Cigarette Smoke Constituents and Nicotine Differentially Affect Cytokine Production by Human Macrophages Stimulated by TLR Ligands In Vitro: Considerations for a Standardised Protocol.

Chronic obstructive pulmonary disease (COPD) is an inflammatory lung condition associated with cigarette (tobacco) smoking. Numerous in vivo animal studies have been conducted to investigate the links between cigarette smoke, nicotine and infection in lung pathology. As an alternative to animal experiments, we used an in vitro system to investigate the effects of cigarette smoke extract (CSE) or nicotine on TNF-α and IL-10 production by monocyte-derived human macrophages. The macrophages were simultaneously stimulated with either poly-IC (as a chemical surrogate for viral infection) or lipopolysaccharide (as a chemical surrogate for Gram-negative bacterial infection). CSE enhanced TNF-α production, whereas nicotine inhibited IL-10 production by the macrophages, particularly when co-stimulated with the microbial chemical surrogates. A system of this type may help to further our understanding of the immunological and inflammatory effects of smoking, without recourse to in vivo studies. Requirements for the optimisation and standardisation of such an in vitro system are also discussed.

Mutations in the binding site of TNFR1 PLAD reduce homologous interactions but can enhance antagonism of wild-type TNFR1 activity.

The tumour necrosis factor receptor superfamily (TNFRSF) members contain cysteine-rich domains (CRD) in their extracellular regions, and the membrane-distal CRD1 forms homologous interactions in the absence of ligand. The CRD1 is therefore termed a pre-ligand assembly domain (PLAD). The role of PLAD-PLAD interactions in the induction of signalling as a consequence of TNF-TNFR binding led to the development of soluble PLAD domains as antagonists of TNFR activation. In the present study, we generated recombinant wild-type (WT) PLAD of TNFR1 and mutant forms with single alanine substitutions of amino acid residues thought to be critical for the formation of homologous dimers and/or trimers of PLAD (K19A, T31A, D49A and D52A). These mutated PLADs were compared with WT PLAD as antagonists of TNF-induced apoptosis or the activation of inflammatory signalling pathways. Unlike WT PLAD, the mutated PLADs showed little or no homologous interactions, confirming the importance of particular amino acids as contact residues in the PLAD binding region. However, as with WT PLAD, the mutated PLADs functioned as antagonists of TNF-induced TNFR1 activity leading to induction of cell death or NF-κB signalling. Indeed, some of the mutant PLADs, and K19A PLAD in particular, showed enhanced antagonistic activity compared with WT PLAD. This is consistent with the reduced formation of homologous multimers by these PLAD mutants effectively increasing the concentration of PLAD available to bind and antagonize WT TNFR1 when compared to WT PLAD acting as an antagonist. This may have implications for the development of antagonistic PLADs as therapeutic agents.

The role of CD8 + T lymphocytes in chronic obstructive pulmonary disease: a systematic review.

OBJECTIVE AND DESIGN: This systematic review aims to establish the role of CD8 + T lymphocytes in COPD. METHODS: Forty-eight papers published in the last 15 years were identified for inclusion. RESULTS: CD8 + T-cells are increased in the lungs of patients with COPD (17 studies, 16 positive) whereas in the circulation, findings were inconclusive. Activation of CD8 + T-cells was enhanced in lungs (four studies, three positive) but cell phenotype was unclear. There was substantial evidence of a higher proportion of type 1 CD8 + (Tc1) cells in COPD (11 studies, 9 positive), though the population of type 2 (Tc2) cells was also increased (5 studies, 4 positive). CD8 + T-cells in COPD exhibited greater expression of cytotoxic proteins (five studies, five positive). Studies assessed a variety of questions so evidence was insufficient to draw firm conclusions. The role of CD8 + T-cells at acute exacerbation of COPD and also their contribution to alveolar destruction can only be hypothesised at this stage. CONCLUSIONS: Not only is the number of CD8 + T-cells increased in COPD, these cells have increased capacity to exert effector functions and are likely to contribute to disease pathogenesis. Several mechanisms highlighted show promise for future investigation to consolidate current knowledge.

Extracellular vesicles and asthma: A review of the literature.

Asthma is a chronic, recurrent and incurable allergy-related respiratory disease characterized by inflammation, bronchial hyperresponsiveness and narrowing of the airways. Extracellular vesicles (EVs) are a universal feature of cellular function and can be detected in different bodily fluids. Recent evidence has shown the possibility of using EVs in understanding the pathogenesis of asthma, including their potential as diagnostic and therapeutic tools. Studies have reported that EVs released from key cells involved in asthma can induce priming and activation of other asthma-associated cells. A literature review was conducted on all current research regarding the role and function of EVs in the pathogenesis of asthma via the PRISMA statement method. An electronic search was performed using EMBASE and PubMed through to November 2018. The EMBASE search returned 76 papers, while the PubMed search returned 211 papers. Following duplicate removal, titles and abstracts were screened for eligibility with a total of 34 studies included in the final qualitative analysis. The review found evidence of association between the presence of EVs and physiological changes characteristic of asthma, suggesting that EVs are involved in the pathogenesis, with the weight of evidence presently favouring deleterious effects of EVs in asthma. Numerous studies highlighted differences in exosomal contents between EVs of healthy and asthmatic individuals, which could be employed as potential diagnostic markers. In some circumstances, EVs were also found to be suppressive to disease, but more often promote inflammation and airway remodelling. In conclusion, EVs hold immense potential in understanding the pathophysiology of asthma, and as diagnostic and therapeutic markers. While more research is needed for definitive conclusions and their application in medical practice, the literature presented in this review should encourage further research and discovery within the field of EVs and asthma.

Electronic cigarette vapour moderately stimulates pro-inflammatory signalling pathways and interleukin-6 production by human monocyte-derived dendritic cells.

Dendritic cells (DCs) are professional antigen presenting cells that play a critical role in bridging innate and adaptive immunity. Numerous studies have shown that tobacco constituents present in conventional cigarettes affect the phenotype and function of DCs; however, no studies have examined the effects of vapour from E-cigarettes on human DCs. Here, the effects of E-cigarette vapour extract (ECVE) on the phenotype and function of DCs were investigated by creating an in vitro cell culture model using human monocyte-derived DCs (MoDCs). Immature DCs were generated from peripheral blood monocytes and mature DCs were then produced by treatment with LPS or Poly I:C for 24 h. For LPS-matured DCs, 3% ECVE treatment slightly suppressed HLA-DR and CD86 expression, whereas 1% ECVE treatment enhanced IL-6 production. The overall expression of 29 signalling molecules and other cytoplasmic proteins (mainly associated with DC activation) was significantly upregulated in immature DCs by 1% ECVE, and in LPS-treated DCs by 3% ECVE. In particular, the condition that induced IL-6 production also upregulated MAPK pathway activation. These findings indicate that E-cigarette vapour moderately affects human DCs, but the effects are less pronounced than those reported for tobacco smoke.

Proximate underwater soundscape of a North Sea offshore petroleum exploration jack-up drilling rig in the Dogger Bank.

Little is known about localized, near-field soundscapes during offshore hydrocarbon drilling campaigns. In the Dogger Bank, North Sea, underwater noise recordings were made 41-60 m from the drill stem of the Noble Kolskaya jack-up exploration drilling rig. The aims were to document noise received levels (RLs) and frequency characteristics of rig-associated near-field noise. The rig produced sound pressure levels (SPLs) of 120 dB re 1 µPa in the frequency range of 2-1400 Hz. Over transient periods, RLs varied by 15-20 dB between softest (holding) and noisiest (drilling) operations. Tonal components at different frequencies varied with depth. Support vessel noise was significantly louder than the jack-up rig at frequencies <1 kHz, even in its noisiest "boulder-drilling" phase, though radiated noise levels were higher above 2 kHz. Rig SPLs fell rapidly above 8 kHz. Marine mammals, such as harbor porpoise (Phocoena phocoena) forage regularly near offshore oil and gas rigs and platforms, and it is predicted that animals experience different noise regimes while traversing the water column and can potentially detect the higher-frequency components of drilling noise to a distance of 70 m from the source; however, while levels were unlikely to cause auditory injury, effects on echolocation behavior are still unknown.

Positive mood on the day of influenza vaccination predicts vaccine effectiveness: A prospective observational cohort study.

Influenza vaccination is estimated to only be effective in 17-53% of older adults. Multiple patient behaviors and psychological factors have been shown to act as 'immune modulators' sufficient to influence vaccination outcomes. However, the relative importance of such factors is unknown as they have typically been examined in isolation. The objective of the present study was to explore the effects of multiple behavioral (physical activity, nutrition, sleep) and psychological influences (stress, positive mood, negative mood) on the effectiveness of the immune response to influenza vaccination in the elderly. A prospective, diary-based longitudinal observational cohort study was conducted. One hundred and thirty-eight community-dwelling older adults (65-85years) who received the 2014/15 influenza vaccination completed repeated psycho-behavioral measures over the two weeks prior, and four weeks following influenza vaccination. IgG responses to vaccination were measured via antigen microarray and seroprotection via hemagglutination inhibition assays at 4 and 16weeks post-vaccination. High pre-vaccination seroprotection levels were observed for H3N2 and B viral strains. Positive mood on the day of vaccination was a significant predictor of H1N1 seroprotection at 16weeks post-vaccination and IgG responses to vaccination at 4 and 16weeks post-vaccination, controlling for age and gender. Positive mood across the 6-week observation period was also significantly associated with post-vaccination H1N1 seroprotection and IgG responses to vaccination at 16weeks post-vaccination, but in regression models the proportion of variance explained was lower than for positive mood on the day of vaccination alone. No other factors were found to significantly predict antibody responses to vaccination. Greater positive mood in older adults, particularly on the day of vaccination, is associated with enhanced responses to vaccination.

Autoantibodies of IgM and IgG classes show differences in recognition of multiple autoantigens in chronic obstructive pulmonary disease.

Autoimmunity occurs in chronic obstructive pulmonary disease (COPD). We describe an antigen microarray for detecting serum autoantibodies (AAbs) to determine how IgM, as well as IgG, AAbs distinguish patients with COPD from controls with a history of smoking without COPD. All COPD patients' sera contained elevated levels of AAbs to some of 30 autoantigens. There were significant differences in the autoantigenic specificities of IgM AAbs compared to IgG AAbs in the COPD sera: for example, AAbs to histone and scl-70 were mainly IgG, whereas AAbs to CENP-B and La/ssB were mainly IgM; by contrast, IgM and IgG AAbs to collagen-V were equally prevalent. Thus, a combination of IgM and IgG AAbs specific for multiple autoantigens are detected in all cases of COPD at a level at which all non-COPD controls are negative for AAbs. This highlights the importance of different classes of AAbs to a range of autoantigens in COPD.

A signalome screening approach in the autoinflammatory disease TNF receptor associated periodic syndrome (TRAPS) highlights the anti-inflammatory properties of drugs for repurposing.

TNF receptor associated periodic syndrome (TRAPS) is an autoinflammatory disease caused by mutations in TNF Receptor 1 (TNFR1). Current therapies for TRAPS are limited and do not target the pro-inflammatory signalling pathways that are central to the disease mechanism. Our aim was to identify drugs for repurposing as anti-inflammatories based on their ability to down-regulate molecules associated with inflammatory signalling pathways that are activated in TRAPS. This was achieved using rigorously optimized, high through-put cell culture and reverse phase protein microarray systems to screen compounds for their effects on the TRAPS-associated inflammatory signalome. 1360 approved, publically available, pharmacologically active substances were investigated for their effects on 40 signalling molecules associated with pro-inflammatory signalling pathways that are constitutively upregulated in TRAPS. The drugs were screened at four 10-fold concentrations on cell lines expressing both wild-type (WT) TNFR1 and TRAPS-associated C33Y mutant TNFR1, or WT TNFR1 alone; signalling molecule levels were then determined in cell lysates by the reverse-phase protein microarray. A novel mathematical methodology was developed to rank the compounds for their ability to reduce the expression of signalling molecules in the C33Y-TNFR1 transfectants towards the level seen in the WT-TNFR1 transfectants. Seven high-ranking drugs were selected and tested by RPPA for effects on the same 40 signalling molecules in lysates of peripheral blood mononuclear cells (PBMCs) from C33Y-TRAPS patients compared to PBMCs from normal controls. The fluoroquinolone antibiotic lomefloxacin, as well as others from this class of compounds, showed the most significant effects on multiple pro-inflammatory signalling pathways that are constitutively activated in TRAPS; lomefloxacin dose-dependently significantly reduced expression of 7/40 signalling molecules across the Jak/Stat, MAPK, NF-κB and PI3K/AKT pathways. This study demonstrates the power of signalome screening for identifying candidates for drug repurposing.

Peripheral killer cells do not differentiate between asthma patients with or without fixed airway obstruction.

OBJECTIVE: The three main types of killer cells - CD8+ T cells, NK cells and NKT cells - have been linked to asthma and chronic obstructive pulmonary disease (COPD). However, their role in a small subset of asthma patients displaying fixed airway obstruction (FAO), similar to that seen in COPD, has not been explored. The objective of the present study was to investigate killer cell numbers, phenotype and function in peripheral blood from asthma patients with FAO, asthma patients without FAO, and healthy individuals. METHODS: Peripheral CD8+ T cells (CD8+CD3+CD56-), NK cells (CD56+CD3-) and NKT-like cells (CD56+CD3+) of 14 asthma patients with FAO (post-bronchodilator FEV/FVC <0.7, despite clinician-optimised treatment), 7 asthma patients without FAO (post-bronchodilator FEV/FVC ≥ 0.7), and 9 healthy individuals were studied. RESULTS: No significant differences were seen between the number, receptor expression, MAPK signalling molecule expression, cytotoxic mediator expression, and functional cytotoxicity of peripheral killer cells from asthma patients with FAO, asthma patients without FAO and healthy individuals. CONCLUSIONS: Peripheral killer cell numbers or functions do not differentiate between asthma patients with or without fixed airway obstruction.

Tumour necrosis factor receptor I blockade shows that TNF-dependent and TNF-independent mechanisms synergise in TNF receptor associated periodic syndrome.

TNF receptor associated periodic syndrome (TRAPS) is an autoinflammatory disease involving recurrent episodes of fever and inflammation. It is associated with autosomal dominant mutations in TNF receptor superfamily 1A gene localised to exons encoding the ectodomain of the p55 TNF receptor, TNF receptor-1 (TNFR1). The aim of this study was to investigate the role of cell surface TNFR1 in TRAPS, and the contribution of TNF-dependent and TNF-independent mechanisms to the production of cytokines. HEK-293 and SK-HEP-1 cell lines were stably transfected with WT or TRAPS-associated variants of human TNF receptor superfamily 1A gene. An anti-TNFR1 single domain antibody (dAb), and an anti-TNFR1 mAb, bound to cell surface WT and variant TNFR1s. In HEK-293 cells transfected with death domain-inactivated (R347A) TNFR1, and in SK-HEP-1 cells transfected with normal (full-length) TNFR1, cytokine production stimulated in the absence of exogenous TNF by the presence of certain TNFR1 variants was not inhibited by the anti-TNFR1 dAb. In SK-Hep-1 cells, specific TRAPS mutations increased the level of cytokine response to TNF, compared to WT, and this augmented cytokine production was suppressed by the anti-TNFR1 dAb. Thus, TRAPS-associated variants of TNFR1 enhance cytokine production by a TNF-independent mechanism and by sensitising cells to a TNF-dependent stimulation. The TNF-dependent mechanism requires cell surface expression of TNFR1, as this is blocked by TNFR1-specific dAb.

Multiple Circulating Cytokines Are Coelevated in Chronic Obstructive Pulmonary Disease.

Inflammatory biomarkers, including cytokines, are associated with COPD, but the association of particular circulating cytokines with systemic pathology remains equivocal. To investigate this, we developed a protein microarray system to detect multiple cytokines in small volumes of serum. Fourteen cytokines were measured in serum from never-smokers, ex-smokers, current smokers, and COPD patients (GOLD stages 1-3). Certain individual circulating cytokines (particularly TNFα and IL-1ß) were significantly elevated in concentration in the serum of particular COPD patients (and some current/ex-smokers without COPD) and may serve as markers of particularly significant systemic inflammation. However, numerous circulating cytokines were raised such that their combined, but not individual, elevation was significantly associated with severity of disease, and these may be further indicators of, and contributors to, the systemic inflammatory manifestations of COPD. The coelevation of numerous circulating cytokines in COPD is consistent with the insidious development, chronic nature, and systemic comorbidities of the disease.

Modifying Hofstee standard setting for assessments that vary in difficulty, and to determine boundaries for different levels of achievement.

BACKGROUND: Fixed mark grade boundaries for non-linear assessment scales fail to account for variations in assessment difficulty. Where assessment difficulty varies more than ability of successive cohorts or the quality of the teaching, anchoring grade boundaries to median cohort performance should provide an effective method for setting standards. METHODS: This study investigated the use of a modified Hofstee (MH) method for setting unsatisfactory/satisfactory and satisfactory/excellent grade boundaries for multiple choice question-style assessments, adjusted using the cohort median to obviate the effect of subjective judgements and provision of grade quotas. RESULTS: Outcomes for the MH method were compared with formula scoring/correction for guessing (FS/CFG) for 11 assessments, indicating that there were no significant differences between MH and FS/CFG in either the effective unsatisfactory/satisfactory grade boundary or the proportion of unsatisfactory graded candidates (p > 0.05). However the boundary for excellent performance was significantly higher for MH (p < 0.01), and the proportion of candidates returned as excellent was significantly lower (p < 0.01). MH also generated performance profiles and pass marks that were not significantly different from those given by the Ebel method of criterion-referenced standard setting. CONCLUSIONS: This supports MH as an objective model for calculating variable grade boundaries, adjusted for test difficulty. Furthermore, it easily creates boundaries for unsatisfactory/satisfactory and satisfactory/excellent performance that are protected against grade inflation. It could be implemented as a stand-alone method of standard setting, or as part of the post-examination analysis of results for assessments for which pre-examination criterion-referenced standard setting is employed.

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.

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.

Development and validation of protein microarray technology for simultaneous inflammatory mediator detection in human sera.

Biomarkers, including cytokines, can help in the diagnosis, prognosis, and prediction of treatment response across a wide range of disease settings. Consequently, the recent emergence of protein microarray technology, which is able to quantify a range of inflammatory mediators in a large number of samples simultaneously, has become highly desirable. However, the cost of commercial systems remains somewhat prohibitive. Here we show the development, validation, and implementation of an in-house microarray platform which enables the simultaneous quantitative analysis of multiple protein biomarkers. The accuracy and precision of the in-house microarray system were investigated according to the Food and Drug Administration (FDA) guidelines for pharmacokinetic assay validation. The assay fell within these limits for all but the very low-abundant cytokines, such as interleukin- (IL-) 10. Additionally, there were no significant differences between cytokine detection using our microarray system and the "gold standard" ELISA format. Crucially, future biomarker detection need not be limited to the 16 cytokines shown here but could be expanded as required. In conclusion, we detail a bespoke protein microarray system, utilizing well-validated ELISA reagents, that allows accurate, precise, and reproducible multiplexed biomarker quantification, comparable with commercial ELISA, and allowing customization beyond that of similar commercial microarrays.

Role of interleukin-6 in a patient with tumor necrosis factor receptor-associated periodic syndrome: assessment of outcomes following treatment with the anti-interleukin-6 receptor monoclonal antibody tocilizumab.

In this report, we describe treatment outcomes in the first case of a patient with tumor necrosis factor receptor-associated periodic syndrome (TRAPS) treated with the anti-interleukin-6 (anti-IL-6) receptor monoclonal antibody tocilizumab. Since IL-6 levels are elevated in TRAPS, we hypothesized that tocilizumab might be effective. The patient, a 52-year-old man with lifelong TRAPS in whom treatment with etanercept and anakinra had failed, was administered tocilizumab for 6 months, and the therapeutic response was assessed by measurement of monocyte CD16 expression and cytokine levels. Following treatment, the evolving acute attack was aborted and further attacks of TRAPS were prevented. The patient did not require corticosteroids and showed significant clinical improvement in scores for pain, stiffness, and well-being. Moreover, the acute-phase response diminished significantly with treatment. Monocyte CD16 expression was reduced and the numbers of circulating CD14+CD16+ and CD14++CD16- monocytes were transiently decreased. However, cytokine levels were not reduced. This case supports the notion of a prominent role for IL-6 in mediating the inflammatory attacks in TRAPS, but blockade of IL-6 did not affect the underlying pathogenesis. These preliminary findings require confirmation.

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.

Array-based measurements of aero-allergen-specific IgE correlate with skin-prick test reactivity in asthma regardless of specific IgG4 or total IgE measurements.

Skin prick testing (SPT) and measurement of serum allergen-specific IgE (sIgE) are used to investigate asthma and other allergic conditions. Measurement of serum total IgE (tIgE) and allergen-specific IgG4 (sIgG4) may also be useful. The aim was to ascertain the correlation between these serological parameters and SPT. Sera from 60 suspected asthmatic patients and 18 healthy controls were assayed for sIgE and sIgG4 reactivity against a panel of 70 SPT allergen preparations, and for tIgE. The patients were also assessed by skin prick tests for reactivity to cat, dog, house dust mite and grass allergens. Over 50% of the patients had tIgE levels above the 75th percentile of the controls. 58% of patients and 39% of controls showed sIgE reactivity to ≥1 allergen. The mean number of allergens detected by sIgE was 3.1 in suspected asthma patients and 0.9 in controls. 58% of patients and 50% of controls showed sIgG4 reactivity to ≥1 allergen. The mean number of allergens detected by sIgG4 was 2.5 in patients and 1.7 in controls. For the patients, a strong correlation was observed between clinical SPT reactivity and serum sIgE levels to cat, dog, house dust mite (HDM) and grass allergens. SPT correlations using sIgE/sIgG4 or sIgE/tIgE ratios were not markedly higher. The measurement of serum sIgE by microarray using SPT allergen preparations showed good correlation with clinical SPT reactivity to cat, dog, HDM and grass allergens. This concordance was not improved by measuring tIgE or sIgG4.