Whole blood stimulation provides preliminary evidence of altered immune function following SRC.

PURPOSE: To implement an approach combining whole blood immune stimulation and causal modelling to estimate the impact of sport-related concussion (SRC) on immune function. METHODS: A prospective, observational cohort study was conducted on athletes participating across 13 university sports at a single academic institute; blood was drawn from 52 athletes, comprised of 22 athletes (n = 11 male, n = 11 female) within seven days of a physician-diagnosed SRC, and 30 healthy athletes (n = 18 female, n = 12 male) at the beginning of their competitive season. Blood samples were stimulated for 24 h under two conditions: (1) lipopolysaccharide (lps, 100ng/mL) or (2) resiquimod (R848, 1uM) using the TruCulture® system. The concentration of 45 cytokines and chemokines were quantitated in stimulated samples by immunoassay using the highly sensitive targeted Proximity Extension Assays (PEA) on the Olink® biomarker platform. A directed acyclic graph (DAG) was used as a heuristic model to make explicit scientific assumptions regarding the effect of SRC on immune function. A latent factor analysis was used to derive two latent cytokine variables representing immune function in response to LPS and R848 stimulation, respectively. The latent variables were then modelled using student-t regressions to estimate the total causal effect of SRC on immune function. RESULTS: There was an effect of SRC on immune function in males following SRC, and it varied according to prior concussion history. In males with no history of concussion, those with an acute SRC had lower LPS reactivity compared to healthy athletes with 93% posterior probability (pprob), and lower R848 reactivity with 77% pprob. Conversely, in males with a history of SRC, those with an acute SRC had higher LPS reactivity compared to healthy athletes with 85% pprob and higher R848 reactivity with 82%. In females, irrespective of concussion history, SRC had no effect on LPS reactivity. However, in females with no concussion history, those with an acute SRC had higher R848 reactivity compared to healthy athletes with 86% pprob. CONCLUSION: Whole blood stimulation can be used within a causal framework to estimate the effect of SRC on immune function. Preliminary evidence suggests that SRC affects LPS and R848 immunoreactivity, that the effect is stronger in male athletes, and differs based on concussion history. Replication of this study in a larger cohort with a more sophisticated causal model is necessary.

A Distinct Metabolite Signature in Military Personnel Exposed to Repetitive Low-Level Blasts.

Military Breachers and Range Staff (MBRS) are subjected to repeated sub-concussive blasts, and they often report symptoms that are consistent with a mild traumatic brain injury (mTBI). Biomarkers of blast injury would potentially aid blast injury diagnosis, surveillance and avoidance. Our objective was to identify plasma metabolite biomarkers in military personnel that were exposed to repeated low-level or sub-concussive blast overpressure. A total of 37 military members were enrolled (18 MBRS and 19 controls), with MBRS having participated in 8-20 breaching courses per year, with a maximum exposure of 6 blasts per day. The two cohorts were similar except that the number of blast exposures were significantly higher in the MBRS, and the MBRS cohort suffered significantly more post-concussive symptoms and poorer health on assessment. Metabolomics profiling demonstrated significant differences between groups with 74% MBRS classification accuracy (CA). Feature reduction identified 6 metabolites that resulted in a MBRS CA of 98%, and included acetic acid (23.7%), formate (22.6%), creatine (14.8%), acetone (14.2%), methanol (12,7%), and glutamic acid (12.0%). All 6 metabolites were examined with individual receiver operating characteristic (ROC) curve analyses and demonstrated areas-under-the-curve (AUCs) of 0.82-0.91 (P ≤ 0.001) for MBRS status. Several parsimonious combinations of three metabolites increased accuracy of ROC curve analyses to AUCs of 1.00 (P < 0.001), while a combination of volatile organic compounds (VOCs; acetic acid, acetone and methanol) yielded an AUC of 0.98 (P < 0.001). Candidate biomarkers for chronic blast exposure were identified, and if validated in a larger cohort, may aid surveillance and care of military personnel. Future point-of-care screening could be developed that measures VOCs from breath, with definitive diagnoses confirmed with plasma metabolomics profiling.

Virtual Reality-Based Treatment for Military Members and Veterans With Combat-Related Posttraumatic Stress Disorder: Protocol for a Multimodular Motion-Assisted Memory Desensitization and Reconsolidation Randomized Controlled Trial.

BACKGROUND: Military members are at elevated risk of operational stress injuries, including posttraumatic stress disorder (PTSD) and moral injury. Although psychotherapy can reduce symptoms, some military members may experience treatment-resistant PTSD. Multimodular motion-assisted memory desensitization and reconsolidation (3MDR) has been introduced as a virtual reality (VR) intervention for military members with PTSD related to military service. The 3MDR intervention incorporates exposure therapy, psychotherapy, eye movement desensitization and reconsolidation, VR, supportive counselling, and treadmill walking. OBJECTIVE: The objective of this study is to investigate whether 3MDR reduces PTSD symptoms among military members with combat-related treatment-resistant PTSD (TR-PTSD); examine the technology acceptance and usability of the Computer Assisted Rehabilitation ENvironment (CAREN) and 3MDR interventions by Canadian Armed Forces service members (CAF-SMs), veterans, 3MDR clinicians, and operators; and evaluate the impact on clinicians and operators of delivering 3MDR. METHODS: This is a mixed-methods waitlist controlled crossover design randomized controlled trial. Participants include both CAF-SMs and veterans (N=40) aged 18-60 years with combat-related TR-PTSD (unsuccessful experience of at least 2 evidence-based trauma treatments). Participants will also include clinicians and operators (N=12) who have been trained in 3MDR and subsequently utilized this intervention with patients. CAF-SMs and veterans will receive 6 weekly 90-minute 3MDR sessions. Quantitative and qualitative data will be collected at baseline and at 1, 3, and 6 months postintervention. Quantitative data collection will include multiomic biomarkers (ie, blood and salivary proteomic and genomic profiles of neuroendocrine, immune-inflammatory mediators, and microRNA), eye tracking, electroencephalography, and physiological data. Data from outcome measures will capture self-reported symptoms of PTSD, moral injury, resilience, and technology acceptance and usability. Qualitative data will be collected from audiovisual recordings of 3MDR sessions and semistructured interviews. Data analysis will include univariate and multivariate approaches, and thematic analysis of treatment sessions and interviews. Machine learning analysis will be included to develop models for the prediction of diagnosis, symptom severity, and treatment outcomes. RESULTS: This study commenced in April 2019 and is planned to conclude in April 2021. Study results will guide the further evolution and utilization of 3MDR for military members with TR-PTSD and will have utility in treating other trauma-affected populations. CONCLUSIONS: The goal of this study is to utilize qualitative and quantitative primary and secondary outcomes to provide evidence for the effectiveness and feasibility of 3MDR for treating CAF-SMs and veterans with combat-related TR-PTSD. The results will inform a full-scale clinical trial and stimulate development and adaptation of the protocol to mobile VR apps in supervised clinical settings. This study will add to knowledge of the clinical effectiveness of 3MDR, and provide the first comprehensive analysis of biomarkers, technology acceptance and usability, moral injury, resilience, and the experience of clinicians and operators delivering 3MDR. TRIAL REGISTRATION: ISRCTN Registry 11264368; http://www.isrctn.com/ISRCTN11264368. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/20620.

High-Intensity Interval Training Is Associated With Alterations in Blood Biomarkers Related to Brain Injury.

Purpose: Blood biomarkers are a useful tool to study concussion. However, their interpretation is complicated by a number of potential biological confounds, including exercise. This is particularly relevant in military and athletic settings where injury commonly occurs during physical exertion. The impact of high-intensity interval training (HIIT) on putative brain injury biomarkers remains under-examined. The purpose of this study was to observe the effects of HIIT on a panel of blood biomarkers associated with brain injury. Methods: Eleven healthy, recreationally active males (median age = 29.0, interquartile range = 26.0-31.5) performed HIIT on a bicycle ergometer (8-12 × 60-s intervals at 100% of peak power output, interspersed by 75-s recovery at 50 W) three times/week for 2 weeks. Peripheral blood samples were collected before and immediately after HIIT during the first and last training sessions. Plasma concentrations of s100 calcium-binding protein beta (S100B), glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), brain-derived neurotrophic factor (BDNF), neurogranin (NRGN), peroxiredoxin (PRDX)-6, creatine kinase-BB isoenzyme (CKBB), visinin-like protein (VILIP)-1, von Willebrand factor (vWF), monocyte chemoattractant protein (MCP)-1, matrix metalloproteinase (MMP)-9, and total tau (T-tau) were quantitated by high-sensitivity MULTI-SPOT® immunoassay, on the MesoScale Diagnostics electrochemiluminescence detection platform. Differences in biomarker concentrations in response to HIIT were evaluated by partial least squares discriminant analysis (PLSDA) within a repeated-measures bootstrapped framework. Results: Ten of 12 biomarkers were increased pre-to-post HIIT; VILIP-1 remained unchanged, and GFAP was not statistically evaluated due to insufficient detectability. After 2 weeks of HIIT, T-tau was no longer significantly elevated pre-to-post HIIT, and significant attenuation was noted in the acute responses of NRGN, PRDX-6, MMP-9, and vWF. In addition, compared to session 1, session 6 pre-exercise concentrations of NSE and VILIP-1 were significantly lower and higher, respectively. Conclusion: Blood biomarkers commonly associated with brain injury are significantly elevated in response to a single bout of HIIT. After a 2-week, six-session training protocol, this response was attenuated for some, but not all markers. While biomarkers continue to provide promise to concussion research, future studies are necessary to disentangle the common biological sequelae to both exercise and brain injury.

Human hair follicle transcriptome profiling: a minimally invasive tool to assess molecular adaptations upon low-volume, high-intensity interval training.

High-intensity interval training (HIIT) has become a popular fitness training approach under both civilian and military settings. Consisting of brief and intense exercise intervals, HIIT requires less time commitment yet is able to produce the consistent targeted physical adaptations as conventional endurance training. To effectively characterize and monitor HIIT-induced cellular and molecular responses, a highly accessible yet comprehensive biomarker discovery source is desirable. Both gene differential expression (DE) and gene set (GS) analyses were conducted using hair follicle transcriptome established from pre and postexercise subjects upon a 10-day HIIT program by RNA-Seq, Comparing between pre and posttraining groups, differentially expressed protein coding genes were identified. To interpret the functional significance of the DE results, a comprehensive GS analysis approach featuring multiple algorithms was used to enrich gene ontology (GO) terms and KEGG pathways. The GS analysis revealed enriched themes such as energy metabolism, cell proliferation/growth/survival, muscle adaptations, and cytokine-cytokine interaction, all of which have been previously proposed as HIIT responses. Moreover, related cell signaling pathways were also measured. Specifically, G-protein-mediated signal transduction, phosphatidylinositide 3-kinases (PI3K) - protein kinase B (PKB) and Janus kinase (JAK) - Signal Transducer and Activator of Transcription (STAT) signaling cascades were over-represented. Additionally, the RNA-Seq analysis also identified several HIIT-responsive microRNAs (miRNAs) that were involved in regulating hair follicle-specific processes, such as miR-99a For the first time, this study demonstrated that both existing and new biomarkers like miRNA can be explored for HIIT using the transcriptomic responses exhibited by the hair follicle.

Sympathoadrenal Activation is Associated with Acute Traumatic Coagulopathy and Endotheliopathy in Isolated Brain Injury.

BACKGROUND: Acute coagulopathy after traumatic brain injury (TBI) involves a complex multifactorial hemostatic response that is poorly characterized. OBJECTIVES: To examine early posttraumatic alterations in coagulofibrinolytic, endothelial, and inflammatory blood biomarkers in relation to sympathetic nervous system (SNS) activation and 6-month patient outcomes, using multivariate partial least-squares (PLS) analysis. PATIENTS AND METHODS: A multicenter observational study of 159 adult isolated TBI patients admitted to the emergency department at an urban level I trauma center, was performed. Plasma concentrations of 6 coagulofibrinolytic, 10 vascular endothelial, 19 inflammatory, and 2 catecholamine biomarkers were measured by immunoassay on admission and 24 h postinjury. Neurological outcome at 6 months was assessed using the Extended Glasgow Outcome Scale. PLS-discriminant analysis was used to identify salient biomarker contributions to unfavorable outcome, whereas PLS regression analysis was used to evaluate the covariance between SNS correlates (catecholamines) and biomarkers of coagulopathy, endotheliopathy, and inflammation. RESULTS: Biomarker profiles in patients with an unfavorable outcome displayed procoagulation, hyperfibrinolysis, glycocalyx and endothelial damage, vasculature activation, and inflammation. A strong covariant relationship was evident between catecholamines and biomarkers of coagulopathy, endotheliopathy, and inflammation at both admission and 24 h postinjury. CONCLUSIONS: Biomarkers of coagulopathy and endotheliopathy are associated with poor outcome after TBI. Catecholamine levels were highly correlated with endotheliopathy and coagulopathy markers within the first 24 h after injury. Further research is warranted to characterize the pathogenic role of SNS-mediated hemostatic alterations in isolated TBI.

Inflammatory cytokine and chemokine profiles are associated with patient outcome and the hyperadrenergic state following acute brain injury.

BACKGROUND: Traumatic brain injury (TBI) elicits intense sympathetic nervous system (SNS) activation with profuse catecholamine secretion. The resultant hyperadrenergic state is linked to immunomodulation both within the brain and systemically. Dysregulated inflammation post-TBI exacerbates secondary brain injury and contributes to unfavorable patient outcomes including death. The aim of this study was to characterize the early dynamic profile of circulating inflammatory cytokines/chemokines in patients admitted for moderate-to-severe TBI, to examine interrelationships between these mediators and catecholamines, as well as clinical indices of injury severity and neurological outcome. METHODS: Blood was sampled from 166 isolated TBI patients (aged 45 ± 20.3 years; 74.7 % male) on admission, 6-, 12-, and 24-h post-injury and from healthy controls (N = 21). Plasma cytokine [interleukin (IL)-1ß, -2, -4, -5, -10, -12p70, -13, tumor necrosis factor (TNF)-α, interferon (IFN)-γ] and chemokine [IL-8, eotaxin, eotaxin-3, IFN-γ-induced protein (IP)-10, monocyte chemoattractant protein (MCP)-1, -4, macrophage-derived chemokine (MDC), macrophage inflammatory protein (MIP)-1ß, thymus activation regulated chemokine (TARC)] concentrations were analyzed using high-sensitivity electrochemiluminescence multiplex immunoassays. Plasma catecholamines [epinephrine (Epi), norepinephrine (NE)] were measured by immunoassay. Neurological outcome at 6 months was assessed using the extended Glasgow outcome scale (GOSE) dichotomized as good (>4) or poor (≤4) outcomes. RESULTS: Patients showed altered levels of IL-10 and all chemokines assayed relative to controls. Significant differences in a number of markers were evident between moderate and severe TBI cohorts. Elevated IL-8, IL-10, and TNF-α, as well as alterations in 8 of 9 chemokines, were associated with poor outcome at 6 months. Notably, a positive association was found between Epi and IL-1ß, IL-10, Eotaxin, IL-8, and MCP-1. NE was positively associated with IL-1ß, IL-10, TNF-α, eotaxin, IL-8, IP-10, and MCP-1. CONCLUSIONS: Our results provide further evidence that exaggerated SNS activation acutely after isolated TBI in humans may contribute to harmful peripheral inflammatory cytokine/chemokine dysregulation. These findings are consistent with a potentially beneficial role for therapies aimed at modulating the inflammatory response and hyperadrenergic state acutely post-injury.

Resuscitation of traumatic hemorrhagic shock patients with hypertonic saline-without dextran-inhibits neutrophil and endothelial cell activation.

Posttraumatic inflammation and excessive neutrophil activation cause multiple organ dysfunction syndrome (MODS), a major cause of death among hemorrhagic shock patients. Traditional resuscitation strategies may exacerbate inflammation; thus, novel fluid treatments are needed to reduce such posttraumatic complications. Hypertonic resuscitation fluids inhibit inflammation and reduce MODS in animal models. Here we studied the anti-inflammatory efficacy of hypertonic fluids in a controlled clinical trial. Trauma patients in hypovolemic shock were resuscitated in a prehospital setting with 250 mL of either 7.5% hypertonic saline (HS; n = 9), 7.5% hypertonic saline + 6% dextran 70 (HSD; n = 8), or 0.9% normal saline (NS; n = 17). Blood samples were collected on hospital admission and 12 and 24 h after resuscitation. Multicolor flow cytometry was used to quantify neutrophil expression of cell-surface activation/adhesion (CD11b, CD62L, CD64) and degranulation (CD63, CD66b, CD35) markers as well as oxidative burst activity. Circulating concentrations of soluble intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVACM-1), P- and E-selectins, myeloperoxidase (MPO), and matrix metalloproteinase 9 (MMP-9) were assessed by immunoassay. Multiple organ dysfunction syndrome, leukocytosis, and mortality were lower in the HS and HSD groups than in the NS group. However, these differences were not statistically significant. Hypertonic saline prevented priming and activation and neutrophil oxidative burst and CD11b and CD66b expression. Hypertonic saline also reduced circulating markers of neutrophil degranulation (MPO and MMP-9) and endothelial cell activation (sICAM-1, sVCAM-1, soluble E-selectin, and soluble P-selectin). Hypertonic saline + 6% dextran 70 was less capable than HS of suppressing the upregulation of most of these activation markers. This study demonstrates that initial resuscitation with HS, but neither NS nor HSD, can attenuate posttraumatic neutrophil and endothelial cell activation in hemorrhagic shock patients. These data suggest that hypertonic resuscitation without dextran may inhibit posttraumatic inflammation. However, despite this effect, neither HS nor HSD reduced MODS in trauma patients with hemorrhagic shock.