ObjectiveTo validate a modified algometer method for assessing mechanical nociceptive threshold (MNT) in lambs before and after husbandry procedures (marking) under field conditions by testing the precision, intra-operator reliability and agreement, and minimal detectable change (MDC).AnimalsForty-six randomly allocated Merino lambs: 6 control (sham handled), 13 hot knife tail docked, 13 rubber ring tail docked, 7 hot knife tail docked and rubber ring castrated, and 7 rubber ring tail docked and castrated.MethodsMNT (mean triplicate measurements at each replicate) was assessed at the tail base, using three replicate measurements before, and three hours after marking to assess precision, reliability, agreement, and MDC. A single operator performed all testing, with lambs restrained by one of three handlers (randomly) for each replicate. Within-animal/-occasion (pre- or post-marking) relative and absolute precision were evaluated using the coefficient of variation (CV) and a combination of standard deviation (SD) and dispersion modelling, respectively. Agreement was assessed using Bland-Altman plots, and linear mixed effects regression was used to determine the intra-class correlation coefficient (ICC) for intra-operator reliability, and any systematic effects of replicate order or handler. MDC was calculated from the standard error of measurement. Responsiveness to marking was analyzed by comparing MNT change from pre- to post-marking using linear mixed effects regression.ResultsThe overall median MNT was 3.07 kgf (IQR = 2.00–3.84, range = 1.23–5.00). Greater body weight was associated with significantly higher absolute imprecision (p = 7 × 10−5). Median CV was 12.5% (IQR = 7.29–18.16, range = 1.5–55.4) and was significantly higher post-marking (p = 0.0003), likely related to lower post-marking MNT. The agreement between replicates was good with small mean differences (0.02–0.26 kgf), although there were wide Bland-Altman limits of agreement (2.27–3.51 kgf). There was good intra-operator reliability [ICC (95% CI) = 0.83 (0.81–0.94)]. The minimum detectable change was 1.38 kgf. There was a significant reduction in MNT after marking for all marking groups (p < 0.01).ConclusionsModified algometer MNT measurement in lambs around the time of marking was feasible with sufficient precision and responsiveness to demonstrate a reduction in nociceptive threshold following husbandry procedures.

The growing frequency of emerging infectious diseases, antimicrobial resistance, and accidental or deliberate biological threat releases underscores the urgent need for early detection of biological threat exposure. Current diagnostic approaches primarily rely on identification of specific pathogens or toxins, often after clinical symptoms have emerged. An alternative approach could exploit the body's conserved psychoneuroimmunological (PNI) response to biological threat. This approach could provide a host-centred framework capable of detecting real-time deviations from baseline PNI status, even in response to previously unencountered threats. Using lipopolysaccharide (LPS) as a well-characterised model of immune challenge, we examine experimental work in rodents to evaluate how early-phase sickness behaviour, and the neuroimmune interactions that drive this response, can inform the development of wearable early-warning systems. Drawing on anatomical, molecular, electroencephalography (EEG), and behavioural studies, we identify early PNI alterations with potential utility for real-time threat detection and discuss analytical frameworks through which these signals may be leveraged. Converging evidence indicates that peripheral immune challenges rapidly engage central immunosensory neural circuits, followed by recruitment of autonomic, stress-related systems, and higher-order integrative centres. This response is strongly modulated by interindividual and contextual factors, producing marked heterogeneity in response type, timing, and magnitude, underscoring the need for continuous, personalised monitoring. We discuss how advances in wearable biosensing, combined with machine-learning-based approaches, could leverage autonomic, EEG, and behavioural signals to detect subtle deviations from an individual's PNI baseline, and outline knowledge gaps to translate mechanistic insights into real-time surveillance systems.

Equine gastric ulcer syndrome (EGUS) is highly prevalent in horses. Despite its presumed painful nature, evaluation of objective biomarkers associated with EGUS presence and severity remains limited. The objective of this study was to investigate the potential of interleukin-1 beta (IL-1β) release by peripheral blood mononuclear cells (PBMCs) following ex vivo stimulation of toll-like receptors 2 and 4 (TLR2 and TLR4) as a potential biomarker for EGUS status, with gastric lesions used as a proxy for presumed EGUS-associated pain. Venous blood was collected from 77 horses, PBMCs were isolated and stimulated with increasing concentrations of TLR2 and TLR4 agonists (Pam3CSK4 and lipopolysaccharide (LPS)) to assess IL-1β production. Fisher's exact test compared IL-1β responder proportion between horses with and without EGUS. Cochrane-Armitage test assessed trends across severity groups, and diagnostic potential was assessed via Receiver Operating Characteristic (ROC) curve analysis. Statistical significance was set at p < 0.05. Stimulation of TLR2 with Pam3CSK4 was unsuccessful (all <LLOQ) in all horses and was removed from final analysis. After exclusions for lameness and systemic inflammation, 25% of horses were LPS responders and EGUS was present in 92.2% of horses. No associations were identified between IL-1β response and EGUS presence (OR (95% CI) = 0.47 (0.049-6.2); p = 0.6). IL-1β AUC lacked diagnostic ability to differentiate between horses with and without EGUS (area under the ROC curve [AUROC] (95% CI) = 0.39 (0.11-0.67)), Equine Squamous Gastric Disease (ESGD) (AUROC = 0.30 (0.10-0.49)) and Equine Glandular Gastric Disease (EGGD) (AUROC = 0.56 (0.43-0.68)). The tested LPS-induced PBMC IL-1β release protocol was not associated with EGUS status in this cohort.

Introduction Knee osteoarthritis is a leading cause of pain and disability and frequently results in total knee arthroplasty (TKA). Decisions about surgery and postoperative management rely largely on subjective pain scales and patient-reported outcome measures (PROMs), and 10–15% of patients remain dissatisfied after TKA despite technically successful surgery. Evidence suggests that pain is partly reflected in peripheral immune signalling, yet this neuroimmune interface has not been studied in patients with joint pain due to knee osteoarthritis. This study will explore whether peripheral immune responses (interleukin-1 beta, IL-1β) are associated with pain and may serve as objective pain biomarkers in patients with painful knee osteoarthritis compared with pain-free controls and how these markers relate to pain and psychological (anxiety, depression and pain catastrophising) PROMs. This will be the first study to correlate pain markers (peripheral immune responses) with subjective pain levels in orthopaedic patients with symptomatic knee osteoarthritis.Methods and analysis This is a protocol for a prospective cross-sectional matched-subject observational study. We will include 20 adults undergoing unilateral primary TKA for painful advanced knee osteoarthritis, their contralateral pain-free knees as internal controls and 20 age and sex matched healthy controls without joint pain or functional limitation. All participants will undergo standardised clinical assessment and complete pain and psychological PROMs. In patients with TKA, venous blood will be collected pre-operatively. During TKA surgery, synovial fluid will be aspirated from the painful operated knee and the contralateral pain-free knee. Healthy controls will provide a single venous blood sample at a hospital visit. Peripheral blood mononuclear cells and synovial fluid mononuclear cells will be stimulated ex vivo with toll-like receptor 2 and 4 agonists to quantify IL-1β release. In parallel, hyperspectral imaging will characterise unstimulated immune cell phenotypes. Multivariable statistical and machine-learning approaches will relate biomarker profiles to pain and psychological PROMs.Ethics and dissemination The study has been approved by the Southern Adelaide Local Health Network (SALHN) HREC (references: 2024/HRE00253, SSA 2024/SSA00641). Written informed consent will be obtained from all participants. Study results will be disseminated through peer-reviewed publications and presentations at national and international scientific conferences.Trial number registration ACTRN12626000084381.

Childhood dementias are a group of paediatric neurodegenerative disorders characterised by neurocognitive decline, and in many cases underpinned by pathophysiological mechanisms similar to adult-onset dementias. In this study, we use patient-derived induced pluripotent stem cells (iPSCs) from children with one of the most prevalent childhood dementias, Mucopolysaccharidosis Type IIIA (MPS IIIA), also known as Sanfilippo syndrome. The derived cortical cultures exhibit lysosomal dysfunction, heparan sulfate accumulation, progressive neurodegeneration and astrocytic reactivity, recapitulating prototypical in-vivo phenotypes. Using a multimodal drug screening platform that integrates machine learning, high-content confocal imaging, single-nuclei transcriptomics and electrophysiology, we identify at least nine repurposed compounds that significantly mitigate these adverse effects within two weeks of treatment in vitro, demonstrating potential for rapid clinical translation. This human preclinical model for MPS IIIA, coupled with a robust multimodal therapeutic interrogation platform, serves as an exemplar for advancing drug discovery for childhood dementias and the broader neurodegenerative disease spectrum. Here authors show patient-derived neural models of childhood dementia exhibit key disease features. A drug screening platform integrating machine learning, imaging, transcriptomics and electrophysiology, identifies repurposed compounds that mitigate adverse effects.

Peripheral neuropathy and gastrointestinal dysfunction are frequent, debilitating side effects of the neurotoxic myeloma drug bortezomib that reduce quality of life and adherence to optimal therapy. Therapeutic strategies to manage these complications are limited. Here, we have developed a mouse model of bortezomib side effects to investigate the role of the gut microbiota in their development. C57BL/6 specific pathogen-free (SPF), germ-free (GF) and ex-GF mice (colonised with healthy gut microbiota via faecal microbiota transplantation [FMT]) were treated with bortezomib (1 mg/kg) twice-weekly for two weeks. Neurotoxicity and neuroimmune signalling were assessed by serum neurofilament light chain (NfL) quantitation and real-time qPCR analysis of inflammatory markers in sensory and autonomic nerve ganglia, respectively. Faecal microbiota composition was characterised using 16S rRNA gene sequencing. Bortezomib side effects were assessed using behavioural phenotyping, von Frey mechanical sensitivity and rotarod testing, and FITC-dextran gut permeability and Evans Blue dye transit assays. Bortezomib-treated SPF mice displayed altered spontaneous behaviour and developed acute gastric retention, altered gastrointestinal motility and increased intestinal permeability, in the absence of intestinal micro-architecture changes. Concurrently, bortezomib induced sensory loss in, and increased grooming of, paws, and reduced motor performance, indicative of peripheral neuropathy. Bortezomib neurotoxicity was evidenced by elevated serum NfL levels and neuroimmune signalling in sciatic nerve dorsal root and vagal nerve nodose ganglia. In SPF mice, bortezomib altered gut microbiota composition with an acute decrease in microbial diversity and expansion of Lactobacillus gasseri. Notably, GF mice developed a milder symptom profile following bortezomib treatment compared with SPF mice, while FMT mice did not develop overt bortezomib side effects despite displaying evidence of nerve damage. This is the first study to model gastrointestinal side effects of bortezomib in rodents, implicating neuroimmune dysregulation of the vagus nerve. Our data show that the gut microbiota is not a primary driver of bortezomib side effects. However, the absence of a symptom profile in FMT mice suggests that, in GF mice, the gut microbiota beneficially modulates the host to protect against bortezomib side effects. Further studies are required to determine whether this can be harnessed to mitigate clinical complications of bortezomib.

Pain in humans and animals is a complex experience that is influenced by numerous biological, psychological and social factors. It is increasingly recognised that contributors to pain exist across multiple dimensions, including time and space. This chapter examines the concept of four-dimensional contributors to pain and emphasises the multiscale nature of biopsychosocial factors that contribute to chronic pain development in particular. At the nanoscale, there are molecular and cellular processes that contribute to chronic pain development across all species. Importantly contributors to pain also exist across multiple timescales, from nanoseconds to years. For example, early life molecular events, stress and trauma can exert long-lasting effects on the developing brain and increase the risk of developing chronic pain later in life. Similarly, social support, coping strategies and expectations can also play important roles in shaping the pain experience. Understanding the multidimensional nature of pain is critical for developing effective interventions that address the diverse factors that contribute to chronic pain. By taking a multiscale and multidimensional approach to pain, targeted interventions can be developed that address the unique needs of individual patients. Mainly focusing on pain studies conducted in rodents and humans, this chapter will focus on the role of early life events in shaping the experience of pain later in life. Although similarities in the mechanisms underlying pain are expected across species, unique contributors to pain (e.g. prior experience, environmental and genetic influences) are likely to result in nuanced differences. Nevertheless, a convergent understanding of pain across species will rapidly advance knowledge and technology transfer between human medicine and animal veterinary industries to address the unmet challenges of under-diagnosed and untreated pain.

Alterations in synaptic homeostasis are linked to cognitive and behavioural impairments in brain disorders. However, synaptic dysfunction in childhood dementia is poorly understood. Here, we generate human cortical circuits from induced pluripotent stem cells (iPSCs) derived from donors with Mucopolysaccharidosis Type IIIA (MPS IIIA), also known as Sanfilippo syndrome, a common form of childhood-onset dementia. Action potential firing capacity and morphology of MPS IIIA patient neurons in culture are similar to those of neurons from neurotypical donors. However, long-term neural maturation reveals excitation/inhibition imbalances caused by hyperactive excitatory synapses, disrupted network dynamics, and dysregulated gene expression linked to synaptic homeostasis. This study validates in vitro human neural models to detect neurophysiological phenotypes in childhood dementias and supports drug discovery strategies that target synaptic dysfunction to improve cognition in MPS IIIA and related brain disorders.

Discovery 5.0 heralds a human-centric, sustainable, and resilient research paradigm, mirroring Industry 5.0's transformation of manufacturing. It reimagines laboratory practice as a circular, stakeholder-driven ecosystem valuing social benefit and planetary boundaries alongside scientific novelty. This is illustrated by fiber-optic MEMS sensors quantifying neuroimmune pain signatures, with a planned bidirectional translation from human to animal health and back. This fit-for-purpose design, fostered by networked communities and enabled by digital twins, creates publicly trusted, personalized health technologies. As open scorecards redefine impact, success will belong to research that maximizes human benefit, safeguards the planet, and bolsters system resilience.

Background The immune system protects against invading pathogens and helps maintain homeostasis. Other pivotal roles include the regulation of tissue health through interactions with the nervous system. Understanding how these neuroimmune interactions may go awry in musculoskeletal conditions and how they can be targeted therapeutically may optimise patient care. Methods We conducted a clinically focused narrative review of the role of the immune and nervous systems in musculoskeletal health and conditions such as neck pain, back pain and osteoarthritis and how psychosocial and behavioural factors impact these conditions via interacting with neuroimmune functioning. Results The interplay between the immune and nervous system is involved in both the physiology and pathology of musculoskeletal tissues, including bone, joint, nerve, muscle and tendon. We describe this at the local tissue, whole nervous system, and systemic (blood) level and how psychosocial and behavioural factors impact immune activity and influence outcomes. We also highlight recent advances in medical imaging and multi-omics that shed new light on the interplay between the immune and nervous systems in musculoskeletal conditions. Advances in understanding these relationships provide promising new treatment avenues for musculoskeletal conditions and important insights into how psychosocial- and behavioural-based therapies such as exercise and cognitive behavioural therapy work and can be optimised to improve outcomes. Conclusions This review provides clinicians with a foundation in the neuroimmunology of musculoskeletal conditions. It also explores how the immune and nervous systems, and their interplay can be modulated to improve prevention and management strategies.

Neuropathic pain involves disruptions in sensory, cognitive, and affective processing, with microglial reactivity playing a crucial role in its development. While spinal microglial changes post-injury are well-documented, the time-dependent patterns of microglial reactivity in the brain remain unclear. This study aimed to characterize microglial morphological changes over time following peripheral nerve injury. Adult male and female Sprague-Dawley rats underwent chronic constriction injury (CCI) or sham surgery to the sciatic nerve, and brains were collected at 7 or 28 days post-surgery. CD11b immunostaining was used to visualize microglia across 52 brain regions linked to sensory, affective, and cognitive pain modalities. Morphological measures-including reactivity score, area, and length-were quantified with HALO software. A repeated-measures linear mixed model revealed significant effects of injury and timepoint on all parameters. Post hoc analyses identified region-specific changes (FDR-adjusted) in areas such as the anterior cingulate cortex, central amygdala, dorsal raphe, and dorsomedial hypothalamus, highlighting spatial specificity of microglial responses. A functional circuit-wide correlation network analysis showed a dynamic reorganization of microglial morphology following injury. Initially, at day 7, the primary motor cortex emerged as a hub, reflecting acute sensorimotor changes. By day 28, network hubs had shifted to the dorsomedial hypothalamus and ventral tegmental area, suggesting engagement of homeostatic and reward circuits in chronic pain. Graph-theoretic metrics revealed a progressive decline in global network connectivity over time, supporting the view that chronic neuropathic pain alters central microglial signaling in a region- and circuit-specific manner, relevant to pain chronification and its comorbidities. PERSPECTIVE: This article presents a comprehensive mapping of microglial morphology in brain areas associated with pain processing. The findings will guide further investigations into the ways in which microglia contribute to neuropathic pain and its comorbidities.

Psychological distress predicts the onset and worsening of persistent pain, but the mechanisms that underpin this influence are poorly understood. Proinflammatory signalling is a plausible link, given its known connections to distress, pain, and neural upregulation. Sustained distress may prime the inflammatory system to respond more strongly to a phasic noxious challenge, supporting neuroimmune upregulation of central nociceptive signalling and persistent pain. This cross-sectional study tested the hypotheses that in vitro endotoxin-provoked expression of typically proinflammatory cytokines (IL1β, IL6) is a partial mediator between distress and persistent pain, and that it is associated with experimentally induced secondary hyperalgesia, in people with suppressed HIV. Study participants were 99 adults (mean [range] age: 43 [28-64 y/o; 72 females]) with either no pain (n = 54) or persistent pain (n = 45), mostly of black South African ethnicity, low socio-economic status, and with high social support. The results replicated previous reports that distress is associated with persistent pain status and pain severity, and distress was associated with the anatomical extent of pain. However, distress was not associated with provoked cytokine expression, nor was provoked cytokine expression associated with secondary hyperalgesia. The conflict between our findings and prior evidence could reflect the influence of differentially trained immune systems or a more complex relationship arising from diverse psychoneuroimmunological interactions in this sample. This sample's combination of HIV status, African genetic ancestry, financial impoverishment, and rich social interconnectedness is poorly represented in current research and provides an opportunity to deepen insight into psychoneuroimmunological interactions in persistent pain.

Toll-like receptor 4 (TLR4) plays a pivotal role in the innate immune system by recognizing pathogens and initiating immune responses. Despite extensive research over three decades, current methods lack the resolution to measure ligand-induced TLR4 receptor dynamics at the earliest stages of signaling, relying instead on downstream outputs such as gene expression and cytokine secretion. Here, we present the illuminating TLR4 (iTLR4) assay, a novel Bioluminescence Resonance Energy Transfer (BRET)-based platform that provides real-time insights into TLR4 receptor-level events in live cells. The iTLR4 assay demonstrates, for the first time, that lipopolysaccharide (LPS) induces stable interactions between intracellular domains of TLR4 monomers, with an EC50 of 660 EU/mL. Kinetic analysis revealed a gradual, sustained increase in the BRET signal over time. Additionally, the assay uncovered subtle mechanistic differences among functional antagonists. While all antagonists completely abolished LPS-induced IL-8 secretion, the assay demonstrated that at the receptor level LPS-RS completely inhibited the LPS-induced BRET signal, TAK-242 partially inhibited it and (+)-naloxone potentiated it. The assay also identified potential regulatory roles for CD14 and MD2 in naloxone stereoisomer activity, marking the first report of such mechanistic differences. These findings highlight the unique capabilities of the iTLR4 assay to track nuanced TLR4 receptor dynamics, enabling high-throughput screening of TLR4-specific modulators. This platform provides critical insights into ligand-induced signaling, paving the way for the development of novel therapeutics targeting TLR4-related diseases and advancing our understanding of innate immune responses.

Persistent pain represents a significant global health challenge, necessitating innovative biomarker technologies that facilitate personalised prediction, prevention, and treatment. Recent advances in omics, encompassing genomics, proteomics, transcriptomics, lipidomics, epigenomics, and metabolomics, now permit high-resolution mapping of neuroimmune pathways implicated in pain chronification. Yet, biomarkers must transcend isolated molecular or sensory indicators, integrating emotional, cognitive, functional, and social dimensions of pain. Emerging quantum sensing technologies, such as diamond nitrogen-vacancy sensors and portable magnetoencephalography systems, promise precise and wearable tools capable of real-time, multimodal assessment of pain. Concurrently, transparent machine learning methods combining explainable artificial intelligence with physiologically informed modelling are crucial for managing the vast data complexity inherent to these multidimensional omics approaches. Ultimately, achieving economically viable, environmentally sustainable, and universally accessible pain management solutions requires strategically streamlined methods. Here, we outline a visionary framework of measurement-enabled understanding that enables precision pain medicine with rapid feedback that points toward actionable clinical outcomes, harnessing interdisciplinary innovation to address persistent pain comprehensively, just as genomics and immunotherapy have transformed cancer care.

Dr. Linda R. Watkins, a Distinguished Professor at the University of Colorado Boulder, fundamentally altered the understanding of pain and neuroimmune signaling. As she concludes her tenure as Associate Editor of Brain, Behavior, and Immunity, this tribute reflectson her revolutionary discoveries. She pioneered the concept that glial cells actively participate in pain states, challenging neuron-centric dogma. Her work elucidated the roles of cytokines like IL-1β and IL-10, the chemokine fractalkine (CX3CL1), and the Toll-Like Receptor 4 (TLR4) in glial reactivity, sickness behavior, and unwanted opioid effects (tolerance, hyperalgesia). As a dedicated mentor and collaborator, particularly with Steve Maier, she fosters interdisciplinary research. Watkins champions translational science, co-founding Xalud Therapeutics to develop immune therapies like IL-10 gene therapy, leaving a profound legacy in neuroscience.

Preclinical research supports a critical role for nervous system glia in pain pathophysiology. This systematic review of human trials of potential glia-modulating drugs for the prevention or treatment of pain followed a predefined search strategy and protocol registration. We searched for English language, randomized, double-blind trials comparing putative glia-modulating drugs to placebo or other comparators. The primary outcomes included validated participant-reported measures of pain intensity or relief and, in studies of opioid administration, measures of opioid consumption and/or opioid-related adverse effects. Twenty-six trials (2132 participants) of glial modulators (12 minocycline, 11 pentoxifylline, and 3 ibudilast) were included. Because of clinical heterogeneity related to study drug, participant population, outcome measures, and trial design, no meta-analysis was possible. Only 6 trials reported a positive effect of the treatment (pentoxifylline-4 trials; minocycline-2 trials), whereas 11 trials reported mixed results and 9 trials reported no effect. This review does not provide convincing evidence of efficacy of current pharmacological targets of nervous system glial function for pain treatment or prevention. However, in light of ample preclinical evidence of the importance of neuroimmune signalling and glial functions in pain pathophysiology, continued strategic human research is anticipated to identify (1) drugs with maximal activity as selectively targeted glial modulators, (2) the necessary timing and duration of pharmacological glial modulation needed for pain prevention or treatment for specific injuries or pain conditions, and (3) the best design of future clinical trials of glial-targeted drugs for pain treatment and/or prevention.

The recent SARS-CoV-2 pandemic has underscored the significance of viral infections, affecting billions of lives and costing trillions of dollars globally. Even beyond SARS-CoV-2, common infections with viruses like influenza, HIV, and herpesviruses have profound impacts beyond their typical manifestations, often triggering acute and chronic pain syndromes that can be life-altering. These virally induced pain states can arise through direct viral replication within neurons, or indirectly, via immune responses to infection in both the contexts of afferent signaling in the dorsal root ganglion (DRG) or subsequent higher order integration in intracranial systems. Varicella-zoster virus (VZV), influenza virus, and SARS-CoV-2 each provide a unique lens through which to examine the interplay between viral activity and pain. This perspective paper is not meant to be an exhaustive review of virally-induced neuropathic pain states. It seeks to explore curated aspects of the complexities of these pain states, identify research gaps, and suggest solutions using nanoscale molecular understanding and psychoneuroimmunological and biopsychosocial frameworks. Each subheading is accompanied by a list of related issues for study which we think will lead to advances in our understanding of the vexing pain phenotype associated with viral infection.

Adversity in childhood elevates the risk of persistent pain in adulthood. Neuroimmune interactions are a candidate mechanistic link between childhood adversity and persistent pain. We aimed to clarify whether immune reactivity is associated with provoked differences in nociceptive processing in adults with a range of childhood adversity. Pain-free adults (n = 96; 61 female; median [range] age: 23 [18-65] years old) with a history of mild to severe childhood adversity underwent psychophysical assessments before and after in vivo neural provocation (high-frequency electrical stimulation) and, separately, before and after in vivo immune provocation (influenza vaccine administration). Psychophysical assessments included the surface area of secondary hyperalgesia after neural provocation and change in conditioned pain modulation (test stimulus: pressure pain threshold; conditioning stimulus: cold water immersion) after immune provocation. Immune reactivity was operationalised as interleukin-6 and tumour necrosis factor-α expression after in vitro lipopolysaccharide provocation of whole blood. We hypothesised associations between immune reactivity and (1) childhood adversity, (2) induced secondary hyperalgesia, and (3) vaccine-associated change in conditioned pain modulation. We found that provoked expression of proinflammatory cytokines was not statistically associated with childhood adversity, induced secondary hyperalgesia, or vaccine-associated change in conditioned pain modulation. The current findings from a heterogenous sample cast doubt on 2 prominent ideas: that childhood adversity primes the inflammatory system for hyper-responsiveness in adulthood and that nociceptive reactivity is linked to inflammatory reactivity. This calls for the broader inclusion of heterogeneous samples in fundamental research to investigate the psychoneuroimmunological mechanisms underlying vulnerability to persistent pain.