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OBJECTIVES The primary end point of this study was to evaluate the impact of bile acids on severity of laryngo-pharyngeal reflux (LPR) and the possible correlation with esophagitis and upper airway malignancies. The second end point was to evaluate if salivary bile acids and molecules other than pepsin might serve as diagnostic biomarkers of LPR. DESIGN Observational prospective comparative study. SETTING Otorhinolaryngology unit of a tertiary hospital. PARTICIPANTS Sixty-two consecutive adult outpatients suspected of LPR. MAIN OUTCOME MEASURES Bile acids, bilirubin and pepsinogen I-II were measured in saliva. Patients underwent pH metry and based on the results of bile acids were subdivided as acid, mixed and alkaline LPR. RESULTS Significantly higher Reflux Findings Score (RFS) and Reflux Symptoms Index (RSI) were seen in patients with alkaline and mixed LPR compared to acid LPR. Salivary bile acids >1 µmol/L seem to be a reliable indicator of the severity of LPR. Compared to those without, patients with esophagitis or a history of upper airway malignancy have high concentrations of bile acids in saliva. Among the molecules studied, bile acids were the most suitable for diagnosis of LPR, with a sensitivity of 86% and a positive predictive value of 80.7%. CONCLUSIONS Our data suggest that high concentrations of bile acids are associated with higher values of RSI and RFS in LPR as well as a higher risk of esophagitis and history of upper airway malignancies. We finally observed that bile acids provided the best biometric parameters for diagnosis of LPR among the molecules tested.

Objective: The aim of our study was to analyze the montelukast effectiveness in improving oculonasal symptoms, patient-reported outcomes (PROs), and eosinophilic biomarkers in patients with nonallergic rhinitis eosinophilic syndrome (NARES).Methods: We enrolled prospectively 80 symptomatic patients treated with 10 mg once a day of montelukast in mono-therapy for 2 months. All patients were investigated before and after treatment. Nasal symptoms (nasal obstruction, rhinor-rhoea, sneezing, nasal itching), ocular symptoms (redness/puffiness, watery eyes), and other PROs (olfactory dysfunction,difficulty going to sleep, nighttime awakenings, and nasal congestion on awakening) were scored by visual analogic scale. Thefollowing clinical scores were assessed: Total Nasal Symptom Score (T4NSS), Total Ocular Symptom Score (T2OSS), TotalSymptom Score of Patient-Reported Outcomes (TSS-PROs), and a Composite Symptoms Score (CSS). Patients were classified asresponders when a reduction of at least 50% of the CSS was observed. Before and after treatment, the eosinophilic biomarkersin nasal lavage were analyzed: nasal eosinophilia (number of eosinophils per high power field), eotaxin-1 and eotaxin-2.Results: Aft er tre atment, s ig nificant reductions were observed for all the symptom scores. Forty-two of 78 patients were con-sidered responders. A significant reduction of eosinophils in nasal mucosa and of levels of eotaxin-1 and eotaxin-2 in nasal lavagewere observed after treatment in responder patients. Patients with asthma had an increased probability to be responders.Conclusion: NARES patients may benefit from treatment with montelukast. In particular, the presence of concomitantasthma may be predictive of a greater efficacy.

Background We evaluated the prognostic value of nasal cytology and clinical factors in predicting nasal polyp (NP) development in patients with history of nonallergic chronic sinonasal inflammation. Methods This was a retrospective case-control study of 295 patients followed at our institution for a mean of 85.70 ± 19.41 months. According to the inclusion criteria we enrolled 84 cases with persistent eosinophilic nonallergic sinonasal inflammation (group A) and 106 cases with neutrophilic inflammation (group B), both without evidence of NPs at the baseline. We considered as controls 105 patients affected by nonallergic noninfectious vasomotor rhinitis without evidence of inflammation at nasal cytology (group C). Patients were checked every 6 months for NPs. Temporal analyses was performed by Kaplan-Mayer curves and odds ratios were evaluated by logistic regression analyses. Results The percentage of patients that developed NPs was higher in group A (29/84 [34.52%]) than in group B (17/106 [16.03%]) and group C (5/104 [4.7%]) (p < 0.05). Logistic regression analyses showed that eosinophilic patients had a higher risk of NP development over the years than neutrophilic patients compared to controls (odds ratio [OR], 10.55 vs 3.2). We also demonstrated that hypereosinophilia, asthma, and aspirin intolerance may increase the OR differently in eosinophilic patients. Conclusion Our data suggest that early identification of inflammatory patterns and associated clinical factors in patients affected by chronic nonallergic sinonasal inflammation have a prognostic value that can help to identify patients with different risks of NP development. Our data confirm that detection of nasal eosinophilic inflammation represents an early marker for identification of a more aggressive inflammatory phenotype.

Objective To describe a skin–SCID mouse chimeric model of systemic sclerosis (SSc; scleroderma) fibrosis based on engraftment of ex vivo–bioengineered skin using skin cells derived either from scleroderma patients or from healthy donors. Methods Three-dimensional bioengineered skin containing human keratinocytes and fibroblasts isolated from skin biopsy specimens from healthy donors or SSc patients was generated ex vivo and then grafted onto the backs of SCID mice. The features of the skin grafts were analyzed by immunohistochemistry, and the functional profile of the graft fibroblasts was defined before and after treatment with IgG from healthy controls or SSc patients. Two procedures were used to investigate the involvement of platelet-derived growth factor receptor (PDGFR): 1) nilotinib, a tyrosine kinase inhibitor, was administered to mice before injection of IgG from SSc patient sera (SSc IgG) into the grafts, and 2) human anti-PDGFR monoclonal antibodies were injected into the grafts. Results Depending on the type of bioengineered skin grafted, the regenerated human skin exhibited either the typical scleroderma phenotype or the healthy human skin architecture. Treatment of animals carrying healthy donor skin grafts with SSc IgG resulted in the appearance of a bona fide scleroderma phenotype, as confirmed by increased collagen deposition and fibroblast activation markers. Results of the experiments involving administration of nilotinib or monoclonal antibodies confirmed the involvement of PDGFR. Conclusion Our results provide the first in vivo demonstration of the fibrotic properties of anti-PDGFR agonistic antibodies. This bioengineered skin–humanized mouse model can be used to test in vivo the progression of the disease and to monitor response to antifibrotic drugs.

Objective Reactive oxygen species (ROS) contribute to the pathogenesis of fibrosis in systemic sclerosis (SSc; scleroderma), and NADPH oxidase (NOX) is an important source of ROS. Since the role of single NOX isoforms has not been previously investigated in SSc, this study was undertaken to assess the expression of NOX in SSc fibroblasts compared to normal healthy cells and to analyze their role in cell activation. Methods Expression of NOX isoforms in dermal fibroblasts from patients with SSc and healthy control subjects was analyzed by real-time polymerase chain reaction, immunoblotting, and immunofluorescence. NOX isoforms were silenced using small interfering RNA. Production of ROS was measured by fluorometry and confocal microscopy. Results Scleroderma fibroblasts showed up-regulation of NOX-2 and NOX-4 protein and messenger RNA (mRNA) expression. Treatment of the cells with diphenyleneiodonium, a nonselective inhibitor of flavin-containing enzymes, and silencing of NOX2 and NOX4 decreased the production of ROS as well as the expression of type I collagen and α-smooth muscle actin in SSc fibroblasts. ROS generated by NOX-2 and NOX-4 were involved in DNA damage and activation of a DNA repair checkpoint. Incubation of healthy control fibroblasts with platelet-derived growth factor (PDGF) or with IgG isolated from SSc patient serum enhanced the expression of NOX2 and NOX4 mRNA, via ROS, in a time-dependent manner. Treatment with actinomycin D, a transcription inhibitor, reversed the effects of PDGF stimulation but not the effects of SSc IgG. Conclusion Both NOX2 and NOX4 generate ROS in SSc fibroblasts and play a critical role in cell activation and DNA damage. Expression of NOX-2 and NOX-4 in SSc fibroblasts is maintained by a ROS-mediated loop.

Background The aim of the present study was to measure levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) in nasal lavage of patients affected by chronic eosinophilic sinonasal inflammation to clarify the relationship with eosinophilic tissue infiltration and clinical features. Methods Between November 2012 and June 2013, we selected 70 patients with chronic eosinophilic inflammation (average age 41.8 years) who were classified into the following groups: persistent allergic rhinitis (group 1), noninfectious non-allergic rhinitis with eosinophilia syndrome (group 2) and chronic rhinosinusitis with polyps (group 3). Finally, we enrolled 20 healthy subjects as controls (group 4). All patients underwent symptoms score questionnaire based on a visual analogue scale, nasal endoscopy and/or computed tomography (CT) scan, and allergy testing. Nasal cytology by scraping of the mucosa and GM-CSF assays in nasal lavage were performed in all subjects. Results Detectable levels of GM-CSF were found in 34 of 70 (48.57%) patients, with an average concentration of 2.67 ± 0.8 pg/mL, whereas in controls only 1 of 20 individuals showed detectable GM-CSF levels. Eosinophil infiltration was significantly higher in patients with detectable GM-CSF compared to those with undetectable levels (49.4% vs 39.2%, respectively; p < 0.05). Furthermore, significant weakly-moderate correlation was found between GM-CSF levels and percentage of eosinophil infiltration in tissue (p < 0.05). Correlation between symptom scores and GM-CSF levels was significant only in group 2, which showed higher average concentrations of GM-CSF compared to groups 1 and 3 (2.9 pg/mL vs 1.6 pg/mL and 1.8 pg/mL, respectively; p < 0.05). Conclusion Our data confirm that GM-CSF is more frequently detectable in nasal lavages of patients affected by chronic sinonasal eosinophilic inflammation than in controls. Statistical analyses revealed a significant weakly-moderate correlation between GM-CSF levels in nasal lavage of all patients and percentage of eosinophil infiltration of nasal mucosa.

Objective To explore the role of reactive oxygen species (ROS) in the in vitro activation of skin fibroblasts from patients with systemic sclerosis (SSc). Methods Fibroblasts were obtained from involved skin of patients with limited or diffuse SSc. Oxidative activity imaging in living cells was carried out using confocal microscopy. Levels of O2− and H2O2 released from fibroblasts were estimated by the superoxide dismutase (SOD)–inhibitable cytochrome c reduction and homovanilic acid assays, respectively. To verify NADPH oxidase activation, the light membrane of fibroblasts was immunoblotted with an anti-p47phox–specific antibody. Fibroblasts were stimulated with various cytokines and growth factors to determine whether any of these factors modulate ROS generation. Cell proliferation was estimated by 3H-thymidine incorporation. Northern blot analysis was used to study α1 and α2 type I collagen gene expression. Results Unstimulated skin fibroblasts from SSc patients released more O2− and H2O2 in vitro through the NADPH oxidase complex pathway than did normal fibroblasts, since incubation of SSc fibroblasts with diphenylene iodonium, a flavoprotein inhibitor, suppressed the generation of ROS. This suppression was not seen with rotenone, a mitochondrial oxidase inhibitor, or allopurinol, a xanthine oxidase inhibitor. Furthermore, the cytosolic component of NADPH oxidase, p47phox, was translocated to the plasma membrane of resting SSc fibroblasts. A transient increase in ROS production was induced in normal but not in SSc fibroblasts by interleukin-1β (IL-1β), platelet-derived growth factor type BB (PDGF-BB), transforming growth factor β1 (TGFβ1), and H2O2. Treatment of normal and SSc fibroblasts with tumor necrosis factor α (TNFα), IL-2, IL-4, IL-6, IL-10, interferon-α (IFNα), IFNγ, granulocyte–macrophage colony-stimulating factor (GM-CSP), G-CSF, or connective tissue growth factor (CTGF) had no effect on ROS generation. Constitutive ROS production by SSc fibroblasts was not inhibited when these cells were treated with catalase, SOD, IL-1 receptor antagonist, or antibodies blocking the effect of TGFβ1, PDGF-BB, and other agonists (IL-4, IL-6, TNFα, CTGF). In contrast, treatment of SSc fibroblasts with the membrane-permeant antioxidant N-acetyl-L-cysteine inhibited ROS production, and this was accompanied by decreased proliferation of these cells and down-regulation of α1(I) and α2(I) collagen messenger RNA. Conclusion The constitutive intracellular production of ROS by SSc fibroblasts derives from the activation of an NADPH oxidase–like system and is essential to fibroblast proliferation and expression of type I collagen genes in SSc cells. Our results also exclude O2−, H2O2, IL-1β, TGFβ1, PDGF-BB, IL-4, IL-6, TNFα, or CTGF as mediators of a positive, autocrine feedback mechanism of ROS generation.

The purpose of the present study was to evaluate the effects of MUFA vs PUFA enriched diets on the plasma and LDL lipid profile and antioxidant contents in mild hypercholesterolemic and triglyceridemic subjects. The study was divided in two consecutive diet periods. Two groups of 11 dyslipidemic patients each (type IIb and type IV) were recruited and during the first period (lasting four weeks) received a linoleic rich diet while during the following four weeks took an oleate rich diet. Both groups showed no significant changes in cholesterol and TG concentration either in plasma or in LDL. Coenzyme Q10 and vitamin E were also unaffected by the dietary treatments. LDL proneness to be oxidatively modified increased after dietary PUFA administration and markedly decreased following the virgin olive oil enriched diet. In fact, LDL from hypertrigliceridemic subjects on a oleate-enriched diet displayed a 26% (p < 0.05) longer lag-phase in conjugated dienes generation than during linoleate-enriched diet and at recruitment. In hypercholesterolemic subjects similar results were obtained: the lag-phase was 28% longer after MUFA diet that after PUFA diet. No differences were found in the maximum propagation rate and maximum concentration of conjugated dienes among dietary periods and at recruitment. Since we found that the vit. E and CoQ10 levels in plasma and in LDL particles remained unchanged during the course of the study, we may conclude that LDL proneness to undergo oxidative modifications is mainly the result of compositional change due to the enrichment from the different diets of the relative fats.

1. In the present study we have investigated the effects of the relatively low plasma concentrations of gemfibrozil (GFZ) found in clinical practice on the oxygen dissociation curve (ODC) of erythrocytes. 2. ODCs were measured at 30 degrees C and 37 degrees C and at pH 7.4: a) both on HbA solution and erythrocytes incubated in vitro with gemfibrozil and clofibric acid; b) on erythrocytes from healthy volunteers treated with a single oral dose of gemfibrozil. 3. These experiments showed a significant drug-induced shift of the ODC towards lower O2 affinity values without any significant modification of metabolic parameters of erythrocytes such as intracellular pH and intraerythrocytic levels of ATP and DPG. 4. In our experimental conditions gemfibrozil appears to lower both in vitro and in vivo, the partial pressure of oxygen required to give 50% of the haemes saturated with oxygen (P50) of erythrocytes from the control value of 24 +/- 0.5 mm Hg to 29 +/- 0.5 mm Hg (mean +/- s.d.; P < 0.02 by ANOVA). 5. These data clearly indicate that therapeutic doses of gemfibrozil may influence the oxygen transport properties of red cells. This effect could have relevant pharmacological and toxicological implications.