Martha Tingle

Clinical recovery from surgery correlates with single-cell immune signatures

Single-cell mass cytometry revealed immune correlates of patient-associated variability in surgical recovery. Signaling Surgical Recovery The speed and ease of recovery after surgery differ for every patient, and determining the mechanisms that drive recovery could lead to patient-specific recovery protocols. Gaudilliere et al. used mass cytometry to characterize postsurgical immunological insult at a single-cell level and found a surgical immune signature that correlated with clinical recovery across patients. Specifically, cell signaling responses, but not cell frequency, were linked to recovery. Moreover, the correlated signaling responses occurred most notably in CD14+ monocytes, suggesting that these cells may play a predominant role in surgical recovery. The consistency of this signature across patients suggests a tightly regulated immune response to surgical trauma, which, if validated, may form the basis of a diagnostic guideline for personalized postsurgical care. Delayed recovery from surgery causes personal suffering and substantial societal and economic costs. Whether immune mechanisms determine recovery after surgical trauma remains ill-defined. Single-cell mass cytometry was applied to serial whole-blood samples from 32 patients undergoing hip replacement to comprehensively characterize the phenotypic and functional immune response to surgical trauma. The simultaneous analysis of 14,000 phosphorylation events in precisely phenotyped immune cell subsets revealed uniform signaling responses among patients, demarcating a surgical immune signature. When regressed against clinical parameters of surgical recovery, including functional impairment and pain, strong correlations were found with STAT3 (signal transducer and activator of transcription), CREB (adenosine 3′,5′-monophosphate response element–binding protein), and NF-κB (nuclear factor κB) signaling responses in subsets of CD14+ monocytes (R = 0.7 to 0.8, false discovery rate <0.01). These sentinel results demonstrate the capacity of mass cytometry to survey the human immune system in a relevant clinical context. The mechanistically derived immune correlates point to diagnostic signatures, and potential therapeutic targets, that could postoperatively improve patient recovery.

Cytokine profile in human skin in response to experimental inflammation, noxious stimulation, and administration of a COX-inhibitor: a microdialysis study.

Abstract Animal studies have documented a critical role for cytokines in cell signaling events underlying inflammation and pain associated with tissue injury. While clinical reports indicate an important role of cytokines in inflammatory pain, methodological limitations have made systematic human studies difficult. This study examined the utility of a human in vivo bioassay combining microdialysis with multiplex immunoassay techniques for measuring cytokine arrays in tissue. The first experiment measured cytokines in interstitial fluid collected from non‐inflamed and experimentally inflamed skin (UVB). The effects of noxious heat on cytokine release were also assessed. The second experiment examined whether anti‐hyperalgesic effects of the COX‐inhibitor ibuprofen were associated with decreased tissue levels of the pro‐inflammatory cytokines IL‐1β and IL‐6. In the first experiment, inflammation significantly increased IL‐1β, IL‐6, IL‐8, IL‐10, G‐CSF, and MIP‐1β. Noxious heat but not experimental inflammation significantly increased IL‐7 and IL‐13. In the second experiment, an oral dose of 400 and 800 mg ibuprofen produced similar anti‐hyperalgesic effects suggesting a ceiling effect. Tissue levels of IL‐1β and IL‐6 were not affected after the 400 mg dose but decreased significantly (44 ± 32% and 38 ± 13%) after the 800 mg dose. These results support the utility of explored method for tracking cytokines in human tissue and suggest that anti‐hyperalgesic and anti‐inflammatory effects of ibuprofen are at least partially dissociated. The data further suggest that high clinical doses of ibuprofen exert anti‐inflammatory effects by down‐regulating tissue cytokine levels. Explored human bioassay is a promising tool for studying the pathology and pharmacology of inflammatory and chronic pain conditions.

Pain sensitivity and opioid analgesia: a pharmacogenomic twin study.

TOC Summary This twin study demonstrates significant genetic and familial effects for pain sensitivity and opioid analgesia and provides a strong rationale for more detailed genetic studies. ABSTRACT Opioids are the cornerstone medication for the management of moderate to severe pain. Unfortunately, vast inter‐individual differences in dose requirements complicate their effective and safe clinical use. Mechanisms underlying such differences are incompletely understood, are likely multifactorial, and include genetic and environmental contributions. While accumulating evidence suggests that variants of several genes account for some of the observed response variance, the relative contribution of these factors remains unknown. This study used a twin paradigm to provide a global estimate of the genetic and environmental contributions to inter‐individual differences in pain sensitivity and analgesic opioid effects. Eighty one monozygotic and 31 dizygotic twin pairs successfully underwent a computer‐controlled infusion with the μ‐opioid agonist alfentanil in a single occasion, randomized, double‐blind and placebo‐controlled study design. Pain sensitivity and analgesic effects were assessed with experimental heat and cold pressor pain models along with important covariates including demographic factors, depression, anxiety, and sleep quality. Significant heritability was detected for cold pressor pain tolerance and opioid‐mediated elevations in heat and cold pressor pain thresholds. Genetic effects accounted for 12–60% of the observed response variance. Significant familial effects accounting for 24–32% of observed variance were detected for heat and cold pressor pain thresholds and opioid‐mediated elevation in cold pressor pain tolerance. Significant covariates included age, gender, race, education, and anxiety. Results provide a strong rationale for more detailed molecular genetic studies to elucidate mechanisms underlying inter‐individual differences in pain sensitivity and analgesic opioid responses. Such studies will require careful consideration of the studied pain phenotype.

No evidence for the development of acute tolerance to analgesic, respiratory depressant and sedative opioid effects in humans

ABSTRACT It is widely accepted that chronic opioid therapy is associated with the development of pharmacological tolerance. More controversial is the question as to whether acute opioid administration can result in “acute tolerance.” The aim of this double‐blind, placebo‐controlled study in thirty‐six healthy human volunteers was to examine whether a 3‐h intravenous infusion delivering two different but clinically relevant doses of the μ‐opioid receptor agonist remifentanil would result in tolerance to analgesic, respiratory depressant and/or sedative opioid effects. The blood remifentanil concentration versus opioid effect relationship was determined before and after the 3‐h infusion. Tolerance was inferred if the potency of remifentanil was significantly lower after the 3‐h infusion. Opioid analgesia was assessed with the aid of the cold pressor test and models of electrical and heat pain. Respiratory depression was assessed by measuring arterial pCO2 and minute ventilation. Subjective sedation scores were assessed on a visual analogue scale. Mixed effects modeling was used to relate the steady‐state blood remifentanil concentration to each pharmacodynamic assessment. Neither dose of remifentanil produced detectable tolerance to any of the measured opioid effects following a 3‐h infusion. The study was adequately powered to detect a decrease in potency of 5–24% for analgesia, 20–48% for respiratory depression, and 32% for sedative effects. These results suggest that short‐term administration of clinically useful doses of remifentanil is not associated with the development of significant tolerance to analgesic, respiratory depressant, or sedative opioid effects.

Human umbilical cord plasma proteins revitalize hippocampal function in aged mice

Ageing drives changes in neuronal and cognitive function, the decline of which is a major feature of many neurological disorders. The hippocampus, a brain region subserving roles of spatial and episodic memory and learning, is sensitive to the detrimental effects of ageing at morphological and molecular levels. With advancing age, synapses in various hippocampal subfields exhibit impaired long-term potentiation, an electrophysiological correlate of learning and memory. At the molecular level, immediate early genes are among the synaptic plasticity genes that are both induced by long-term potentiation and downregulated in the aged brain. In addition to revitalizing other aged tissues, exposure to factors in young blood counteracts age-related changes in these central nervous system parameters, although the identities of specific cognition-promoting factors or whether such activity exists in human plasma remains unknown. We hypothesized that plasma of an early developmental stage, namely umbilical cord plasma, provides a reservoir of such plasticity-promoting proteins. Here we show that human cord plasma treatment revitalizes the hippocampus and improves cognitive function in aged mice. Tissue inhibitor of metalloproteinases 2 (TIMP2), a blood-borne factor enriched in human cord plasma, young mouse plasma, and young mouse hippocampi, appears in the brain after systemic administration and increases synaptic plasticity and hippocampal-dependent cognition in aged mice. Depletion experiments in aged mice revealed TIMP2 to be necessary for the cognitive benefits conferred by cord plasma. We find that systemic pools of TIMP2 are necessary for spatial memory in young mice, while treatment of brain slices with TIMP2 antibody prevents long-term potentiation, arguing for previously unknown roles for TIMP2 in normal hippocampal function. Our findings reveal that human cord plasma contains plasticity-enhancing proteins of high translational value for targeting ageing- or disease-associated hippocampal dysfunction.

Aversive and Reinforcing Opioid Effects: A Pharmacogenomic Twin Study

Background: The clinical utility of opioids is limited by adverse drug effects including respiratory depression, sedation, nausea, and pruritus. In addition, abuse of prescription opioids is problematic. Gaining a better understanding of the genetic and environmental mechanisms contributing to an individuals susceptibility to adverse opioid effects is essential to identify patients at risk. Methods: A classic twin study paradigm provided estimates for the genetic and familial (genetic and/or shared environment) contribution to acute adverse and affective opioid responses, all secondary outcomes of a larger dataset. One hundred twenty-one twin pairs were recruited in a single occasion, randomized, double-blind, and placebo-controlled study. The &mgr;-opioid receptor agonist alfentanil and saline placebo were administered as target-controlled infusions under carefully monitored laboratory conditions. Measured outcomes included respiratory depression, sedation, nausea, pruritus, drug liking, and drug disliking. Demographic information was collected, and aspects of mood and sleep were evaluated. Results: Significant heritability was detected for respiratory depression (30%), nausea (59%), and drug disliking (36%). Significant familial effects were detected for sedation (29%), pruritus (38%), dizziness (32%), and drug liking (26%). Significant covariates included age, sex, race, ethnicity, education, mood, and depression. Covariates affected sedation, pruritus, drug liking and disliking, and dizziness. Conclusions: This study demonstrates that large-scale efforts to collect quantitative and well-defined opioid response data are not only feasible but also produce data that are suitable for genetic analysis. Genetic, environmental, and demographic factors work together to control adverse and reinforcing opioid responses, but contribute differently to specific responses.

Patient-specific Immune States before Surgery Are Strong Correlates of Surgical Recovery.

Background:Recovery after surgery is highly variable. Risk-stratifying patients based on their predicted recovery profile will afford individualized perioperative management strategies. Recently, application of mass cytometry in patients undergoing hip arthroplasty revealed strong immune correlates of surgical recovery in blood samples collected shortly after surgery. However, the ability to interrogate a patient’s immune state before surgery and predict recovery is highly desirable in perioperative medicine. Methods:To evaluate a patient’s presurgical immune state, cell-type–specific intracellular signaling responses to ex vivo ligands (lipopolysaccharide, interleukin [IL]-6, IL-10, and IL-2/granulocyte macrophage colony-stimulating factor) were quantified by mass cytometry in presurgical blood samples. Selected ligands modulate signaling processes perturbed by surgery. Twenty-three cell surface and 11 intracellular markers were used for the phenotypic and functional characterization of major immune cell subsets. Evoked immune responses were regressed against patient-centered outcomes, contributing to protracted recovery including functional impairment, postoperative pain, and fatigue. Results:Evoked signaling responses varied significantly and defined patient-specific presurgical immune states. Eighteen signaling responses correlated significantly with surgical recovery parameters (|R| = 0.37 to 0.70; false discovery rate < 0.01). Signaling responses downstream of the toll-like receptor 4 in cluster of differentiation (CD) 14+ monocytes were particularly strong correlates, accounting for 50% of observed variance. Immune correlates identified in presurgical blood samples mirrored correlates identified in postsurgical blood samples. Conclusions:Convergent findings in pre- and postsurgical analyses provide validation of reported immune correlates and suggest a critical role of the toll-like receptor 4 signaling pathway in monocytes for the clinical recovery process. The comprehensive assessment of patients’ preoperative immune state is promising for predicting important recovery parameters and may lead to clinical tests using standard flow cytometry.

Opioid Pharmacogenomics Using a Twin Study Paradigm: Methods and Procedures for Determining Familial Aggregation and Heritability

Opioids are the cornerstone medication for the treatment of moderate to severe pain. However, analgesic opioid requirements and the propensity to suffer from aversive opioid effects, including fatal respiratory depression and addiction, vary widely among patients. The factors underlying the substantial response variance remain largely unknown and need clarification for using opioids more effectively in appropriately selected patients. This ongoing study takes advantage of the twin paradigm to estimate the genetic and environmental contributions to inter-individual differences in opioid responses. Evidence of significant heritability will justify more detailed and extensive genomic studies. The enrollment target is 80 monozygotic and 45 dizygotic twin pairs who undergo a target-controlled infusion of the opioid alfentanil and saline placebo in sequential but randomized order. In a laboratory-type setting, well-defined pharmacodynamic endpoints are measured to quantify pain sensitivity, analgesic opioid effects, and aversive opioid effects including respiratory depression, sedation and reinforcing affective responses. First results obtained in 159 participants provide evidence for the feasibility and utility of this interventional study paradigm to estimate familial aggregation and heritability components of relevant drug effects. Areas highlighted in this report include recruitment strategies, required infrastructure and personnel, selection of relevant outcome measures, drug infusion algorithm minimizing pharmacokinetic variability, and considerations for optimizing data quality and quantity without hampering feasibility. Applying the twin paradigm to complex and potentially harmful studies comprehensively characterizing pharmacological response profiles is without much precedent. Methods and first results including heritability estimates for heat and cold pain sensitivity should be of interest to investigators considering similar studies.

Implementing Mass Cytometry at the Bedside to Study the Immunological Basis of Human Diseases: Distinctive Immune Features in Patients with a History of Term or Preterm Birth

Single‐cell technologies have immense potential to shed light on molecular and biological processes that drive human diseases. Mass cytometry (or Cytometry by Time Of Flight mass spectrometry, CyTOF) has already been employed in clinical studies to comprehensively survey patients’ circulating immune system. As interest in the “bedside” application of mass cytometry is growing, the delineation of relevant methodological issues is called for. This report uses a newly generated dataset to discuss important methodological considerations when mass cytometry is implemented in a clinical study. Specifically, the use of whole blood samples versus peripheral blood mononuclear cells (PBMCs), design of mass‐tagged antibody panels, technical and analytical implications of sample barcoding, and application of traditional and unsupervised approaches to analyze high‐dimensional mass cytometry datasets are discussed. A mass cytometry assay was implemented in a cross‐sectional study of 19 women with a history of term or preterm birth to determine whether immune traits in peripheral blood differentiate the two groups in the absence of pregnancy. Twenty‐seven phenotypic and 11 intracellular markers were simultaneously analyzed in whole blood samples stimulated with lipopolysaccharide (LPS at 0, 0.1, 1, 10, and 100 ng mL−1) to examine dose‐dependent signaling responses within the toll‐like receptor 4 (TLR4) pathway. Complementary analyses, grounded in traditional or unsupervised gating strategies of immune cell subsets, indicated that the prpS6 and pMAPKAPK2 responses in classical monocytes are accentuated in women with a history of preterm birth (FDR<1%). The results suggest that women predisposed to preterm birth may be prone to mount an exacerbated TLR4 response during the course of pregnancy. This important hypothesis‐generating finding points to the power of single‐cell mass cytometry to detect biologically important differences in a relatively small patient cohort.

An immune clock of human pregnancy

High-dimensional analysis reveals the chronology of immunological adaptations during the course of human pregnancies. Following the hand of the immunological clock Immune function is altered during pregnancy to protect the fetus from an immunological attack without disrupting protection against infection. Now, Aghaeepour et al. use mass cytometry to examine the precise timing of these pregnancy-induced changes in immune function and regulation. They developed an algorithm that captures the immunological timeline during pregnancy that both validates previous findings and sheds new light on immune cell interaction during gestation. By defining this immunological chronology during normal term pregnancy, they can now begin to determine which alterations associate with pregnancy-related pathologies. The maintenance of pregnancy relies on finely tuned immune adaptations. We demonstrate that these adaptations are precisely timed, reflecting an immune clock of pregnancy in women delivering at term. Using mass cytometry, the abundance and functional responses of all major immune cell subsets were quantified in serial blood samples collected throughout pregnancy. Cell signaling–based Elastic Net, a regularized regression method adapted from the elastic net algorithm, was developed to infer and prospectively validate a predictive model of interrelated immune events that accurately captures the chronology of pregnancy. Model components highlighted existing knowledge and revealed previously unreported biology, including a critical role for the interleukin-2–dependent STAT5ab signaling pathway in modulating T cell function during pregnancy. These findings unravel the precise timing of immunological events occurring during a term pregnancy and provide the analytical framework to identify immunological deviations implicated in pregnancy-related pathologies.