Perioperative redox changes in patients undergoing hepato-pancreatico-biliary cancer surgery.

BACKGROUND: Tissue injury induces inflammation and the surgical stress response, which are thought to be central to the orchestration of recovery or deterioration after surgery. Enhanced formation of reactive oxygen and nitrogen species accompanies the inflammatory response and triggers separate but integrated reduction/oxidation (redox) pathways that lead to oxidative and/or nitrosative stress (ONS). Quantitative information on ONS in the perioperative period is scarce. This single-centre exploratory study investigated the effects of major surgery on ONS and systemic redox status and their potential associations with postoperative morbidity. METHODS: Blood was collected from 56 patients at baseline, end of surgery (EoS) and the first postoperative day (day-1). Postoperative morbidity was recorded using the Clavien-Dindo classification and further categorised into minor, moderate and severe. Plasma/serum measures included markers of lipid oxidation (thiobarbituric acid-reactive substances; TBARS, 4-hydroxynonenal; 4-HNE, 8-iso-prostaglandin F2⍺; 8-isoprostanes). Total reducing capacity was measured using total free thiols (TFTs) and ferric-reducing ability of plasma (FRAP). Nitric oxide (NO) formation/metabolism was measured using cyclic guanosine monophosphate (cGMP), nitrite, nitrate and total nitroso-species (RxNO). Interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-⍺) were measured to evaluate inflammation. RESULTS: Both oxidative stress (TBARS) and nitrosative stress (total nitroso-species) increased from baseline to EoS (+14%, P = 0.003 and +138%, P < 0.001, respectively), along with an increase in overall reducing capacity (+9%, P = 0.03) at EoS and protein-adjusted total free thiols (+12%, P = 0.001) at day-1 after surgery. Nitrite, nitrate and cGMP concentrations declined concomitantly from baseline to day-1. Baseline nitrate was 60% higher in the minor morbidity group compared to severe (P = 0.003). The increase in intraoperative TBARS was greater in severe compared to minor morbidity (P = 0.01). The decline in intraoperative nitrate was more marked in the minor morbidity group compared to severe (P < 0.001), whereas the cGMP decline was greatest in the severe morbidity group (P = 0.006). CONCLUSION: In patients undergoing major HPB surgery, intraoperative oxidative and nitrosative stress increased, with a concomitant increase in reductive capacity. Baseline nitrate was inversely associated with postoperative morbidity, and the hallmarks of poor postoperative outcome include changes in both oxidative stress and NO metabolism.

Sputum processing by mechanical dissociation: A rapid alternative to traditional sputum assessment approaches.

BACKGROUND: Sputum cytology is currently the gold standard to evaluate cellular inflammation in the airways and phenotyping patients with airways diseases. Sputum eosinophil proportions have been used to guide treatment for moderate to severe asthma. Furthermore, raised sputum neutrophils are associated with poor disease control and impaired lung function in both asthma and COPD and small airways disease in cystic fibrosis. However, induced-sputum analysis is subjective and resource heavy, requiring dedicated specialist processing and assessment; this limits its utility in most clinical settings. Indirect blood eosinophil measures have been adopted in clinical care. However, there are currently no good peripheral blood biomarkers of airway neutrophils. A resource-light sputum processing approach could thus help integrate induced sputum more readily into routine clinical care. New mechanical disruption (MD) methods can rapidly obtain viable single cell suspensions from sputum samples. AIMS: The aim of this study was to compare MD sputum processing to traditional methods for cell viability, granulocyte proportions and sputum cytokine analysis. METHODS: Sputum plugs were split and processed using traditional methods and the MD method, and samples were then compared. RESULTS: The MD method produced a homogeneous cell suspension in 62 s; 70 min faster than the standard method used. No significant difference was seen between the cell viability (p = 0.09), or the concentration of eosinophils (p = 0.83), neutrophils (p = 0.99) or interleukin-8 (p = 0.86) using MD. CONCLUSION: This cost-effective method of sputum processing could provide a more pragmatic, sustainable means of directly monitoring the airway milieu. Therefore, we recommend this method be taken forward for further investigation.

Hair cortisol concentration in anxiety disorders: exploration of relationships with symptom severity and inflammatory markers.

BACKGROUND: Hair cortisol concentration (HCC) can be used to periodically assess hypothalamic-pituitary-adrenal (HPA) axis function, and appears correlated with prolonged exposure to stress. METHODS: Serial assessment (at Baseline, Week 6 and Week 12) of participants (n = 35) with anxiety disorders by psychopathological rating scales, with assays of HCC and levels of peripheral anti- and pro-inflammatory cytokines. Patients underwent antidepressant treatment for an initial 6 weeks, followed by cyclo-oxygenase inhibitor-2 (COX-2) inhibitor (celecoxib) augmentation or 'treatment as usual' for a further 6 weeks. RESULTS: At Baseline (n = 35), HCC was elevated in patients with single-episode but not recurrent-episode anxiety disorders, mean IL-12p70 levels were low, and mean TNF-α levels were elevated. Following 6 weeks of antidepressant treatment (n = 33), mean HCC was within the normal range but mean IL-2 level was low. Celecoxib augmentation (n = 18) was associated with a reduction in anxiety symptoms and normalisation of mean IL-2 levels. LIMITATIONS: Small sample size. Not all participants were assessed at all time points. CONCLUSION: Serial assessment of HCC is practicable in patients with anxiety disorders. These preliminary findings warrant further investigation in larger samples.

Peripheral immunophenotype in dementia with Lewy bodies and Alzheimer's disease: an observational clinical study.

BACKGROUND: Inflammation plays a key role in the aetiology and progression of Alzheimer's disease (AD). However, the immunophenotype of the second most common neurodegenerative cause of dementia, dementia with Lewy bodies (DLB), remains unclear. To date there have been no studies examining peripheral inflammation in DLB using multiplex immunoassay and flow cytometry concomitantly. We hypothesised that, using blood biomarkers, DLB would show an increased proinflammatory profile compared with controls, and that there would be a distinct profile compared with AD. METHODS: 93 participants (31 with DLB, 31 with AD and 31 healthy older controls) completed a single study visit for neuropsychiatric testing and phlebotomy. Peripheral blood mononuclear cells were quantified for T and B cell subsets using flow cytometry, and serum cytokine concentrations were measured using multiplex immunoassay. RESULTS: We detected reduced relative numbers of helper T cells and reduced activation of B cells in DLB compared with AD. Additionally, interleukin (IL)-1ß was detected more frequently in DLB and the serum concentration of IL-6 was increased compared with controls. CONCLUSIONS: Peripheral inflammation is altered in DLB compared with AD, with T cell subset analysis supporting a possible shift towards senescence of the adaptive immune system in DLB. Furthermore, there is a proinflammatory signature of serum cytokines in DLB. Identification of this unique peripheral immunophenotype in DLB could guide development of an immune-based biomarker and direct future work exploring potential immune modulation as a novel treatment.

Epithelial IL-6 trans-signaling defines a new asthma phenotype with increased airway inflammation.

BACKGROUND: Although several studies link high levels of IL-6 and soluble IL-6 receptor (sIL-6R) to asthma severity and decreased lung function, the role of IL-6 trans-signaling (IL-6TS) in asthmatic patients is unclear. OBJECTIVE: We sought to explore the association between epithelial IL-6TS pathway activation and molecular and clinical phenotypes in asthmatic patients. METHODS: An IL-6TS gene signature obtained from air-liquid interface cultures of human bronchial epithelial cells stimulated with IL-6 and sIL-6R was used to stratify lung epithelial transcriptomic data (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes [U-BIOPRED] cohorts) by means of hierarchical clustering. IL-6TS-specific protein markers were used to stratify sputum biomarker data (Wessex cohort). Molecular phenotyping was based on transcriptional profiling of epithelial brushings, pathway analysis, and immunohistochemical analysis of bronchial biopsy specimens. RESULTS: Activation of IL-6TS in air-liquid interface cultures reduced epithelial integrity and induced a specific gene signature enriched in genes associated with airway remodeling. The IL-6TS signature identified a subset of patients with IL-6TS-high asthma with increased epithelial expression of IL-6TS-inducible genes in the absence of systemic inflammation. The IL-6TS-high subset had an overrepresentation of frequent exacerbators, blood eosinophilia, and submucosal infiltration of T cells and macrophages. In bronchial brushings Toll-like receptor pathway genes were upregulated, whereas expression of cell junction genes was reduced. Sputum sIL-6R and IL-6 levels correlated with sputum markers of remodeling and innate immune activation, in particular YKL-40, matrix metalloproteinase 3, macrophage inflammatory protein 1ß, IL-8, and IL-1ß. CONCLUSIONS: Local lung epithelial IL-6TS activation in the absence of type 2 airway inflammation defines a novel subset of asthmatic patients and might drive airway inflammation and epithelial dysfunction in these patients.

Primary ciliary dyskinesia ciliated airway cells show increased susceptibility to Haemophilus influenzae biofilm formation.

Non-typeable Haemophilus influenzae (NTHi) is the most common pathogen in primary ciliary dyskinesia (PCD) patients. We hypothesised that abnormal ciliary motility and low airway nitric oxide (NO) levels on airway epithelial cells from PCD patients might be permissive for NTHi colonisation and biofilm development.We used a primary epithelial cell co-culture model to investigate NTHi infection. Primary airway epithelial cells from PCD and non-PCD patients were differentiated to ciliation using an air-liquid interface culture and then co-cultured with NTHi.NTHi adherence was greater on PCD epithelial cells compared to non-PCD cells (p<0.05) and the distribution of NTHi on PCD epithelium showed more aggregated NTHi in biofilms (p<0.001). Apart from defective ciliary motility, PCD cells did not significantly differ from non-PCD epithelial cells in the degree of ciliation and epithelial integrity or in cytokine, LL-37 and NO production. Treatment of PCD epithelia using exogenous NO and antibiotic significantly reduced NTHi viability in biofilms compared with antibiotic treatment alone.Impaired ciliary function was the primary defect in PCD airway epithelium underlying susceptibility to NTHi biofilm development compared with non-PCD epithelium. Although NO responses were similar, use of targeted NO with antibiotics enhanced killing of NTHi in biofilms, suggesting a novel therapeutic approach.

Peripheral inflammatory cytokines and immune balance in Generalised Anxiety Disorder: Case-controlled study.

INTRODUCTION: Previous investigations have demonstrated that major depression is associated with particular patterns of cytokine signalling. The primary aim of this study was to examine peripheral pro-inflammatory and anti-inflammatory cytokines and immune balance in Generalised Anxiety Disorder (GAD). METHODS: A case-controlled cross-sectional study design was employed: 54 patients with GAD and 64 healthy controls were recruited. Participants completed self-report measures of anxiety and depression. Two pro-inflammatory and two anti-inflammatory cytokines were measured using multiplex technology. RESULTS: Case-control logistic regression analyses revealed significant differences in serum levels of IL-10, TNF-α, and IFN-γ between GAD and control groups after adjusting for age, gender, body mass index, smoking and alcohol consumption: these group differences were independent of the presence or degree of depression. Comparison of pro-inflammatory to anti-inflammatory cytokine ratios indicated that there were significantly higher ratios of TNF-α/IL10, TNF-α/IL4, IFN-γ/IL10, and IFN-γ/IL4 in the GAD group compared to the control group. CONCLUSIONS: This study is the first to investigate both pro- and anti-inflammatory cytokines and their balance in patients with GAD in comparison to healthy controls. The findings indicate a relatively increased pro-inflammatory response and decreased anti-inflammatory response and provide the first demonstration of an altered cytokine balance in GAD. Serum cytokine levels in GAD were independent of the presence of depression.

Common airborne fungi induce species-specific effects on upper airway inflammatory and remodelling responses.

OBJECTIVE: Whilst the exact cause of chronic rhinosinusitis (CRS) remains elusive, it is clear that both inflammation and remodelling are key disease processes. Environmental fungi have been linked to airway inflammation in CRS; however, their role in the pathogenesis of this condition remains controversial. The current consensus suggests that whilst fungi may not be directly causative, it is likely that CRS patients have deficits in their innate and potentially acquired immunity, which in turn may modify their ability to react to fungi. This study used a nasal polyp explant tissue stimulation model to study the inflammatory and remodelling responses related to challenge with common airborne fungal species. METHODS: Ex vivo nasal polyp tissue from six well phenotyped CRSwNP patients undergoing functional endoscopic sinus surgery was stimulated with 1, 10 and 100 µg/ml of Alternaria alternata, Aspergillus niger, Cladosporium sphaerospermum and Penicillium notatum and compared with unchallenged polyp tissue as control. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of pro-inflammatory cytokines interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF) and tumour necrosis factor-α (TNF-α); and pro-remodelling cytokines transforming growth factor-b1 (TGF-b1), and basic fibroblast growth factor (bFGF) in the polyp supernatant. RESULTS: Aspergillus niger stimulation increased pro-inflammatory cytokines TNF-α, GM-CSF and IL-6 whilst having little effect on the remodelling cytokines bFGF and TGF-b1. In contrast, stimulation with Cladosporium sphaerospermum, Alternaria alternata and Penicillium notatum reduced pro-inflammatory cytokines TNF-α and IL-6, but induced a dose-dependent increase in remodelling cytokines TGF-b1 and bFGF. CONCLUSIONS: This study shows that common airborne fungi induce species-specific effects on the upper airway inflammatory and remodelling responses. These findings provide further immunological evidence of a disease-modifying role for fungi in CRS.

EGF-induced bronchial epithelial cells drive neutrophil chemotactic and anti-apoptotic activity in asthma.

Chronic damage and repair of the bronchial epithelium are features of asthma. We have previously reported that ex vivo stimulation of normal bronchial epithelial cells with epidermal growth factor (EGF), a key factor of epithelial repair, enhances the mechanisms of neutrophil accumulation, thereby promoting neutrophil defences during acute injury but potentially enhancing inflammation in chronic airway diseases. We have now sought to (i) determine whether this EGF-dependent pro-neutrophil activity is increased in asthma, where EGF and its epithelial receptor are over-expressed, and (ii) elucidate some of the mechanisms underlying this asthmatic epithelial-neutrophil interaction. Primary bronchial epithelial cells (PBEC) from healthy subjects, mild asthmatics and moderate-to-severe asthmatics (Mod/Sev) were stimulated with EGF, a model that mimics a repairing epithelium. Conditioned culture media (EGF-CM) were assessed for neutrophil chemotactic and anti-apoptotic activities and inflammatory mediator production. EGF induced the epithelium to produce soluble mediators with neutrophil chemotactic (p<0.001) and pro-survival (p = 0.021) activities which were related to the clinical severity of asthma (trend p = 0.010 and p = 0.009, respectively). This was associated with enhanced IL-6, IL-8, GM-CSF and TNF-α release, and cytokine-neutralising experiments using EGF-CM from Mod/Sev asthmatics demonstrated a role for GM-CSF in neutrophil survival (p<0.001). Pre-treatment of neutrophils with specific inhibitors of the myeloid-restricted class I phosphatidylinositol-3-OH kinase (PI(3)K) isoforms showed that the EGF-CM from Mod/Sev asthmatics depended on the γ (p<0.021) but not δ isoforms, while neutrophil survival required multiple class I PI(3)Ks. The EGF-induced chemotactic, but not pro-survival activity, involved RhoA signaling in neutrophils (p = 0.012). EGF whose activity is upregulated in asthma induces ex vivo the epithelium from asthmatic patients to produce pro-neutrophil activities; these are related to asthma severity and, in moderate-to-severe asthmatics, involves class IB PI(3)Kγ signaling, providing a potential therapeutic target for neutrophilic forms of asthma.

Resistin-like molecule-ß is induced following bronchoconstriction of asthmatic airways.

BACKGROUND AND OBJECTIVE: Resistin-like molecule-ß (RELM-ß) is a necessary and sufficient stimulus for airway remodelling in animal models of asthma, but until recently, its role in human disease had not been investigated. The hypothesis that RELM-ß expression would increase with increasing asthma severity and further increase following acute bronchoconstrictor challenges has been examined. METHODS: Bronchial biopsies from healthy subjects and patients with mild and severe asthma were immunostained for RELM-ß, as were airway biopsies obtained in mild asthmatics before and 4 days after repeated inhalation challenges with either allergen, methacholine or methacholine preceded by salbutamol as a control. Bronchial brushings were also evaluated for RELM-ß mRNA. RESULTS: RELM-ß immunoreactivity, which co-localized to airway epithelial cells, increased with disease severity; healthy volunteers, median per cent epithelial area 1.98%, mild asthma 3.49% and severe asthma 5.89% (P < 0.001 between groups). RELM-ß immunoreactivity significantly and inversely correlated in asthma with forced expiratory volume in 1 s % predicted (P = 0.005). Acute changes in immunoexpression were evident after repeated inhalation challenge with allergen (2.15 % to 4.35 % (P = 0.01)) and methacholine (4.21 % to 6.16 % (P = 0.01)) but did not change in the salbutamol/methacholine challenge group. These changes correlated with change in basement membrane thickness (r = 0.38, P = 0.02). Epithelial RELM-ß gene expression was not altered in asthma. CONCLUSIONS: RELM-ß may play an important role not only in animal models of airway remodelling, but also in human airway pathology.

Tissue factor-bearing exosome secretion from human mechanically stimulated bronchial epithelial cells in vitro and in vivo.

BACKGROUND: Tissue factor (TF), a primary initiator of blood coagulation, also plays a pivotal role in angiogenesis. TF expression in the airways is associated with asthma, a disease characterized in part by subepithelial angiogenesis. OBJECTIVES: To determine potential sources of TF and the mechanisms of its availability in the lung microenvironment. METHODS: Normal human bronchial epithelial cells grown in air-liquid interface culture were subjected to a compressive stress of 30 cm H(2)O; this is comparable to that generated in the airway epithelium during bronchoconstriction in asthma. Conditioned media and cells were harvested to measure TF mRNA and TF protein. We also tested bronchoalveolar lavage fluid and airway biopsies from asthmatic patients and healthy controls for TF. RESULTS: TF mRNA was upregulated 2.2-fold after 3 hours of stress compared with unstressed cells. Intracellular and secreted TF proteins were enhanced 1.6-fold and more than 50-fold, respectively, compared with those of control cells after the onset of compression. The amount of TF in the bronchoalveolar lavage fluid from patients with asthma was found at mean concentrations that were 5 times greater than those of healthy controls. Immunohistochemical staining of endobronchial biopsies identified epithelial localization of TF with increased expression in asthma. Exosomes isolated from the conditioned media of normal human bronchial epithelial cells and the bronchoalveolar lavage fluid of asthmatic subjects by ultracentrifugation contained TF. CONCLUSIONS: Our in vitro and in vivo studies show that mechanically stressed bronchial epithelial cells are a source of secreted TF and that exosomes are potentially a key carrier of the TF signal.

Effect of bronchoconstriction on airway remodeling in asthma.

BACKGROUND: Asthma is characterized pathologically by structural changes in the airway, termed airway remodeling. These changes are associated with worse long-term clinical outcomes and have been attributed to eosinophilic inflammation. In vitro studies indicate, however, that the compressive mechanical forces that arise during bronchoconstriction may induce remodeling independently of inflammation. We evaluated the influence of repeated experimentally induced bronchoconstriction on airway structural changes in patients with asthma. METHODS: We randomly assigned 48 subjects with asthma to one of four inhalation challenge protocols involving a series of three challenges with one type of inhaled agent presented at 48-hour intervals. The two active challenges were with either a dust-mite allergen (which causes bronchoconstriction and eosinophilic inflammation) or methacholine (which causes bronchoconstriction without eosinophilic inflammation); the two control challenges (neither of which causes bronchoconstriction) were either saline alone or albuterol followed by methacholine (to control for nonbronchoconstrictor effects of methacholine). Bronchial-biopsy specimens were obtained before and 4 days after completion of the challenges. RESULTS: Allergen and methacholine immediately induced similar levels of bronchoconstriction. Eosinophilic inflammation of the airways increased only in the allergen group, whereas both the allergen and the methacholine groups had significant airway remodeling not seen in the two control groups. Subepithelial collagen-band thickness increased by a median of 2.17 µm in the allergen group (interquartile range [IQR], 0.70 to 3.67) and 1.94 µm in the methacholine group (IQR, 0.37 to 3.24) (P<0.001 for the comparison of the two challenge groups with the two control groups); periodic acid-Schiff staining of epithelium (mucus glands) also increased, by a median of 2.17 percentage points in the allergen group (IQR, 1.03 to 4.77) and 2.13 percentage points in the methacholine group (IQR, 1.14 to 7.96) (P=0.003 for the comparison with controls). There were no significant differences between the allergen and methacholine groups. CONCLUSIONS: Bronchoconstriction without additional inflammation induces airway remodeling in patients with asthma. These findings have potential implications for management.

Double-stranded RNA induces disproportionate expression of thymic stromal lymphopoietin versus interferon-beta in bronchial epithelial cells from donors with asthma.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that strongly activates dendritic cells and can initiate allergic inflammation. Since exposure to rhinovirus or double-stranded (ds) RNA (a surrogate of viral infection) induces TSLP expression in bronchial epithelial cells (BECs), this cytokine may link innate antiviral responses and the type 2 adaptive immune response. OBJECTIVE: As BECs from donors with asthma have a deficient interferon (IFN) response to rhinovirus infection, a study was undertaken to test the hypothesis that their antiviral response shows a bias towards TSLP production. METHODS: Primary BECs were grown from subjects with asthma and healthy volunteers. After exposure to dsRNA, interleukin (IL)-8, IFNbeta and TSLP mRNA and protein expression were measured by RT-qPCR and ELISA, respectively. RESULTS: dsRNA dose-dependently increased IL-8 expression in BECs with no significant difference between the groups. However, BECs from subjects with asthma expressed less IFNbeta and more TSLP mRNA and protein in response to dsRNA than BECs from those without asthma (median (IQR) 57 (38-82) pg/ml vs 106 (57-214) pg/ml for IFNbeta (p<0.05) and 114 (86-143) pg/ml vs 65 (32-119) pg/ml for TSLP (p<0.05) in response to 10 microg/ml dsRNA for 24 h). Induction of TSLP mRNA by dsRNA was blocked by Toll-like receptor 3 or protein kinase inhibitors or by preventing de novo protein synthesis, but not by neutralisation of type I IFN receptors. CONCLUSION: BECs from subjects with asthma are biased towards higher TSLP and lower IFNbeta production in response to dsRNA, suggesting that viral infection in asthma may lead to an altered mediator profile that biases towards a Th2 immune response.