Post-fracture serum cytokine levels are not associated with a later diagnosis of complex regional pain syndrome: a case-control study nested in a prospective cohort study.

BACKGROUND: Complex Regional Pain Syndrome (CRPS) is a disabling pain disorder that is most common after a distal limb fracture. While the acute systemic immune response to the injury is thought to play a role in the development of CRPS, this hypothesis has never been tested directly. Thus, we evaluated whether elevated levels of circulating pro-inflammatory cytokines early after a fracture were associated with the development of CRPS. METHODS: We conducted a case-control study nested within a prospective cohort study. Individuals with wrist and/or hand fractures were recruited from specialist hand units. Baseline clinical data were obtained from participants within 28 days of fracture. CRPS status was determined 16 weeks after the fracture using a two-stage diagnostic process. Cytokine assays were obtained from all cases (defined using the Budapest criteria) and a random sample of those who did not have CRPS at 16 weeks. We calculated odds ratios with 95% confidence intervals to determine the risk of CRPS associated with the expression of each of 25 cytokines. RESULTS: Baseline data were collected for 702 consenting participants, of whom 535 provided blood samples. Follow-up at 16 weeks was 97.2%. 15 (2.2% of the cohort) met the Budapest CRPS criteria and 69 (including those who met the Budapest criteria; 9.8%) met the International Association for the Study of Pain (IASP) CRPS criteria. In all of the primary analyses (using Budapest criteria) and 49/50 secondary analyses (using IASP criteria), 95% confidence intervals for the association between cytokine levels and the risk of subsequently developing CRPS included the null value (OR = 1). However, the confidence intervals were wide. CONCLUSION: There was no evidence that early post-injury expression of systemic cytokines was associated with a CRPS diagnosis 16 weeks after injury. This study does not provide support for the hypothesis that innate immune activation has a determinative role in the development of CRPS.

Evidence for abnormal cytokine expression in Gulf War Illness: A preliminary analysis of daily immune monitoring data.

BACKGROUND: Gulf War Illness (GWI) is a clinically heterogeneous chronic condition that affects many veterans of the 1990-1991 Persian Gulf War. One of the most prevalent and debilitating symptoms of GWI is abnormal fatigue. The mechanisms underlying GWI generally, and fatigue symptoms specifically, have yet to be conclusively identified, although immune system abnormalities are suspected to be involved. The first goal of this immune monitoring study was to determine if GWI is associated with higher absolute levels and daily variability of pro-inflammatory immune factors. The second goal was to explore the relationship between day-to-day immune marker fluctuations and daily self-reported fatigue severity. METHODS: We recruited veterans with GWI and healthy veteran control (HV) participants to provide self-reported fatigue severity data and blood samples, over 25 consecutive days. We profiled inflammatory processes by using a longitudinal, daily immune-monitoring approach. For each day, serum cytokine and chemokine concentrations were determined using multiplex assays. RESULTS: Seven veterans with GWI and eight healthy veteran control (HV) participants completed the study protocol. We found that GWI was associated with higher variability in the expression of eotaxin-1 (p < 0.001). For GWI participants, higher fatigue severity days were associated with greater IL-1ß (p = 0.008) and IL-15 (p < 0.001). CONCLUSIONS: Our findings provide preliminary evidence that the immune system is involved in the pathophysiology of GWI. Longitudinal immune profiling approaches may be helpful in discovering targets for novel therapies in conditions such as GWI.

MeCP2 Interacts with the Super Elongation Complex to Regulate Transcription.

Loss-of-function mutations in methyl-CpG binding protein 2 ( MECP2 ) cause Rett syndrome, a postnatal neurodevelopmental disorder that occurs in ∼1/10,000 live female births. MeCP2 binds to methylated cytosines across genomic DNA and recruits various partners to regulate gene expression. MeCP2 has been shown to repress transcription in vitro and interacts with co-repressors such as the Sin3A and NCoR complexes. Based on these observations, MeCP2 has been largely considered as a repressor of transcription. However, a mouse model of RTT displays many down-regulated genes, and those same genes are up-regulated in a MECP2 duplication mouse model. Furthermore, TCF20, which has been associated with transcriptional activation, have recently been identified as a protein interactor of MeCP2. These data broaden the potential functions of MeCP2 as a regulator of gene expression. Yet, the molecular mechanisms underlying MeCP2-dependent gene regulation remain largely unknown. Here, using a human MECP2 gain-of-function Drosophila model, we screened for genetic modifiers of MECP2 -induced phenotypes. Our approach identified several subunits of the Drosophila super elongation complex, a P-TEFb containing RNA polymerase II (RNA pol II) elongation factor required for the release of promoter-proximally paused RNA pol II, as genetic interactors of MECP2 . We discovered that MeCP2 physically interacts with the SEC in human cells and in the mouse brain. Furthermore, we found that MeCP2 directly binds AFF4, the scaffold of the SEC, via the transcriptional repression domain. Finally, loss of MeCP2 in the mouse cortex caused reduced binding of AFF4 specifically on a subset of genes involved in the regulation of synaptic function, which also displayed the strongest decrease in RNA pol II binding in the genebody. Taken together, our study reveals a previously unrecognized mechanism through which MeCP2 regulates transcription, providing a new dimension to its regulatory role in gene expression.

Multiplex cytokine concentration measurement: how much do the medium and handling matter?

Cytokine concentrations are thought to be affected by methods of sampling and processing and by storage conditions. In this study we compared 17 cytokine concentrations obtained from plasma and serum at baseline and after a controlled thaw condition. We found that absolute agreement was poor between concentrations of cytokines in plasma and serum, except for MIP1 ß . A thaw condition significantly changed the concentrations of most cytokines, but serum appeared less affected by this than plasma was. Closer examination using Bland-Altman analyses revealed that for each comparison, agreement was moderately good for many cytokine concentrations. This is important because measures of agreement must be interpreted based on the required precision, which may differ between clinical and research demands. We also identified that for some cytokines, the relationship between serum and plasma is affected by concentration, thus advocating for the use of appropriate methods when performing such comparisons in studies such as systematic reviews and meta-analyses.

Altered response to Toll-like receptor 4 activation in fibromyalgia: A low-dose, human experimental endotoxemia pilot study.

In this pilot study, a human intravenous injection of low-dose endotoxin (lipopolysaccharide, LPS) model was used to test if fibromyalgia is associated with altered immune responses to Toll-like receptor 4 (TLR4) activation. Eight women with moderately-severe fibromyalgia and eight healthy women were administered LPS at 0.1 ng/kg in session one and 0.4 ng/kg in session two. Blood draws were collected hourly to characterize the immune response. The primary analytes of interest, leptin and fractalkine, were assayed via commercial radioimmunoassay and enzyme-linked immunosorbent assay kits, respectively. Exploratory analyses were performed on 20 secreted cytokine assays by multiplex cytokine panels, collected hourly. Exploratory analyses were also performed on testosterone, estrogen, and cortisol levels, collected hourly. Additionally, standard clinical complete blood counts with differential (CBC-D) were collected before LPS administration and at the end of the session. The fibromyalgia group demonstrated enhanced leptin and suppressed fractalkine responses to LPS administration. In the exploratory analyses, the fibromyalgia group showed a lower release of IFN-γ, CXCL10, IL-17A, and IL-12 and higher release of IL-15, TARC, MDC, and eotaxin than the healthy group. The results of this study suggest that fibromyalgia may involve an altered immune response to TLR4 activation.

Loss of the batten disease protein CLN3 leads to mis-trafficking of M6PR and defective autophagic-lysosomal reformation.

Batten disease, one of the most devastating types of neurodegenerative lysosomal storage disorders, is caused by mutations in CLN3. Here, we show that CLN3 is a vesicular trafficking hub connecting the Golgi and lysosome compartments. Proteomic analysis reveals that CLN3 interacts with several endo-lysosomal trafficking proteins, including the cation-independent mannose 6 phosphate receptor (CI-M6PR), which coordinates the targeting of lysosomal enzymes to lysosomes. CLN3 depletion results in mis-trafficking of CI-M6PR, mis-sorting of lysosomal enzymes, and defective autophagic lysosomal reformation. Conversely, CLN3 overexpression promotes the formation of multiple lysosomal tubules, which are autophagy and CI-M6PR-dependent, generating newly formed proto-lysosomes. Together, our findings reveal that CLN3 functions as a link between the M6P-dependent trafficking of lysosomal enzymes and lysosomal reformation pathway, explaining the global impairment of lysosomal function in Batten disease.

Longitudinal tracking of human plasma oxytocin suggests complex responses to moral elevation.

Positive social experiences may induce oxytocin release. However, previous studies of moral elevation have generally utilized cross-sectional and simple modeling approaches to establish the relationship between oxytocin and emotional stimuli. Utilizing a cohort of 30 non-lactating women (aged 23.6 ± 5.7 years), we tested whether exposure to a video identified as capable of eliciting moral elevation could change plasma oxytocin levels. Uniquely, we utilized a high-frequency longitudinal sampling approach and multilevel growth curve modeling with landmark registration to test physiological responses. The moral elevation stimulus, versus a control video, elicited significantly greater reports of being "touched/inspired" and "happy/joyful". However, the measured plasma oxytocin response was found to be markedly heterogeneous. While the moral elevation stimulus elicited increased plasma oxytocin as expected, this increase was only modestly larger than that seen following the control video. This increase was also only present in some individuals. We found no relationship between plasma oxytocin and self-report responses to the stimulus. From these data, we argue that future studies of the relationship between oxytocin and emotion need to anticipate heterogeneous responses and thus incorporate comprehensive individual psychological data; these should include evidence-based variables known to be associated with oxytocin such as a history of trauma, and the individual's psychological and emotional state at the time of testing. Given the complexity of physiological oxytocin release, such studies also need to incorporate frequent biological sampling to properly examine the dynamics of hormonal release and response.

Looking on the horizon; potential and unique approaches to developing radiation countermeasures for deep space travel.

Future lunar missions and beyond will require new and innovative approaches to radiation countermeasures. The Translational Research Institute for Space Health (TRISH) is focused on identifying and supporting unique approaches to reduce risks to human health and performance on future missions beyond low Earth orbit. This paper will describe three funded and complementary avenues for reducing the risk to humans from radiation exposure experienced in deep space. The first focus is on identifying new therapeutic targets to reduce the damaging effects of radiation by focusing on high throughput genetic screens in accessible, sometimes called lower, organism models. The second focus is to design innovative approaches for countermeasure development with special attention to nucleotide-based methodologies that may constitute a more agile way to design therapeutics. The final focus is to develop new and innovative ways to test radiation countermeasures in a human model system. While animal studies continue to be beneficial in the study of space radiation, they can have imperfect translation to humans. The use of three-dimensional (3D) complex in vitro models is a promising approach to aid the development of new countermeasures and personalized assessments of radiation risks. These three distinct and unique approaches complement traditional space radiation efforts and should provide future space explorers with more options to safeguard their short and long-term health.

Preclinic group education sessions reduce waiting times and costs at public pain medicine units.

OBJECTIVE: To assess the effects of preclinic group education sessions and system redesign on tertiary pain medicine units and patient outcomes. DESIGN: Prospective cohort study. SETTING: Two public hospital multidisciplinary pain medicine units. PATIENTS: People with persistent pain. INTERVENTIONS: A system redesign from a "traditional" model (initial individual medical appointments) to a model that delivers group education sessions prior to individual appointments. Based on Patient Triage Questionnaires patients were scheduled to attend Self-Training Educative Pain Sessions (STEPS), a two day eight hour group education program, followed by optional patient-initiated clinic appointments. OUTCOME MEASURES: Number of patients completing STEPS who subsequently requested individual outpatient clinic appointment(s); wait-times; unit cost per new patient referred; recurrent health care utilization; patient satisfaction; Global Perceived Impression of Change (GPIC); and utilized pain management strategies. RESULTS: Following STEPS 48% of attendees requested individual outpatient appointments. Wait times reduced from 105.6 to 16.1 weeks at one pain unit and 37.3 to 15.2 weeks at the second. Unit cost per new patient appointed reduced from $1,805 Australian Dollars (AUD) to AUD$541 (for STEPS). At 3 months, patients scored their satisfaction with "the treatment received for their pain" more positively than at baseline (change score=0.88; P=0.0003), GPIC improved (change score=0.46; P<0.0001) and mean number of active strategies utilized increased by 4.12 per patient (P=0.0004). CONCLUSIONS: The introduction of STEPS was associated with reduced wait-times and costs at public pain medicine units and increased both the use of active pain management strategies and patient satisfaction.

Glial PAMPering and DAMPening of Adult Hippocampal Neurogenesis.

Adult neurogenesis represents a mature brain's capacity to integrate newly generated neurons into functional circuits. Impairment of neurogenesis contributes to the pathophysiology of various mood and cognitive disorders such as depression and Alzheimer's Disease. The hippocampal neurogenic niche hosts neural progenitors, glia, and vasculature, which all respond to intrinsic and environmental cues, helping determine their current state and ultimate fate. In this article we focus on the major immune communication pathways and mechanisms through which glial cells sense, interact with, and modulate the neurogenic niche. We pay particular attention to those related to the sensing of and response to innate immune danger signals. Receptors for danger signals were first discovered as a critical component of the innate immune system response to pathogens but are now also recognized to play a crucial role in modulating non-pathogenic sterile inflammation. In the neurogenic niche, viable, stressed, apoptotic, and dying cells can activate danger responses in neuroimmune cells, resulting in neuroprotection or neurotoxicity. Through these mechanisms glial cells can influence hippocampal stem cell fate, survival, neuronal maturation, and integration. Depending on the context, such responses may be appropriate and on-target, as in the case of learning-associated synaptic pruning, or excessive and off-target, as in neurodegenerative disorders.

Mouse Models for Studying Hippocampal Adult Neural Stem Cell Biology.

The mammalian hippocampus shows a remarkable capacity for continued neurogenesis throughout life. Newborn neurons, generated by the radial neural stem cells (NSCs), are important for learning and memory as well as mood control. During aging, the number and responses of NSCs to neurogenic stimuli diminish, leading to decreased neurogenesis and age-associated cognitive decline and psychiatric disorders. Thus, adult hippocampal neurogenesis has been the subject of intense investigation, generating both excitement and controversy. Identifying the core molecular machinery responsible for NSC preservation is of fundamental importance if we are to use neurogenesis to halt or reverse hippocampal age-related pathology. Here, we briefly overview the most frequently used mouse models to study hippocampal neurogenesis and then focus on a unique mouse model that allows NSC-specific studies based on their unique expression of lunatic fringe (Lfng). The Lfng-eGFP and Lfng(BAC)-CreERT2;RCL-tdT transgenic mice provide us with an excellent tool to resolve long-standing questions regarding the properties of NSCs, such as their specific molecular composition, potency, and plasticity, in isolation from any other cell in the hippocampal neurogenic niche.

BASP1 labels neural stem cells in the neurogenic niches of mammalian brain.

The mechanisms responsible for determining neural stem cell fate are numerous and complex. To begin to identify the specific components involved in these processes, we generated several mouse neural stem cell (NSC) antibodies against cultured mouse embryonic neurospheres. Our immunohistochemical data showed that the NSC-6 antibody recognized NSCs in the developing and postnatal murine brains as well as in human brain organoids. Mass spectrometry revealed the identity of the NSC-6 epitope as brain abundant, membrane-attached signal protein 1 (BASP1), a signaling protein that plays a key role in neurite outgrowth and plasticity. Western blot analysis using the NSC-6 antibody demonstrated multiple BASP1 isoforms with varying degrees of expression and correlating with distinct developmental stages. Herein, we describe the expression of BASP1 in NSCs in the developing and postnatal mammalian brains and human brain organoids, and demonstrate that the NSC-6 antibody may be a useful marker of these cells.

Persistent Pain After Wrist or Hand Fracture: Development and Validation of a Prognostic Model.

BACKGROUND: Worldwide, the incidence of wrist fracture is increasing. There are currently no externally validated prognostic models to inform early decision making for these patients. OBJECTIVES: To develop and validate a prognostic model from a comprehensive range of candidate prognostic factors that can identify patients who are at risk of developing persistent pain following wrist or hand fracture. METHODS: We developed and validated a prognostic model using secondary data derived from a prospective cohort study (n = 715), with recruitment sites in 3 metropolitan hospitals in Sydney, Australia. The primary outcome was persistent pain 4 months following the injury. The current study used a backward stepwise regression analysis to develop the model in 2 hospitals (n = 408) and externally validate it in a third hospital (n = 307). To determine the accuracy of the model, we assessed calibration and discrimination in accordance with the PROGnosis RESearch Strategy framework. RESULTS: Complete data were available for 95% of the cohort. Of 14 candidate variables, the final model contained 2 prognostic factors: patient age and pain intensity reported at initial presentation. The area under the receiver operating characteristic curve was 0.63 (95% confidence interval: 0.56, 0.69) in the development sample and 0.61 (95% confidence interval: 0.51, 0.70) in the validation sample. The model systematically overestimated risk (intercept, -1.13; slope, 0.73). CONCLUSION: We developed and externally validated a prognostic model to predict persistent pain 4 months after a wrist or hand fracture. Future studies are needed to assess whether the accuracy of this model can be improved by updating and validating it in local settings. LEVEL OF EVIDENCE: Prognosis, level 1b. J Orthop Sports Phys Ther 2019;49(1):28-35. Epub 12 Sep 2018. doi:10.2519/jospt.2019.8342.

Deficiency of Socs3 leads to brain-targeted EAE via enhanced neutrophil activation and ROS production.

Dysregulation of the JAK/STAT signaling pathway is associated with Multiple Sclerosis (MS) and its mouse model, Experimental Autoimmune Encephalomyelitis (EAE). Suppressors Of Cytokine Signaling (SOCS) negatively regulate the JAK/STAT pathway. We previously reported a severe, brain-targeted, atypical form of EAE in mice lacking Socs3 in myeloid cells (Socs3ΔLysM), which is associated with cerebellar neutrophil infiltration. There is emerging evidence that neutrophils are detrimental in the pathology of MS/EAE, however, their exact function is unclear. Here we demonstrate that neutrophils from the cerebellum of Socs3ΔLysM mice show a hyper-activated phenotype with excessive production of reactive oxygen species (ROS) at the peak of EAE. Neutralization of ROS in vivo delayed the onset and reduced severity of atypical EAE. Mechanistically, Socs3-deficient neutrophils exhibit enhanced STAT3 activation, a hyper-activated phenotype in response to G-CSF, and upon G-CSF priming, increased ROS production. Neutralization of G-CSF in vivo significantly reduced the incidence and severity of the atypical EAE phenotype. Overall, our work elucidates that hypersensitivity of G-CSF/STAT3 signaling in Socs3ΔLysM mice leads to atypical EAE by enhanced neutrophil activation and increased oxidative stress, which may explain the detrimental role of G-CSF in MS patients.

Daily Fluctuations of Progesterone and Testosterone Are Associated With Fibromyalgia Pain Severity.

The purpose of this longitudinal blood sampling study was to examine relationships between sex hormones and fibromyalgia pain. Eight women meeting case definition criteria for fibromyalgia provided venous blood samples and reported their fibromyalgia pain severity over 25 consecutive days. All women exhibited normal menstrual cycles and were not taking oral contraceptives. Cortisol, and the sex hormones estradiol, progesterone, and testosterone, were assayed from serum. A linear mixed model was used to determine if fluctuations of sex hormones were associated with changes in pain severity. In the entire sample, day to day changes in progesterone (P = .002) as well as testosterone (P = .015) were significantly and inversely correlated with pain severity. There was no relationship between estradiol and pain (P = .551) or cortisol and pain (P = .633). These results suggest that progesterone and testosterone play a protective role in fibromyalgia pain severity. Sex and other hormones may serve to increase as well as decrease fibromyalgia pain severity. PERSPECTIVE: Sex hormones fluctuate normally in women with fibromyalgia, but may still contribute to pain severity.

Reduced Pro-Inflammatory Cytokines after Eight Weeks of Low-Dose Naltrexone for Fibromyalgia.

Fibromyalgia (FM) is a complex, multi-symptom condition that predominantly affects women. The majority of those affected are unlikely to gain significant symptomatic control from the few treatments that are approved for FM. In this 10-week, single-blind, crossover trial we tested the immune effects of eight weeks of oral administration of low-dose naltrexone (LDN). We enrolled eight women with an average age of 46 years, symptom severity of 62 out of 100, and symptom duration of 14 years. We found that LDN was associated with reduced plasma concentrations of interleukin (IL)-1ß, IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40, IL-12p70, IL-15, IL-17A, IL-27, interferon (IFN)-α, transforming growth factor (TGF)-α, TGF-ß, tumor necrosis factor (TNF)-α, and granulocyte-colony stimulating factor (G-CSF). We also found a 15% reduction of FM-associated pain and an 18% reduction in overall symptoms. The findings of this pilot trial suggest that LDN treatment in fibromyalgia is associated with a reduction of several key pro-inflammatory cytokines and symptoms. The potential role of LDN as an atypical anti-inflammatory medication should be explored further.

Interdisciplinary Management of Complex Regional Pain Syndrome of the Face.

BACKGROUND AND PURPOSE: Orofacial pain disorders are relatively uncommon and pose a substantial diagnostic challenge. This case report documents the diagnosis and management of hemifacial pain in a patient who was referred to an interdisciplinary pain medicine unit. The purpose of this case report is twofold. First, it presents complex regional pain syndrome (CRPS) as a potential differential diagnosis in cases of facial pain. Second, it describes the successful adaption of contemporary management approaches for distal-extremity CRPS to treat people with CRPS of the facial region. CASE DESCRIPTION: The patient had hemifacial pain and concomitant motor and autonomic symptoms after a small laceration of the eyelid. Extensive specialist medical evaluations were undertaken to exclude an underlying structural pathology. After detailed clinical assessments by a physical therapist, pain physician, and clinical psychologist, a diagnosis of CRPS involving the face was made. OUTCOMES: The patient's pain was largely unresponsive to pharmacological agents. A modified graded motor imagery program, together with desensitization and discrimination training, was commenced by the physical therapist and clinical psychologist. A positive clinical response was indicated by a decrease in allodynia, normalization of motor control, and regained function in activities of daily living. DISCUSSION: Complex regional pain syndrome is an infrequently reported differential diagnosis that can be considered in patients with persistent facial pain. This case report highlights how careful examination and clinical decision making led to the use of an innovative therapeutic strategy to manage a challenging condition.

Psychological Distress Mediates the Relationship Between Pain and Disability in Hand or Wrist Fractures.

Upper limb fracture is a common musculoskeletal injury and can lead to marked pain-related disability. Unlike other common painful musculoskeletal conditions, such as low back pain, little consideration has been given to the role that psychological variables may play in explaining the relationship between pain and disability during early fracture recovery. This cross-sectional study aimed to determine if psychological distress (symptoms of depression, anxiety, and/or stress) mediate the relationship between pain and disability in acute hand/wrist fractures. Self-reported data from a consecutive sample of 594 patients with acute hand/wrist fracture were used. Mediation analyses were conducted to determine the role of depression, anxiety, and stress in the relationship between pain and disability, controlling for relevant demographic and fracture-related variables. Depression and stress, but not anxiety, significantly mediated the relationship between pain and disability. That is, although each psychological distress variable was associated with pain (P < .001), only depression (b1 = 0.27, P = .03) and stress (b3 = .23, P = .02) were significantly associated with disability and fulfilled recommended criteria for establishing a mediating variable. Increased depression and stress, but not anxiety, explain the relationship between pain and disability and may be novel targets for interventions designed to reduce pain-related disability after upper limb fracture. Perspective: This study presents the mediating effect of psychological distress on the relationship between pain and disability in acute upper limb fracture. These factors may be novel targets for interventions designed to reduce pain-related disability after acute fracture.

The use of low-dose naltrexone (LDN) as a novel anti-inflammatory treatment for chronic pain.

Low-dose naltrexone (LDN) has been demonstrated to reduce symptom severity in conditions such as fibromyalgia, Crohn's disease, multiple sclerosis, and complex regional pain syndrome. We review the evidence that LDN may operate as a novel anti-inflammatory agent in the central nervous system, via action on microglial cells. These effects may be unique to low dosages of naltrexone and appear to be entirely independent from naltrexone's better-known activity on opioid receptors. As a daily oral therapy, LDN is inexpensive and well-tolerated. Despite initial promise of efficacy, the use of LDN for chronic disorders is still highly experimental. Published trials have low sample sizes, and few replications have been performed. We cover the typical usage of LDN in clinical trials, caveats to using the medication, and recommendations for future research and clinical work. LDN may represent one of the first glial cell modulators to be used for the management of chronic pain disorders.