow the development of mechanism-based therapies. The identification of autoantibodies against KRT16 as a biomarker in mice and in humans is a critical step towards these goals, and towards redefining CRPS as having an autoimmune etiology.

Hsa-miR-34a mediated repression of corticotrophin releasing hormone receptor 1 regulates pro-opiomelanocortin expression in patients with complex regional pain syndrome.

BACKGROUND: Ketamine provides relief for a subset of patients with complex regional pain syndrome (CRPS). The poor responders had a lower body mass index (BMI) relative to responders. Regulation of proopiomelanocortin (POMC) expression is crucial in normal body weight homeostasis. The main objectives of this study were to investigate the mechanisms underlying lower BMI characterizing CRPS patients responding poorly to intravenous ketamine therapy and identify potential biomarkers for predicting response. METHODS: We investigated POMC transcript levels in blood from CRPS patients grouped as responders and poor responders to ketamine therapy. Plasma levels of ß-endorphin, ACTH and α-MSH were measured by ELISA. We previously identified differential expression of small noncoding microRNA hsa-miR-34a in blood between responders and poor responders. We investigated whether a 11-fold downregulation of hsa-miR-34a in poor responders relative to responders is contributing to the differences in POMC levels by targeting POMC regulator CRHR1. Binding of miR-34a to CRHR1 was assessed using reporter assay; changes in mRNA and protein levels of CRHR1 were used to determine the regulation of CRHR1 by miR-34a. RNA from blood of CRPS and control subjects were used for quantitative PCR for CRHR1. RESULTS: Though ketamine treatment did not alter POMC expression, poor responders had higher levels of POMC mRNA than responders, both before and after treatment. Corticotropin-releasing hormone (CRH) is a key regulator of POMC expression and the biological effects are mediated through its receptor corticotrophin releasing hormone receptor 1 (CRHR1). We show that hsa-miR-34a is a negative regulator of CRHR1; overexpression of hsa-miR-34a in Jurkat cells resulted in reduction of CRH-mediated POMC expression. Poor responders had higher expression of CRHR1 transcripts than responders, indicating a regulatory role for miR-34a. In addition, we found positive correlations between the pretreatment levels of miR-34a to BMI and response to ketamine therapy. CONCLUSIONS: Our findings indicate a mechanism by which hsa-miR-34a can regulate the CRH/CRHR1/POMC axis and may influence BMI. Studies in larger patient cohorts are required to confirm the biomarker utility of circulating hsa-miR-34a levels in predicting treatment response to ketamine therapy.

Identification and Treatment of New Inflammatory Triggers for Complex Regional Pain Syndrome: Small Intestinal Bacterial Overgrowth and Obstructive Sleep Apnea.

Complex regional pain syndrome (CRPS) is evoked by conditions that may be associated with local and/or systemic inflammation. We present a case of long-standing CRPS in a patient with Ehlers-Danlos syndrome in which prolonged remission was attained by directing therapy toward concomitant small intestinal bacterial overgrowth, obstructive sleep apnea, and potential increased microglia activity. We theorize that cytokine production produced by small intestinal bacterial overgrowth and obstructive sleep apnea may act as stimuli for ongoing CRPS symptoms. CRPS may also benefit from the properties of low-dose naltrexone that blocks microglia Toll-like receptors and induces production of endorphins that regulate and reduce inflammation.

Effects of serum immunoglobulins from patients with complex regional pain syndrome (CRPS) on depolarisation-induced calcium transients in isolated dorsal root ganglion (DRG) neurons.

Complex regional pain syndrome (CRPS) is thought to have an auto-immune component. One such target recently proposed from the effects of auto-immune IgGs on Ca(2+) transients in cardiac myocytes and cell lines is the α1-adrenoceptor. We have tested whether such IgGs exerted comparable effects on nociceptive sensory neurons isolated from rat dorsal root ganglia. Depolarisation-induced [Ca(2+)]i transients were generated by applying 30 mM KCl for 2 min and monitored by Fura-2 fluorescence imaging. No IgGs tested (including 3 from CRPS patients) had any significant effect on these [Ca(2+)]i transients. However, IgG from one CRPS patient consistently and significantly reduced the K(+)-induced response of cells that had been pre-incubated for 24h with a mixture of inflammatory mediators (1 µM histamine, 5-hydroxytryptamine, bradykinin and PGE2). Since this pre-incubation also appeared to induce a comparable inhibitory response to the α1-agonist phenylephrine, this is compatible with the α1-adrenoceptor as a target for CRPS auto-immunity. A mechanism whereby this might enhance pain is suggested.

Anti-cytokine autoantibodies in postherpetic neuralgia.

BACKGROUND: The mechanisms by which varicella zoster virus (VZV) reactivation causes postherpetic neuralgia (PHN), a debilitating chronic pain condition, have not been fully elucidated. Based on previous studies identifying a causative role for anti-cytokine autoantibodies in patients with opportunistic infections, we explored this possibility in PHN. METHODS: Sera from herpes zoster (HZ) patients without and with PHN (N = 115 and 83, respectively) were examined for the presence of autoantibodies against multiple cytokines, and other known autoantigens. In addition, a cohort of patients with complex regional pain syndrome or neuropathic pain was tested for autoantibodies against selected cytokines. Antibody levels against VZV, Epstein Barr virus, and herpes simplex virus-2 were also measured in the HZ and PHN patients. Patient sera with high levels of anti-cytokine autoantibodies were functionally tested for in vitro neutralizing activity. RESULTS: Six PHN subjects demonstrated markedly elevated levels of single, autoantibodies against interferon-α, interferon-γ, GM-CSF, or interleukin-6. In contrast, the HZ and the pain control group showed low or no autoantibodies, respectively, against these four cytokines. Further analysis revealed that one PHN patient with high levels of anti-interleukin-6 autoantibodies had a markedly depressed antibody level to VZV, potentially reflecting poor T cell immunity against VZV. In vitro functional testing revealed that three of the five anti-cytokine autoantibody positive PHN subjects had neutralizing autoantibodies against interferon-α, GM-CSF or interleukin-6. In contrast, none of the HZ patients without PHN had neutralizing autoantibodies. CONCLUSIONS: These results suggest the possibility that sporadic anti-cytokine autoantibodies in some subjects may cause an autoimmune immunodeficiency syndrome leading to uncontrolled VZV reactivation, nerve damage and subsequent PHN.

Analgesic Response to Intravenous Ketamine Is Linked to a Circulating microRNA Signature in Female Patients With Complex Regional Pain Syndrome.

Although ketamine is beneficial in treating complex regional pain syndrome (CRPS), a subset of patients respond poorly to therapy. We investigated treatment-induced microRNA (miRNA) changes and their predictive validity in determining treatment outcome by assessing miRNA changes in whole blood from patients with CRPS. Blood samples from female patients were collected before and after 5 days of intravenous ketamine administration. Seven patients were responders and 6 were poor responders. Differential miRNA expression was observed in whole blood before and after treatment. In addition, 33 miRNAs differed between responders and poor responders before therapy, suggesting the predictive utility of miRNAs as biomarkers. Investigation of the mechanistic significance of hsa-miR-548d-5p downregulation in poor responders showed that this miRNA can downregulate UDP-glucuronosyltransferase UGT1A1 mRNA. Poor responders had a higher conjugated/unconjugated bilirubin ratio, indicating increased UGT1A1 activity. We propose that lower pretreatment levels of miR-548d-5p may result in higher UDP-GT activity, leading to higher levels of inactive glucuronide conjugates, thereby minimizing the therapeutic efficacy of ketamine in poor responders. Differences in miRNA signatures can provide molecular insights distinguishing responders from poor responders. Extending this approach to other treatment and outcome assessments might permit stratification of patients for maximal therapeutic outcome. Perspective: This study suggests the usefulness of circulating miRNAs as potential biomarkers. Assessing miRNA signatures before and after treatment demonstrated miRNA alterations from therapy; differences in miRNA signature in responders and poor responders before therapy indicate prognostic value. Mechanistic studies on altered miRNAs can provide new insights into disease.

The prevalence of autoantibodies in complex regional pain syndrome type I.

Autoimmunity has been suggested as one of the pathophysiologic mechanisms that may underlie complex regional pain syndrome (CRPS). Screening for antinuclear antibodies (ANA) is one of the diagnostic tests, which is usually performed if a person is suspected to have a systemic autoimmune disease. Antineuronal antibodies are autoantibodies directed against antigens in the central and/or peripheral nervous system. The aim of this study was to compare the prevalence of these antibodies in CRPS patients with the normal values of those antibodies in the healthy population. Twenty seven (33%) of the 82 CRPS patients of whom serum was available showed a positive ANA test. This prevalence is significantly higher than in the general population. Six patients (7.3%) showed a positive result for typical antineuronal antibodies. This proportion, however, does not deviate from that in the general population. Our findings suggest that autoantibodies may be associated with the pathophysiology of CRPS, at least in a subset of patients. Further research is needed into defining this subset and into the role of autoantibodies in the pathogenesis of CRPS.

Longstanding complex regional pain syndrome is associated with activating autoantibodies against alpha-1a adrenoceptors.

Complex regional pain syndrome (CRPS) is a limb-confined posttraumatic pain syndrome with sympathetic features. The cause is unknown, but the results of a randomized crossover trial on low-dose intravenous immunoglobulins (IVIG) treatment point to a possible autoimmune mechanism. We tested purified serum immunoglobulin G (IgG) from patients with longstanding CRPS for evidence of antibodies interacting with autonomic receptors on adult primary cardiomyocytes, comparing with control IgG from healthy and diseased controls, and related the results to the clinical response to treatment with low-dose IVIG. We simultaneously recorded both single-cell contractions and intracellular calcium handling in an electrical field. Ten of 18 CRPS preparations and only 1/57 control preparations (P<0.0001) increased the sensitivity of the myocytes to the electric field, and this effect was abrogated by preincubation with α-1a receptor blockers. By contrast, effects on baseline calcium were blocked by preincubation with atropine. Interestingly, serum-IgG preparations from all 4 CRPS patients who had responded to low-dose IVIG with meaningful pain relief were effective in these assays, although 4/8 of the nonresponders were also active. To see if there were antibodies to the α-1a receptor, CRPS-IgG was applied to α-1a receptor-transfected rat-1 fibroblast cells. The CRPS serum IgG induced calcium flux, and fluorescence-activated cell sorting showed that there was serum IgG binding to the cells. The results suggest that patients with longstanding CRPS have serum antibodies to α-1a receptors, and that measurement of these antibodies may be useful in the diagnosis and management of the patients.

Osteoprotegerin: a new biomarker for impaired bone metabolism in complex regional pain syndrome?

Osteoprotegerin (OPG) is important for bone remodeling and may contribute to complex regional pain syndrome (CRPS) pathophysiology. We aimed to assess the value of OPG as a biomarker for CRPS and a possible correlation with radiotracer uptake in 3-phase bone scintigraphy (TPBS). OPG levels were analyzed in 23 CRPS patients (17 women; mean age 50±9.0 years; disease duration: 12 weeks [IQR 8-24]), 10 controls (6 women; mean age 58±9.6 years) and 21 patients after uncomplicated fractures (12 women; mean age: 43±15 years; time after fracture: 15 weeks [IQR: 6-22]). The CRPS and control patients also underwent TPBS. OPG in CRPS patients was significantly increased by comparison with both control groups (P=0.001; Kruskal-Wallis test; CRPS patients: 74.1 pg/mL [IQR: 47.1-100.7]; controls: 46.7 pg/mL [IQR: 35.5-55.0]; P=0.004; fracture patients: 45.9 pg/mL [IQR: 37.5-56.7]; P=0.001). As a diagnostic test for CRPS, OPG had a sensitivity of 0.74, specificity of 0.80, positive predictive value of 68% and negative predictive value of 84%. Receiver operating characteristic curve analysis showed an area under the curve of 0.80 (CI: 0.68-0.91). For the CRPS-affected hand, a significant correlation between OPG and TPBS region of interest analysis in phase III was detected (carpal bones; r=0.391; P=0.03). The persistent OPG increase in CRPS indicates enhanced osteoblastic activity shown by increased radiotracer uptake in TPBS phase III. A contribution of bone turnover to CRPS pathophysiology is likely. OPG might be useful as a biomarker for CRPS.