Improved Methods for Quantifying Human Chemokine and Cytokine Biomarker Responses: Ultrasensitive ELISA and Meso Scale Electrochemiluminescence Assays.

ELISAs and similar immunoassays are a backbone of biomedical research and clinical practice. Here we review the major factors to consider in the development and application of ultrasensitive ELISAs for analysis of human immune responses in plasma, serum, urine, or tissue culture supernatants. We focus on cytokine and chemokine biomarkers of health and chronic inflammatory diseases including allergy, asthma, autoimmunity, and cardiovascular disease.

Quantifying Human Innate Cytokine and Chemokine Responses Ex Vivo via Pattern Recognition Receptor Stimulation.

Linkages between human innate immune capacity, the environment in which we live, and the development of clinical tolerance versus a spectrum of disease phenotypes are a major focus of inflammatory disease research. While extensive epidemiologic evidence indicates key roles for the microbiome and other environmental factors, the underlying mechanisms that explain how these stimuli lead to a given clinical phenotype remain speculative. Here we review strategies for characterizing human cytokine production ex vivo in response to innate immune receptor stimulation with defined ligands.

Neurotransmitter signalling via NMDA receptors leads to decreased T helper type 1-like and enhanced T helper type 2-like immune balance in humans.

Given the pivotal roles that CD4 T cell imbalance plays in human immune disorders, much interest centres on better understanding influences that regulate human helper T-cell subset dominance in vivo. Here, using primary CD4 T cells and short-term T helper type 1 (Th1) and Th2-like lines, we investigated roles and mechanisms by which neurotransmitter receptors may influence human type 1 versus type 2 immunity. We hypothesized that N-methyl-d-aspartate receptors (NMDA-R), which play key roles in memory and learning, can also regulate human CD4 T cell function through induction of excitotoxicity.

In vivo immune signatures of healthy human pregnancy: Inherently inflammatory or anti-inflammatory?

Changes in maternal innate immunity during healthy human pregnancy are not well understood. Whether basal immune status in vivo is largely unaffected by pregnancy, is constitutively biased towards an inflammatory phenotype (transiently enhancing host defense) or exhibits anti-inflammatory bias (reducing potential responsiveness to the fetus) is unclear. Here, in a longitudinal study of healthy women who gave birth to healthy infants following uncomplicated pregnancies within the Canadian Healthy Infant Longitudinal Development (CHILD) cohort, we test the hypothesis that a progressively altered bias in resting innate immune status develops.

Class II obese and healthy pregnant controls exhibit indistinguishable pro- and anti-inflammatory immune responses to Caesarian section.

Introduction: Obesity during pregnancy is associated with meta-inflammation and an increased likelihood of clinical complications. Surgery results in intense, acute inflammatory responses in any individual. Because obese individuals exhibit constitutive inflammatory responses and high rates of Caesarian section, it is important to understand the impact of surgery in such populations.

Characteristics and outcomes of Canadian MD/PhD program graduates: a cross-sectional survey.

Background: Combined MD/PhD programs provide a structured path for physician-scientist training, but assessment of their success within Canada is limited by a lack of quantitative data. We collected outcomes data for graduates of Canadian MD/PhD programs.

Methods: We developed and implemented a Web-based survey consisting of 41 questions designed to collect outcomes data for Canadian MD/PhD program alumni from 8 Canadian universities who had graduated before September 2015.

Stability of pro- and anti-inflammatory immune biomarkers for human cohort studies.

Background: Although discovery research has identified the importance of dozens of pro- and anti-inflammatory immune mediators in the pathogenesis, maintenance, exacerbation and resolution of inflammatory diseases, most human cohort studies have incorporated few or no immunological intermediate phenotypes in their analyses. Significant hindrances have been (1) the limited panel of biomarkers known to be readily detected in healthy human populations and (2) the stability, hence utility, of such biomarkers to repeated analysis.

Methods: The frequency and stability of 14 plasma biomarkers linked to in vivo immune regulation of allergic and autoimmune inflammatory disorders was determined in 140 healthy pediatric and adult participants.

High Spinal Anesthesia Enhances Anti-Inflammatory Responses in Patients Undergoing Coronary Artery Bypass Graft Surgery and Aortic Valve Replacement: Randomized Pilot Study.

Background: Cardiac surgery induces many physiologic changes including major inflammatory and sympathetic nervous system responses. Here, we conducted a single-centre pilot study to generate hypotheses on the potential immune impact of adding high spinal anaesthesia to general anaesthesia during cardiac surgery in adults. We hypothesized that this strategy, previously shown to blunt the sympathetic response and improve pain management, could reduce the undesirable systemic inflammatory responses caused by cardiac surgery.

Vitamin D [1,25(OH)2D3] Differentially Regulates Human Innate Cytokine Responses to Bacterial versus Viral Pattern Recognition Receptor Stimuli.

Vitamin D plays multiple roles in regulation of protective and maladaptive immunity. Although epidemiologic studies link poor in vivo 25(OH)D status to increased viral respiratory infections, we poorly understand how vitamin D affects viral pattern recognition receptor (PRR)-driven cytokine production. In this study, we hypothesized that the biologically active metabolite of vitamin D, 1,25(OH)2D3, inhibits human proinflammatory and anti-inflammatory innate cytokine responses stimulated by representative bacterial or viral PRR ligands.

Acute Kidney Injury in Patients Undergoing Open Abdominal Aortic Aneurysm Repair: A Pilot Observational Trial.

Objectives: Acute kidney injury (AKI) is a frequent complication after open repair of abdominal aortic aneurysms (AAA). Little research has been done to determine whether intraoperative hemodynamic events may precipitate AKI. Novel biomarkers also may aid in the earlier diagnosis of AKI.

The Canadian Healthy Infant Longitudinal Development (CHILD) Study: examining developmental origins of allergy and asthma.

The Canadian Healthy Infant Longitudinal Development (CHILD) birth cohort study recruited 3624 pregnant women, most partners and 3542 eligible offspring. We hypothesise that early life physical and psychosocial environments, immunological, physiological, nutritional, hormonal and metabolic influences interact with genetics influencing allergic diseases, including asthma. Environmental and biological sampling, innate and adaptive immune responses, gene expression, DNA methylation, gut microbiome and nutrition studies complement repeated environmental and clinical assessments to age 5.

A randomized controlled trial on the effects of goal-directed therapy on the inflammatory response open abdominal aortic aneurysm repair.

Introduction: Goal-directed therapy (GDT) has been shown in numerous studies to decrease perioperative morbidity and mortality. The mechanism of benefit of GDT, however, has not been clearly elucidated. Targeted resuscitation of the vascular endothelium with GDT might alter the postoperative inflammatory response and be responsible for the decreased complications with this therapy.

Infant gut microbiota and food sensitization: associations in the first year of life.

Background: The gut microbiota is established during infancy and plays a fundamental role in shaping host immunity. Colonization patterns may influence the development of atopic disease, but existing evidence is limited and conflicting.

Objective: To explore associations of infant gut microbiota and food sensitization.

The Canadian healthy infant longitudinal development birth cohort study: biological samples and biobanking.

Background: It is hypothesised that complex interactions between genetic and environmental factors give rise to allergy and asthma in childhood. The Canadian Healthy Infant Longitudinal Development (CHILD) study was designed to explore these factors.

Methods: CHILD is a longitudinal, general population birth cohort study following infants from mid-pregnancy to age 5 years.

Sustained suppression of IL-13 by a vaccine attenuates airway inflammation and remodeling in mice.

We previously reported that a recombinant IL-13 peptide-based virus-like particle vaccine significantly suppressed murine acute airway allergic inflammatory responses. The impact of this strategy on the development of chronic airway inflammation and remodeling has not been investigated. We evaluated whether the vaccine-mediated sustained suppression of IL-13 attenuates features of chronic airway inflammation and remodeling in mice repeatedly challenged with allergen.

Elevated antigen-driven IL-9 responses are prominent in peanut allergic humans.

Food allergies, and peanut allergy in particular, are leading causes of anaphylactic fatalities worldwide. The immune mechanisms that underlie food allergy remain ill-defined and controversial, in part because studies in humans typically focus on analysis of a limited number of prototypical Th1/Th2 cytokines. Here we determine the kinetics and prevalence of a broad panel of peanut-driven cytokine and chemokine responses in humans with current peanut allergy vs those with stable, naturally occurring clinical tolerance to peanut.

Influence of socioeconomic status trajectories on innate immune responsiveness in children.

Objectives: Lower socioeconomic status (SES) is consistently associated with poor health, yet little is known about the biological mechanisms underlying this inequality. In children, we examined the impact of early-life SES trajectories on the intensity of global innate immune activation, recognizing that excessive activation can be a precursor to inflammation and chronic disease.

Methods: Stimulated interleukin-6 production, a measure of immune responsiveness, was analyzed ex vivo for 267 Canadian schoolchildren from a 1995 birth cohort in Manitoba, Canada.

Validation of urinary CXCL10 as a marker of borderline, subclinical, and clinical tubulitis.

Background: Renal allograft injury secondary to subclinical and clinical tubulitis remains an important cause of allograft fibrosis and loss despite modern immunosuppression. The goal of this study was to validate the previously reported use of urinary CXCL10 (interferon-γ-induced protein of 10 kDa) as a noninvasive marker of tubulitis in an independent clinical cohort.

Methods: Urine samples (n=102) from 91 patients with protocol or indication biopsies were assayed for urinary CXCL10 using ELISA.

Maternal distress in early life predicts the waist-to-hip ratio in schoolchildren.

We report on life course stress determinants of overweight in children, using data from the longitudinal follow-up of the nested case-control arm of the SAGE (study of asthma genes and the environment) birth cohort in Manitoba, Canada. Waist and hip measurements were obtained during a clinic visit at age 9-11 years. Multiple linear regression was conducted to determine the relationship between the waist-to-hip ratio and maternal smoking during pregnancy, postpartum maternal distress and stress reactivity in children (cortisol, cortisol-DHEA [dihydroepiandrostrenone] ratio quartiles) following a clinic stressor at age 8-10 years.

TLR4 Asp299Gly and Thr399Ile polymorphisms: no impact on human immune responsiveness to LPS or respiratory syncytial virus.

Background: A broad variety of natural environmental stimuli, genotypic influences and timing all contribute to expression of protective versus maladaptive immune responses and the resulting clinical outcomes in humans. The role of commonly co-segregating Toll-like receptor 4 (TLR4) non-synonymous single nucleotide polymorphisms Asp299Gly and Thr399Ile in this process remains highly controversial. Moreover, what differential impact these polymorphisms might have in at risk populations with respiratory dysfunction, such as current asthma or a history of infantile bronchiolitis, has never been examined.

Early urinary CCL2 is associated with the later development of interstitial fibrosis and tubular atrophy in renal allografts.

Background: Chronic renal allograft injury resulting in progressive interstitial fibrosis and tubular atrophy (IFTA) is a leading cause of graft loss. The goal of this study was to identify early urinary predictors for the subsequent development of IFTA in a prospective cohort of patients (n=111) who underwent serial protocol biopsies at 0, 6, and 24 months.

Methods: The urinary proteins evaluated were CCL2, CXCL9, CXCL10, and alpha1-microglobulin (alpha1M) using ELISA and immunonephelometry.

Are plasma IL-10 levels a useful marker of human clinical tolerance in peanut allergy?

Background: Food allergies are a major component of the burden of allergic disease. Accurate risk assessment for prediction of future clinical reactivity or clinical tolerance is limited by currently available techniques. Recent studies suggest that constitutively elevated global serum levels of IL-10, a cytokine that down-regulates both Th1 and Th2 cytokine production, may be useful in identifying human clinical tolerance to foods.

Lower cortisol levels in children with asthma exposed to recurrent maternal distress from birth.

Background: Existing evidence supports associations between exposure to maternal distress and the development of childhood asthma, between exposure to maternal distress and an increased cortisol response in children, and between childhood asthma and an attenuated cortisol response.

Objective: To investigate the association between children's cortisol levels and the combined predictors of exposure to maternal distress and childhood asthma.

Methods: Serum cortisol levels were examined at age 7 to 10 years in relation to asthma status and exposure to maternal distress in a representative sample of children (n = 503) born in 1995.