Lucy Fairclough

ELISA in the multiplex era: Potentials and pitfalls

Multiplex immunoassays confer several advantages over widely adopted singleplex immunoassays including increased efficiency at a reduced expense, greater output per sample volume ratios and higher throughput predicating more resolute, detailed diagnostics and facilitating personalised medicine. Nonetheless, to date, relatively few protein multiplex immunoassays have been validated for in vitro diagnostics in clinical/point‐of‐care settings. This review article will outline the challenges, which must be ameliorated prior to the widespread integration of multiplex immunoassays in clinical settings: (i) biomarker validation; (ii) standardisation of immunoassay design and quality control (calibration and quantification); (iii) availability, stability, specificity and cross‐reactivity of reagents; (iv) assay automation and the use of validated algorithms for transformation of raw data into diagnostic results. A compendium of multiplex immunoassays applicable to in vitro diagnostics and a summary of the diagnostic products currently available commercially are included, along with an analysis of the relative states of development for each format (namely planar slide based, suspension and planar/microtitre plate based) with respect to the aforementioned issues.

Quantitative Validation and Comparison of Multiplex Cytokine Kits

The focus of biomarker studies is shifting toward deciphering patterns of biomolecules as they provide a more comprehensive depiction of disease than individual biomarkers. Multiplexing technologies are crucial in deciphering such patterns, but it is essential that they are validated for reproducibility and precision to ensure accurate protein identification. Here the authors examine such properties in Cytokine Bead Array (CBA) and Luminex kits and compare concentration measurements to those obtained using enzyme-linked immunosorbent assay (ELISA). Luminex kits were found to be highly reproducible and reliable; however, CBA kits were not due to aberrant standards. Absolute cytokine concentrations were dependent on the detection kit, but correlations with ELISA were good for all technologies.

Enhanced effector function of cytotoxic cells in the induced sputum of COPD patients.

BackgroundWe have previously shown that NK (CD56+CD3-) and NKT-like (CD56+CD3+) cells are reduced in both numbers and cytotoxicity in peripheral blood. The aim of the present study was to investigate their numbers and function within induced sputum.MethodsInduced sputum cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD56+ cells (NK and NKT-like cells) were used in an LDH release assay to determine cytotoxicity.ResultsThe proportion of NK cells and NKT-like cells in smokers with COPD (COPD subjects) was significantly higher (12.7% and 3%, respectively) than in healthy smokers (smokers) (5.7%, p < 0.01; 1%, p < 0.001) and non-smoking healthy subjects (HNS) (4.2%, p < 0.001; 0.8%, p < 0.01). The proportions of NK cells and NKT-like cells expressing both perforin and granzyme B were also significantly higher in COPD subjects compared to smokers and HNS. CD56+ cells from COPD subjects were significantly more cytotoxic (1414 biological lytic activity) than those from smokers (142.5; p < 0.01) and HNS (3.8; p < 0.001) and were inversely correlated to FEV1. (r = -0.75; p = 0.0098).ConclusionWe have shown an increased proportion of NK and NKT-like cells in the induced sputum of COPD subjects and have demonstrated that these cells are significantly more cytotoxic in COPD subjects than smokers and HNS.

Killer cells in chronic obstructive pulmonary disease

COPD (chronic obstructive pulmonary disease) is a treatable and preventable disease state, characterized by progressive airflow limitation that is not fully reversible. It is a current and growing cause of mortality and morbidity worldwide, with the WHO (World Health Organization) projecting that total deaths attributed to COPD will increase by more than 30% in the next 10 years. The pathological hallmarks of COPD are destruction of the lung parenchyma (pulmonary emphysema), inflammation of the central airways (chronic bronchitis) and inflammation of the peripheral airways (respiratory bronchiolitis). The destructive changes and tissue remodelling observed in COPD are a result of complex interactions between cells of the innate and adaptive immune systems. The focus of the present review is directed towards the role of CD8(+) T-lymphocytes, NK (natural killer) cells and NKT cells (NK T-cells). These three classes of killer cell could all play an important part in the pathogenesis of COPD. The observed damage to the pulmonary tissue could be caused in three ways: (i) direct cytotoxic effect against the lung epithelium mediated by the activities of perforin and granzymes, (ii) FasL (Fas ligand)-induced apoptosis and/or (iii) cytokine and chemokine release. The present review considers the role of these killer cells in COPD.

Regulation in chronic obstructive pulmonary disease: the role of regulatory T-cells and Th17 cells

COPD (chronic obstructive pulmonary disease) is an inflammatory disorder of the airways, which is associated with irreversible airway obstruction. The pathological hallmarks of COPD are destruction of the lung parenchyma (pulmonary emphysema), inflammation of the central airways (chronic bronchitis) and inflammation of the peripheral airways (respiratory bronchiolitis). Tobacco smoking is established as the main aetiological factor for COPD. A maladaptive modulation of inflammatory responses to inhalation of noxious particles and gases is generally accepted as being a key central pathogenic process; however, the precise regulatory mechanisms of the disease are poorly understood. Two cell types are known to be important in immune regulation, namely regulatory T-cells and the newly identified Th17 (T-helper 17) cells. Both types of cells are subsets of CD4 T-lymphocytes and modulate the immune response through secretion of cytokines, for example IL (interleukin)-10 and IL-17 respectively. The present review will begin by describing the current understanding of inflammatory cell involvement in the disease process, and then focus on the possible role of subsets of regulatory and helper T-cells in COPD.

Utility, reliability and reproducibility of immunoassay multiplex kits.

Multiplex technologies are becoming increasingly important in biomarker studies as they enable patterns of biomolecules to be examined, which provide a more comprehensive depiction of disease than individual biomarkers. They are crucial in deciphering these patterns, but it is essential that they are endorsed for reliability, reproducibility and precision. Here we outline the theoretical basis of a variety of multiplex technologies: Bead-based multiplex immunoassays (i.e. Cytometric Bead Arrays, Luminex™ and Bio-Plex Pro™), microtitre plate-based arrays (i.e. Mesoscale Discovery (MSD) and Quantsys BioSciences QPlex), Slide-based Arrays (i.e. FastQuant™) and reverse phase protein arrays. Their utility, reliability and reproducibility are discussed.

Altered effector function of peripheral cytotoxic cells in COPD

BackgroundThere is mounting evidence that perforin and granzymes are important mediators in the lung destruction seen in COPD. We investigated the characteristics of the three main perforin and granzyme containing peripheral cells, namely CD8+ T lymphocytes, natural killer (NK; CD56+CD3-) cells and NKT-like (CD56+CD3+) cells.MethodsPeripheral blood mononuclear cells (PBMCs) were isolated and cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD8+ T lymphocytes, NK (CD56+CD3-) and NKT-like (CD56+CD3+) cells were used in an LDH release assay to determine cytotoxicity and cytotoxic mechanisms were investigated by blocking perforin and granzyme B with relevant antibodies.ResultsThe proportion of peripheral blood NKT-like (CD56+CD3+) cells in smokers with COPD (COPD subjects) was significantly lower (0.6%) than in healthy smokers (smokers) (2.8%, p < 0.001) and non-smoking healthy participants (HNS) (3.3%, p < 0.001). NK (CD56+CD3-) cells from COPD subjects were significantly less cytotoxic than in smokers (16.8% vs 51.9% specific lysis, p < 0.001) as were NKT-like (CD56+CD3+) cells (16.7% vs 52.4% specific lysis, p < 0.001). Both cell types had lower proportions expressing both perforin and granzyme B. Blocking the action of perforin and granzyme B reduced the cytotoxic activity of NK (CD56+CD3-) and NKT-like (CD56+CD3+) cells from smokers and HNS.ConclusionIn this study, we show that the relative numbers of peripheral blood NK (CD56+CD3-) and NKT-like (CD56+CD3+) cells in COPD subjects are reduced and that their cytotoxic effector function is defective.

A pro-inflammatory signalome is constitutively activated by C33Y mutant TNF receptor 1 in TNF receptor-associated periodic syndrome (TRAPS)

Mutations in TNFRSF1A encoding TNF receptor 1 (TNFR1) cause the autosomal dominant TNF receptor‐associated periodic syndrome (TRAPS): a systemic autoinflammatory disorder. Misfolding, intracellular aggregation, and ligand‐independent signaling by mutant TNFR1 are central to disease pathophysiology. Our aim was to understand the extent of signaling pathway perturbation in TRAPS. A prototypic mutant TNFR1 (C33Y), and wild‐type TNFR1 (WT), were expressed at near physiological levels in an SK‐Hep‐1 cell model. TNFR1‐associated signaling pathway intermediates were examined in this model, and in PBMCs from C33Y TRAPS patients and healthy controls. In C33Y‐TNFR1‐expressing SK‐Hep‐1 cells and TRAPS patients’ PBMCs, a subtle, constitutive upregulation of a wide spectrum of signaling intermediates and their phosphorylated forms was observed; these were associated with a proinflammatory/antiapoptotic phenotype. In TRAPS patients’ PBMCs, this upregulation of proinflammatory signaling pathways was observed irrespective of concurrent treatment with glucocorticoids, anakinra or etanercept, and the absence of overt clinical symptoms at the time that the blood samples were taken. This study reveals the pleiotropic effect of a TRAPS‐associated mutant form of TNFR1 on inflammatory signaling pathways (a proinflammatory signalome), which is consistent with the variable and limited efficacy of cytokine‐blocking therapies in TRAPS. It highlights new potential target pathways for therapeutic intervention.

Differential Activation of Killer Cells in the Circulation and the Lung: A Study of Current Smoking Status and Chronic Obstructive Pulmonary Disease (COPD)

Background CD8+ T-lymphocytes, natural killer T-like cells (NKT-like cells, CD56+CD3+) and natural killer cells (NK cells, CD56+CD3−) are the three main classes of human killer cells and they are implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Activation of these cells can initiate immune responses by virtue of their production of inflammatory cytokines and chemokines that cause lung tissue damage, mucus hypersecretion and emphysema. The objective of the current study was to investigate the activation levels of human killer cells in healthy non-smokers, healthy smokers, ex-smokers with COPD and current smokers with COPD, in both peripheral blood and induced sputum. Methods/Principal Findings After informed consent, 124 participants were recruited into the study and peripheral blood or induced sputum was taken. The activation states and receptor expression of killer cells were measured by flow cytometry. In peripheral blood, current smokers, regardless of disease state, have the highest proportion of activated CD8+ T-lymphocytes, NKT-like cells and NK cells compared with ex-smokers with COPD and healthy non-smokers. Furthermore, CD8+ T-lymphocyte and NK cell activation is positively correlated with the number of cigarettes currently smoked. Conversely, in induced sputum, the proportion of activated killer cells was related to disease state rather than current smoking status, with current and ex-smokers with COPD having significantly higher rates of activation than healthy smokers and healthy non-smokers. Conclusions A differential effect in systemic and lung activation of killer cells in COPD is evident. Systemic activation appears to be related to current smoking whereas lung activation is related to the presence or absence of COPD, irrespective of current smoking status. These findings suggest that modulating killer cell activation may be a new target for the treatment of COPD.

Immunological basis of reversible and fixed airways disease.

Asthma is characterized by airflow obstruction that is usually completely reversible either spontaneously or in response to treatment. However, a small subset of patients with asthma display FAO (fixed airflow obstruction) despite optimal treatment, a feature more commonly associated with smoking-induced COPD (chronic obstructive pulmonary disease). Why some asthma patients develop FAO is not understood, and it is not clear whether (i) they represent a subset of patients with more severe disease, (ii) they share some characteristics of patients who develop COPD, or (iii) they represent a different disease entity altogether. The present review compares the pulmonary inflammatory profile of asthma patients with FAO with those without FAO, as well as COPD sufferers. The inflammation in asthma patients with FAO can vary from neutrophilic with CD8 T-cell involvement, similar to that of COPD, to eosinophilic with CD4 Th2 cell involvement, akin to that of asthma patients without FAO. Although studies of FAO in asthma sufferers would benefit hugely from consistent inclusion criteria, further research work is also required to shed more light on the immunological processes involved.