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In this article, we tackle the complex relationship between a triangle assessment: energy companies, investment portfolios, and the energy consumption sector. The first objective of this paper was to underline several important features of oil price evolution in the pandemic period, with a special focus both on the 2020 and 2022 periods, in order to reveal some aspects of new passengers’ cars registered, and some important energy sources worldwide. Second, it is highlighted several important features of energy-listed companies on the Bucharest Stock Exchange and of oil prices upon different operators. Third, we analyze important aspects of the influence of the crises regarding the energy industry and several important evolutions upon the stock market. The main findings of this study reveal the nexus between oil shock prices, the energy industry, and the stock market with an empirical focus on constructing an optimal investment portfolio’ by considering several consumption sectors. Future research on this topic will encounter consequently comparisons between several business models of different types of sectors.

The present work analyzes the trade with excise goods and is addressed to traders of excise goods, entrepreneurs, as well as practitioners in the financial and accounting field, who, in the transactions made with excise goods, may violate the relevant legislation, and may be sanctioned as a contravention, or as the case may be, criminal. Given that the sale of excisable products represents a high fiscal risk, the fiscal authorities have introduced a series of laws, acts, and provisions, including the regulation to use electronic invoicing using the SAF-T (Standard Audit File for Taxation) reporting system, E-invoice or Statement 406 in all situations of trading (excisable) products with high fiscal risk starting from July 2022, by issuing electronic invoices, which bring changes regarding the authorization, movement of excisable products and the procedure for carrying out activities with excisable products, to reduce tax evasion, monitoring, compliance and sanctioning of those who do not comply with excise legislation. In the content of the work, through a case study, we will describe a case regarding non-compliance with the legislation by an entity, how it was sanctioned, and the applicable legislation for this act. The present article considers the current implacable reality, which is, that with the appearance of new rules or obstacles in combating fraud, the methods of fraud have also diversified.

In order to better collect taxes and combat fraud and tax evasion by strengthening the national legislative framework, a number of regulations and reports have been put in place for monitoring the declaration of all economic operations between partners in a regulated free market. The provisions adopted in the field of taxation in conjunction with the accounting report provided, respectively reported by the entities to the tax authorities, are applied for the correct assessment of their activity in order to combat the practices of tax avoidance, tax fraud, and tax evasion. This study does not analyze the fiscal policy as a whole, this paper addresses the regime of contraventions and sanctions, as well as the repercussions of non-declaration or incomplete or erroneous declaration of the Recapitulative Statements on intra-Community supplies/acquisitions/services (code D390) and Informative declarations on deliveries/services and purchases made in the national territory (code 394), related to value-added tax.

Abstract: Anaphylaxis is a serious systemic allergic reaction with rapid onset and potentially life‐threatening. We report in detail a case of severe nocturnal anaphylaxis due to pigeon tick bite showing the diagnostic value of the extract and the recombinant allergen in the diagnostic procedures (basophil activation test, IgE immunoblot, and experimental ImmunoCAP). Apart from the presented case, we describe that during the last 10 years, we have collected 28 cases of allergy to Argas reflexus from several European countries. We suspect that this allergy is underdiagnosed because of the lack of diagnostic reagents. Because of the growing number of pigeons in Middle and Southern Europe cities, some cases of idiopathic anaphylaxis could potentially be caused by A. reflexus in those countries. The identification of pigeon ticks as a trigger of anaphylaxis would greatly improve medical care and advice for these patients as the parasite can be exterminated by eradication measures to avoid further incidents. [ABSTRACT FROM AUTHOR]

This paper aims to present gastronomy tourism, which is a new concept and form of tourism, treated independently of other forms of tourism, combining characteristics of cultural, thematic and sustainable tourism, aiming at satisfying and educating tourists in traditional culinary arts, bringing benefits to the local population and maintaining various sectors of the economy. Gastronomy tourism, a new form of tourism, is part of cultural tourism, which addresses the culture of a region, especially through its artistic and traditional values, including tourism in rural areas, with its specific activities such as: agrotourism, outdoor gastronomic festivals, product tasting, etc. Gastronomy tourism, as a distinct form of cultural tourism, is based on the discovery of the traditions and values of local communities and entities, whether it is local tradition, gastronomy or rediscovery of values, folklore or old crafts, 80% of Romanian rural pensions being based on such an approach.

Through this paper, we aim to highlight marketing as the function that distinguishes the needs and aspirations of customers, that challenges the markets that can best be served and to create products, services and programs recommended in servicing these markets. Of all the definitions given to marketing over time, it is clear that the purposes of marketing implementation are: ensuring consumer satisfaction and increasing the profitability of the business. The need for consumption is the primary element of demand, it has an objective character and is determined physiologically, socially, historically and psychologically. Agro-food products are especially designed to meet a physiologically determined need and provide, in addition to oxygen and fragmented water, the supply of nutrients to the human body. The agro-food demand of residents relates to physiological needs and is caused by endogenous and exogenous, equal and individual factors.

Conventions to avoid double taxation are the panacea of tax law, lato sensu, and direct taxation, stricto sensu. Although the current network of double taxation conventions has over 2500 tax treaties concluded by the world’s states, there are still issues that need to be addressed in their application: the anti-abuse provisions to be found in conventions, the practices of the type treaty shopping, LOB clauses, use of arbitration in the application of double taxation avoidance conventions. The case of Romania is analyzed in this article, through the DSSs Râşnov cause vs. ANAF Brasov, in order to highlight the way in which the framework of the double taxation avoidance convention is applied in Romania, if there are differences and divergences between the de jure provisions of the double taxation avoidance conventions and the de facto application, in practice, a state like Romania, which is in the process of catching up with economies in developed countries. The case presented in this article suggests that there is still room for maneuver to improve the framework for double taxation avoidance conventions in Romania and how they are applied in practice, which their provisions are interpreted and respected.

The fiscal policy is an instrument that collects resources for the state budget necessary to perform state functions; stabilize the economy; regulation of the economy and recovery growth. The most important instruments are taxes and taxpayers. Since 1989, with the transition to a market economy, a special tax policy has been known by excise taxes. Analyzing the case of Romania and the evolution of excise duties (hereinafter ED) conformity we created some regressions that illustrate the ED correlation in Romania between 2002 and 2015. The methodology used: three unifactorial regression models showing how ED impacts economic indicators such as GDP, power purchase expressed through the net annual average salary, and household final consumption expenditure. Moreover, each model has been tested and verified using statistic tests to give reliable results. In a first stage, we analyzed the correlation between GDP and consumption as endogen variable and ED, then we created another model that we kept ED as an independent variable, but we changed the dependent variable using the purchasing power as a dependent variable. Because according to the used tests we demonstrated that correlation coefficients are significant, we proceeded to explain them starting from fiscal policy and economic reality, own of these analyzed 14 years. In conclusion we highlighted below as the ED depends on GDP and consumption and the purchasing power can influence the ED. For future discussion and studies we intend to compare the results with other countries in different geographic areas in Europe

Transfer prices are a top field in financial and legal scientific research and practical activity. Although this research field is still in the beginning, due to its complexity, as well as it’s inter-, multi- and transdisciplinarity, it can be noted that empirical studies, as well as practical researches in economic and legal matters, have intensified. Moreover, this field of transfer prices is in close connection with the area of international double taxation, which shows its international character. In this article we sought a holistic approach to the transfer price phenomenon, dealing with economic and legal technical aspects that we believed are important to emphasise. Without addressing the issue of transfer prices in an exhaustive manner, in this article we presented both the legal and the economic framework of transfer prices in Romania. The added value of this article lies in the approach to transfer prices, both legally and economically.

Through this work, we intend to emphasize marketing as that function of the organization thatidentifies customer needs and desires, determines which markets are best served and designsadequate products, services and programs to serve these markets. It is not only a special function,but more - a philosophy that guides the entire organization. The aim of marketing is to satisfycustomers in a profitable manner, by establishing advantageous links with them. Many believe thatmarketing is synonymous with advertising or selling. They are wrong and because of this aspect wedecided to tackle this issue trying to prove that in reality, marketing activity refers not so much tosell, but rather to the knowledge of what must be produced. Submission on the market is the resultof knowing the consumer needs and of finding those solutions that through value, quality andsuperior service delight customers. Neither advertising, nor sale cannot compensate for a possiblecustomer dissatisfaction.

The purpose of this document is desired to brought a significant contribution of the controlactivity, and to discuss new methods to ensure transparency and to eliminate constant disputesbetween the public and private sector. Starting with these aspects, we are looking to highlightinternational fraud as accompanying phenomena of transnational structures and economicrelations, to explain its impact on the Romanian economy and to discus the key to finding themetholodgy to eradicate this phenomenon, and also to indicate our recommendations and opinionsto what we believe is necessary to eradicate this phenomenon. Transfer pricing is basically theprice used in the transfer of tangible and intangible assets and / or transfer of services betweenrelated parties, and this price should be based on market price without influence of the relationshipof affiliation. For services or tangible assets, the transfer price comparison with the market isrelatively easy to perform, but in the case of intangible assets, quantification of future benefits thatit gives to an affiliated person, in comparison to a situation where this would be to an independent,is difficult to determine.

Introduction: Lung cancer is the second most frequent malignancy worldwide, but its aetiology is still unclear. Inflammatory cytokines and Th cells, including Th17, are now emerging as being involved in NSCLC pathways, thus postulating a role of IL-17 in tumour angiogenesis by stimulating the vascular endothelial growth factor and the release of nitric oxide. Despite the fact that many biomarkers are used for chest malignancy diagnosis, data on FeNO levels and inflammatory cytokines in NSCLC are still few. Our study aimed to evaluate the relationship between pulmonary nitric oxide production and VEGF and Th17-related cytokines in the EBC of patients affected by early-stage NSCLC., Methods: FeNO measurement and lung function tests were performed in both patients affected by NCSLC and controls; EBC samples were also taken, and Th1 (IL-1, IL-6, IL-12, IFN-g, TNF-a), Th17 (IL-17, IL-23) and Th2 (IL-4, IL-5, IL-13) related cytokines were measured., Results: Th1 and Th17-related cytokines in EBC, except for IFN-gamma and TNF-alpha, were significantly higher in patients than in healthy controls, whereas no differences were seen for Th2-related cytokines. FeNO at the flow rate of 50 mL/s, JawNO and CalvNO levels were significantly higher in patients affected by NSCLC compared to controls. Significant correlations were found between FeNO 50 mL/s and IL-17, IL-1 and VEGF. JawNO levels positively correlated with IL-6, IL-17 and VEGF. No correlations were found between FeNO and Th2-related cytokines., Conclusion: This is the first report assessing a relationship between FeNO levels and Th17-related cytokines in the EBC of patients affected by early-stage NSCLC. IL-17, which could promote angiogenesis through the VEGF pathway, might be indirectly responsible for the increased lung production of NO in patients with NSCLC.

Introduction: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a common and quality-of-life impacting disorder, with an underlying immunological mechanism similar to other conditions such as eosinophilic asthma or atopic eczema. Areas covered: This review article summarizes the most recent evidence on the main immunological mechanisms involved in the pathogenesis and the perpetuation of CRSwNP, with a particular focus on the key role of epithelium-derived inflammation as a consequence of the interaction with the airborne environment. Expert commentary: The increase in knowledge of the immunology of CRSwNP leads to the development of therapeutical strategies based upon the use of biologic agents that, according to a personalized and precision medicine approach, will provide each single patient with the most suitable immunological treatment.

Thymic stromal lymphopoietin (TSLP) has emerged as an important, but contradictory, player conditioning tumor growth. In certain contexts, by driving T helper (h) 2 responses via tumor-associated OX40 Ligand (OX40L) + dendritic cells (DCs), TSLP may play a pro-tumorigenic role. The study elucidates the importance of TSPL in pancreatic ductal adenocarcinoma (PDAC), by analyzing: i) TSLP levels in PDAC cell-line supernatants and plasma from patients with locally-advanced/metastatic PDAC, pre- and post-treatment with different chemotherapeutic protocols, in comparison with healthy donors; ii) TSLP and OX40L expression in PDAC and normal pancreatic tissues, by immunohistochemistry; iii) OX40L expression on ex vivo -generated normal DCs in the presence of tumor-derived TSLP, by flow cytometry; iv) clinical relevance in terms of diagnostic and prognostic value and influence on treatment modality and response. Some PDAC cell lines, such as BxPC-3, expressed both TSLP mRNA and protein. Normal DCs, generated ex vivo in the presence of TSLP-rich-cell supernatants, displayed increased expression of OX40L, reduced by the addition of a neutralizing anti-TSLP polyclonal antibody. OX40L + cells were detected in pancreatic tumor inflammatory infiltrates. Abnormally elevated TSLP levels were detected in situ in tumor cells and, systemically, in locally-advanced/metastatic PDAC patients. Of the chemotherapeutic protocols applied, gemcitabine plus oxaliplatin (GEMOX) significantly increased circulating TSLP levels. Elevated plasma TSLP concentration was associated with shorter overall survival and increased risk of poor outcome. Plasma TSLP measurement successfully discriminated PDAC patients from healthy controls. These data show that TSLP secreted by pancreatic cancer cells may directly impact PDAC biology and patient outcome., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflict of interest.

Objectives: The management of bronchopulmonary neuroendocrine tumours (BPNETs) is difficult, since imaging, histology and biomarkers have a limited value in diagnosis, predicting outcome and defining therapeutic efficacy. We evaluated a NET multigene blood test (NETest) to diagnose BPNETs, assess disease status and evaluate surgical resection., Methods: (i) Diagnostic cohort: BP carcinoids (n = 118)-typical carcinoid, n = 67 and atypical carcinoid, n = 51; other lung NEN (large-cell neuroendocrine carcinoma and small-cell lung carcinoma, n = 13); adenocarcinoma, (n = 26); squamous cell carcinoma (n = 23); controls (n = 90) and chronic obstructive pulmonary disease (n = 18). (ii) Surgical cohort, n = 28: BP carcinoids (n = 16: typical carcinoid 12; atypical carcinoid 4); large-cell neuroendocrine carcinoma, n = 3; lung adenocarcinoma, n = 8 and squamous cell carcinoma, n = 1. Blood sampling was performed presurgery and 30 days post-surgery. Transcript levels measured by quantitative polymerase chain reaction were calculated as activity scores (0-100% scale: normal < 14%) and compared with chromogranin A (enzyme-linked immunosorbent assay; normal <109 ng/ml)., Results: NETest was significantly elevated in carcinoids (48.7 ± 27%) versus controls (6 ± 6%, P < 0.001) with metrics: sensitivity 93%, specificity 89%, positive predictive value 92% and negative predictive value 91%. NETest differentiated progressive disease (73 ± 22%) from stable disease (36 ± 19%, P < 0.001) and R0 resections (10 ± 5%, P < 0.001, area under the curve: 0.98). Levels in chronic obstructive pulmonary disease and lung cancers were 18-24% while elevated in small-cell lung carcinoma/large-cell neuroendocrine carcinoma (59 ± 10%). In BPNETs on postoperative Day 30, NETest decreased by 60% (P < 0.001). Chromogranin A was elevated in only 40% of carcinoids and not altered by surgery., Conclusions: Blood NET gene levels accurately identified BPNETs (100%) and differentiated these from controls, benign and malignant lung disease. Progressive disease could be identified and surgical resection verified. Chromogranin A had no clinical utility. Monitoring NET transcript levels in blood will facilitate management by detecting residual tumour and identifying progressive disease., (© The Author 2017. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Background: Severe asthma is a heterogeneous disease, which is characterized by airway damage and remodeling. All triggers of asthma, such as allergens, bacteria, viruses, and pollutants, interact with the airway epithelial cells, which drive the airway inflammatory response through the release of cytokines, particularly IL-25, IL-33, and thymic stromal lymphopoietin (TSLP)., Objectives and Methods: To investigate whether the expression of the IL-25, IL-33, and TSLP receptors on the basophil membrane are associated with asthma severity. Twenty-six patients with asthma (11 severe and 15 moderate/mild) and 10 healthy subjects (controls) were enrolled in the study. The results of the basophil activation test and flow cytometry analysis were assessed to investigate basophil membrane expression of IL-25, TSLP, and IL-33 receptors before and after IgE stimulation., Results: IL-25 and IL-33 receptor expression on the basophil membrane at baseline were significantly higher in patients with severe asthma than in those with mild/moderate asthma or healthy subjects, independent of atopy, eosinophilia, asthma control, and exacerbation frequency. Following IgE stimulation, a significantly higher increase in the IL-25 and IL-33 receptors was observed in mild/moderate versus severe asthma., Conclusions: The high expression of the IL-25 and IL-33 receptors on the basophil membrane of patients with severe asthma indicates an overstimulation of basophils by these cytokines in severe asthma. This finding can possibly be used as a biomarker of asthma severity., (© 2018 S. Karger AG, Basel.)

Background: Intervention studies with vitamin D in asthma are inconclusive for several reasons, such as inadequate dosing or duration of supplementation or uncontrolled baseline vitamin D status. Our aim was to evaluate the benefit of long term vitamin D add-on in asthmatic patients with actual vitamin D deficiency, that is a serum 25-hydroxy vitamin D (25-OHD ) below 20 ng/mL., Methods: Serum 25-OHD, asthma exacerbations, spirometry and inhaled corticosteroids (CS) dose were evaluated in a cohort of 119 asthmatic patients. Patients with deficiency were evaluated again after one year vitamin supplementation., Results: 25-OHD was low in 111 patients and was negatively related to exacerbations ( p < 0.001), inhaled CS dose ( p = 0.008) and asthma severity ( p = 0.001). Deficiency was found in 90 patients, 55 of whom took the supplement regularly for one year, while 24 discontinued the study and 11 were not adherent. Patients with vitamin D deficiency after 12 months supplementation showed significant decrease of exacerbations (from 2.6 ± 1.2 to 1.6 ± 1.1, p < 0.001), circulating eosinophils (from 395 ± 330 to 272 ± 212 10⁶/L, p < 0.001), and need of oral CS courses (from 35 to 20, p = 0.007) and improvement of airway obstruction., Conclusions: Asthma exacerbations are favored by vitamin D deficiency and decrease after long-term vitamin D replacement. Patients who are vitamin D deficient benefit from vitamin D supplementation., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Objectives: The impact of skip N2 metastases (i.e. N2 lymph node metastases without N1) on survival in surgically resected non-small lung cancer remains an intriguing and rarely investigated topic. The goal of our study was to elucidate (i) skip N2 influence on overall survival (OS) and time to recurrence (TTR) in patients with resected lung adenocarcinoma and (ii) its link with epidermal growth factor receptor ( EGFR ) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog ( KRAS ) mutations., Methods: A retrospective analysis of 279 consecutive patients with lung pN2 adenocarcinoma, operated in two institutions between 2003 and 2013, was conducted. OS and TTR were calculated using the Kaplan-Meier method. Crude and multivariable-adjusted comparisons by skip N2 for OS and TTR were performed using the Cox method with shared frailty (accounting for the within-centre correlation). Associations between skip N2 metastasis, clinicopathological characteristics and EGFR and KRAS mutations were investigated using the Fisher exact test and Cramér's V -test., Results: The mean age at the time of surgery was 63 years (±12), and the median follow-up time was 36 months (min 3; max 101). Skip N2 was observed in 54 patients (19%). EGFR mutations were observed in 38 patients (14%); KRAS mutations were seen in 86 patients (31%). Patients with skip N2 metastasis were predominantly non-smokers ( P  =   0.001), underwent segmentectomy or limited resections ( P  =   0.004) and were not submitted to adjuvant therapy ( P  =   0.022). Moreover, there was a correlation between EGFR mutations and skip N2 (Cramér's V : 0.25, P  <   0.001). Indeed, EGFR mutations were significantly more frequent in skip N2 tumours (33%) compared with non-skip tumours (10%), P  <   0.001. No correlation between skip N2 and KRAS mutations was observed (Cramér's V : 0.05, P  =   0.46). The multivariable-adjusted model showed a significant skip N2 protective effect on OS (hazard ratio, HR 0.503; P  =   0.014; 95% confidence interval, CI: 0.291-0.8704) but not on TTR (HR 0.788; P  =   0.446; 95% CI: 0.427-1.454)., Conclusions: In our series, lung adenocarcinoma skip N2 metastasis demonstrated a favourable prognosis. The presence of EGFR mutations could have significance in the better survival and in the specific anatomic pathway of lymphatic metastases exhibited by skip N2 tumours., (© The Author 2016. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Objective: Complete separation of upper and lower respiratory tract after total laryngectomy results in permanent effects on nasal cavities and tracheo-bronchial airways. Aim of this study is evaluating nasal and tracheal cytological alterations of mucosa in laryngectomy long-term survivors, analyzing the feasibility of scraping for cytological examination of tracheal mucosa., Methods: Twenty-five laryngectomy patients underwent symptoms' evaluation, endoscopic fiber optic examination, prick tests, and nasal and tracheal scraping for cytological exam. Twenty-five healthy subjects underwent the same assessment, except for tracheal scraping. Eleven laryngectomy patients accepted inferior turbinate biopsy for histological examination., Results: Nasal cytological analysis demonstrated mucous cell metaplasia in 20% of laryngectomized patients, but it was absent in all healthy subjects; no squamous cell metaplasia was found in both groups. In 15 patients (60%), bacteria were present, without inflammatory infiltrate. Tracheal cytological analysis demonstrated a quite high rate of squamous cell metaplasia (24%), neutrophilic infiltrate (32%), and presence of bacteria (40%). Histological examination of inferior turbinate showed submucosal stromal fibrosis in all patients and submucosal inflammatory infiltrate in 1 case (9%)., Conclusion: Nasal cavities and trachea of laryngectomy patients undergo long-term cytological and histological changes of mucosa and submucosa, probably due to airflow modifications.

Exercise-induced dyspnea is common among adolescents and young adults and often originates from exercise-induced bronchoconstriction (EIB). Sometimes, dyspnea corresponds to exercise-induced laryngospasm (EILO), which is a paradoxical decrease in supraglottic/glottic area. Vitamin D deficiency, which occurs frequently at northern latitudes, might favor laryngospasm by impairing calcium transport and slowing striate muscle relaxation. The aim of this study was to evaluate whether vitamin D status has an influence on bronchial and laryngeal responses to exercise in young, healthy athletes. EIB and EILO were investigated during winter in 37 healthy competitive rowers (24 males; age range 13-25 years), using the eucapnic voluntary hyperventilation test (EVH). EIB was diagnosed when forced expiratory volume in the first second decreased by 10%, EILO when maximum mid-inspiratory flow (MIF50) decreased by 20%. Most athletes (86.5%) had vitamin D deficiency (below 30 ng/mL), 29 mild-moderate (78.4%) and 3 severe (8.1%). EVH showed EIB in 10 subjects (27%), EILO in 16 (43.2%), and combined EIB and EILO in 6 (16.2%). Athletes with EILO had lower vitamin D (19.1 ng/mL vs. 27.0 ng/mL, p < 0.001) and higher parathyroid hormone (30.5 pg/mL vs. 19.2 pg/mL, p = 0.006) levels. The degree of laryngoconstriction (post-EVH MIF50 as a percentage of pre-EVH MIF50) was related directly with vitamin D levels (r = 0.51; p = 0.001) and inversely with parathyroid hormone levels (r = -0.53; p = 0.001). We conclude that vitamin D deficiency is common during winter in young athletes living above the 40th parallel north and favors laryngospasm during exercise, probably by disturbing calcium homeostasis. This effect may negatively influence athletic performance.

Objective: Complete separation of upper and lower respiratory tract after total laryngectomy results in loss of physiological nasal functions and presence of "unconditioned" inspired air in lower airways. Aim of this study is evaluating the presence of a microbial colonization of nasal cavities and trachea in laryngectomized long-term survivors., Methods: Twenty-five laryngectomized patients underwent symptoms' anamnestic evaluation, endoscopic fiber optic nasal and tracheal examination, specimen collection for microbiological exam, and culture. Enrolled patients had at least a 2-year follow-up period in order to evaluate long-term microflora., Results: Gram positive polimicrobic flora represented the main finding in nasal cavities and trachea (92% and 48% of patients, respectively). Other bacteria were non-fermenters Gram negative bacteria, Enterobacteriaceae and Staphylococcus aureus. The same microflora was demonstrated in nasal cavity and trachea in 5 patients (20%), while sterile nasal cavity and trachea were seen in 3 (12%) and 4 (16%) cases, respectively. No fungi were observed in nasal cavity and trachea., Conclusion: Nasal cavities and trachea of laryngectomized patients are colonized by nonpathogenic and/or potentially pathogenic bacteria, in absence of signs and symptoms of infection. Colonizer microflora should be kept in mind when a culture from nasal or tracheal swabs is needed in daily practice., (© The Author(s) 2015.)

Introduction: Bio-repositories are invaluable resources to implement translational cancer research and clinical programs. They represent one of the most powerful tools for biomolecular studies of clinically annotated cohorts, but high quality samples are required to generate reliable molecular readouts and functional studies. The objective of our study was to define the impact of cancer tissue ischemia time on RNA and DNA quality, and for the generation of Patient-Derived Xenografts (PDXs)., Methods: One-hundred thirty-five lung cancer specimens were selected among our Institutional BioBank samples. Associations between different warm (surgical) and cold (ex-vivo) ischemia time ranges and RNA quality or PDXs engraftment rates were assessed. RNA quality was determined by RNA integrity number (RINs) values. Fresh viable tissue fragments were implanted subcutaneously in NSG mice and serially transplanted., Results: RNAs with a RIN>7 were detected in 51% of the sample (70/135), with values of RIN significantly lower (OR 0.08, P = 0.01) in samples preserved for more than 3 hours before cryopreservation. Higher quality DNA samples had a concomitant high RIN. Sixty-three primary tumors (41 adenocarcinoma) were implanted with an overall engraftment rate of 33%. Both prolonged warm (>2 hours) and ex-vivo ischemia time (>10 hours) were associated to a lower engraftment rate (OR 0.09 P = 0.01 and OR 0.04 P = 0.008, respectively)., Conclusion: RNA quality and PDXs engraftment rate were adversely affected by prolonged ischemia times. Proper tissue collection and processing reduce failure rate. Overall, NSCLC BioBanking represents an innovative modality, which can be successfully executed in routine clinical settings, when stringent Standard Operating Procedures are adopted.

Background . T2 inflammation of chronic rhinosinusitis with nasal polyps (CRSwNP) may be influenced by epithelial cytokines release (TSLP, IL-25, and IL-33). We investigated the release of TSLP, IL-25, and IL-33 by epithelial CRSwNP cells compared to epithelial sinus mucosa cells of patients with chronic rhinosinusitis without nasal polyps (CRSsNP). Methods . IL-25, IL-33, and TSLP were measured by ELISA in the supernatant of cell cultures derived by CRSwNP (9 patients, 6 atopic) and CRSsNP (7 patients, 2 atopic) in baseline condition and following stimulation with Dermatophagoides pteronyssinus (DP), Aspergillus fumigatus (AF), and poly(I:C). Results . CRSwNP epithelial cells released increased levels of IL-25 (from 0.12 ± 0.06 pg/ml to 0.27 ± 0.1 pg/ml, p < 0.01) and TSLP (from 0.77 ± 0.5 pg/ml to 2.53 ± 1.17 pg/ml, p < 0.001) following poly(I:C) stimulation, while CRSsNP epithelial cells released increased levels of IL-25 and IL-33 following AF and DP stimulation, respectively (IL-25: from 0.18 ± 0.07 pg/ml to 0.51 ± 0.1 pg/ml, p < 0.001; IL-33: from 2.57 ± 1.3 pg/ml to 5.7 ± 3.1 pg/ml, p < 0.001). Conclusions . CRSwNP epithelial cells release TSLP and IL-25 when stimulated by poly(I:C) but not by DP or AF, suggesting that viral infection may contribute to maintain and amplify the T2 immune response seen in CRSwNP., Competing Interests: The authors declare that they have no conflict of interests.

Background: Ciliopathies refer to a wide variety of diseases in which mutations in the genes encoding proteins involved in ciliogenesis or protein transport to the primary cilia play pathogenetic roles, and in such diseases, retinal involvement may be present. Nitric oxide (NO) plays an important role in airway physiology, including regulation of ciliary motility and host defense. In primary ciliary dyskinesia, a syndromic ciliopathy, nasal NO (nNO) levels were reported to be extremely low compared with controls, possibly reflecting molecular defects leading to structural and functional ciliary abnormalities. We investigated whether decreased nitric levels were also present in patients with retinal inherited dystrophies., Methods: Nasal NO was measured in a group of patients with syndromic and nonsyndromic inherited retinal dystrophies., Results: Patients with inherited retinal dystrophies, both syndromic and nonsyndromic, had mean nNO levels that were lower than healthy controls. Seven patients had particularly low levels of nNO: 3 patients with retinitis pigmentosa and 4 individual patients with Mainzer-Saldino syndrome, Bardet-Biedl syndrome, Usher syndrome, and cone-rod disease., Conclusions: These findings provide evidence that there is an underlying abnormal ciliary function involving the nasal epithelium in some patients with inherited retinal dystrophies.

Background: Radiation therapy is a cornerstone in nasopharyngeal cancer treatment. However, it can induce acute and long-term adverse effects, such as acute mucositis and late submucosal fibrosis. Late toxicities could not only affect submucosa but also mucosal cells, determining long-term cytological changes., Objective: Evaluation of delayed nasal cytological alterations in patients who underwent radiation therapy for nasopharyngeal carcinoma (NPC)., Methods: In this case-control study, we analyzed 30 healthy subjects and 30 patients treated with chemotherapy and radiotherapy for NPC between 2003 and 2011, with a median follow-up of 59 months. All subjects underwent symptoms anamnestic evaluation (rhinorrea, nasal obstruction), endoscopic fiber optic nasal examination, skin-prick tests, and nasal scraping for cytological exam., Results: A higher percentage of rhinorrhea, nasal obstruction, mucosal hyperemia, and presence of nasopharyngeal secretions at fiber optic endoscopic exam was found in radiated subjects (p < 0.05). Nasal cytology analysis demonstrated a higher percentage of neutrophilic inflammation and squamous cell metaplasia and mucous cell metaplasia in treated patients (p < 0.05). No cytological atypia was seen. No statistically significant correlation between nasal cytological changes and objective findings, patients' age, tobacco smoking, and gastroesophageal reflux has been found in the radiotherapy group (p > 0.05)., Conclusion: Radiation therapy induces late nasal mucosal changes, which may be related to clinical consequences, such as abundant mucus production and its consequent endonasal stagnation. In the future, detailed knowledge of cytological changes in patients' nasal mucosa could represent a key prerequisite for the choice of effective interventions for late radiation-induced rhinitis.

The aims of this study were to investigate OX40 ligand expression in sinus tissue from patients with nasal polyposis compared with patients with chronic rhinosinusitis without nasal polyps (NPs), and to determine if OX40 ligand expression is related to eosinophilic sinus infiltration. Twenty patients with chronic rhinosinusitis (11 with and nine without NPs) and seven controls were enrolled in the study. The mRNA expression of OX40 ligand and thymic stromal lymphopoietin and its receptor were analyzed. The immunoreactivity score for OX40 ligand and the eosinophil count were obtained. The mRNA expression and immunoreactivity score of OX40 ligand were higher in patients with nasal polyposis than in patients without NPs, as well as healthy controls. The mRNA expression of thymic stromal lymphopoietin and its receptor was significantly higher in nasal polyposis than in the control, but not significantly higher than in chronic rhinosinusitis without NPs. A correlation between the number of OX40 ligand-positive cells and the number of eosinophils in sinus biopsies was found only in patients with nasal polyposis. In conclusion, the thymic stromal lymphopoietin/OX40 ligand axis is up-regulated in nasal polyposis and is related to the intensity of eosinophilic inflammation., (© The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

Background: To investigate the modulation of B-cell-activating factor (BAFF) expression on the basophil membrane of allergic patients. BAFF is an important regulator of B-cell activation, proliferation and immunoglobulin production, which may play a role in respiratory allergic diseases in promoting the production of IgE by B cells., Methods: Peripheral blood samples of 10 patients with allergic rhinitis, 3 with severe asthma and fungal sensitization (SAFS), 3 with allergic bronchopulmonary aspergillosis (ABPA) and 11 healthy controls were assessed regarding BAFF (CD257) expression using the basophil activation test before and after stimulation with IgE and allergens, as well IgE-independent stimuli, like fMLP, lipotheichoic acid from Staphylococcus aureus (LTA-SA) and lipopolysaccharide (LPS)., Results: BAFF membrane expression did not change after IgE and allergen stimulation both in patients and controls, while it was upregulated by Aspergillus stimulation, both in sensitized patients and controls. In both patients and controls, BAFF expression was significantly upregulated following LTA-SA and β-1,3-glucan exposure (toll-like receptor-2 ligands), but not following LPS stimulation., Conclusions: Basophils from allergic and healthy subjects constitutively express membrane BAFF, which is not upregulated by IgE or specific allergens but by TLR-2 ligands (LTA-SA and β-1,3-glucan). Aspergillus fumigatus stimulation was able to upregulate BAFF expression on the basophils of sensitized asthmatic patients, but not via IgE-dependent mechanisms, since results did not differ between the patient and control groups. These findings suggest that basophils may contribute to the polyclonal production of IgE commonly observed in patients with SAFS and ABPA., (© 2015 S. Karger AG, Basel.)

Inflammation mediated by the immune system is known to be important in carcinogenesis and, specifically, T helper 17 cells have been reported to play a role in tumor progression by promoting neo-angiogenesis. The aim of this study was to investigate whether inflammatory cytokines and vascular endothelial growth factor (VEGF) levels in exhaled breath condensate (EBC) and in serum were related to tumor size in patients with non-small cell lung cancer (NSCLC). Il-6, IL-17, TNF-α and VEGF levels were measured in EBC and serum of 15 patients with stage I-IIA NSCLC and in 30 healthy controls by immunoassay. The tumor size was measured by a CT scan. The concentrations of IL-6, IL-17 and VEGF were significantly higher in EBC of patients with lung cancer, compared with controls, while only serum IL-6 concentration was higher in patients compared to controls. A significant correlation (r = 0.78, p = 0.001) was observed between EBC levels of IL-6 and IL-17; IL-17 was also correlated to EBC levels of the VEGF (r = 0.83, p < 0.001) and TNF-α (r = 0.62, p = 0.014). The tumor diameter was significantly correlated with EBC concentrations of VEGF (r = 0.58, p = 0.039), IL-6 (r = 0.67, p = 0.013) and IL-17 (r = 0.66, p = 0.017). Our results show a significant relationship between inflammatory and angiogenic markers, measured in EBC by a non-invasive method, and tumor mass.

In eosinophilic granulomatosis with polyangiitis (EGPA) clonally expanded T cells might concur in granuloma formation and vascular injury. The TCR β-variable (BV) chain repertoire and third complementarity determining region (CDR3) of peripheral CD4+ and CD8+ cells in EGPA patients and age-matched controls and the expression of cytokines and chemokine receptors were investigated. The CD8+ lymphocytes of EGPA patients showed an increased frequency of BV expansions with a skewed profile of BV CDR3 lengths, increased CCR5 and CXCR3 expression and increased INFγ and TNFα production. In two patients, the TCR CDR3 cDNA sequences of the expanded BV family were identified. The CD4+ lymphocytes of EGPA patients revealed a higher expression of CRTH2 and increased production of IL-5. In conclusion, CD4+ T cells display a Th2 profile and CD8+ T cells are clonally expanded in EGPA and have a proinflammatory phenotype, suggesting their pathogenic role in vasculitic damage., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Cardiovascular disease (CVD) is a common comorbidity in patients with chronic airway obstruction, and is associated with systemic inflammation and airway obstruction. The aim of this study was to evaluate the predictors of CVD in two different conditions causing chronic airway obstruction, asthma and COPD., Methods: Lung function tests, clinical and echocardiographic data were assessed in 229 consecutive patients, 100 with asthma and 129 with COPD. CVD was classified into: pressure overload (PO) and volume overload (VO). Sub-analysis of patients with ischemic heart disease (IHD) and pulmonary hypertension (PH) was also performed., Results: CVD was found in 185 patients (81%: 51% COPD and 30% asthmatics) and consisted of PO in 42% and of VO in 38% patients. COPD patients, as compared to asthmatics, had older age, more severe airway obstruction, higher prevalence of males, of smokers, and of CVD (91% vs 68%), either PO (46% vs 38%) or VO (45% vs 30%). CVD was associated with older age and more severe airway obstruction both in asthma and COPD. In the overall patients the predictive factors of CVD were age, COPD, and male sex; those of PO were COPD, BMI, VC, FEV1 and MEF50 and those of VO were age, VC and MEF50. In asthma, the predictors of CVD were VC, FEV1, FEV1 /VC%, and PaO2, those of PO were VC, FEV1 and FEV1 /VC%, while for VO there was no predictor. In COPD the predictors of CVD were age, GOLD class and sex, those of VO age, VC and MEF50, and that of PO was BMI. Sub-analysis showed that IHD was predicted by COPD, age, BMI and FEV1, while PH (found only in 25 COPD patients), was predicted by VO (present in 80% of the patients) and FEV1. In subjects aged 65 years or more the prevalence of CVD, PO and VO was similar in asthmatic and COPD patients, but COPD patients had higher prevalence of males, smokers, IHD, PH, lower FEV1 and higher CRP., Conclusions: The results of this study indicate that cardiovascular diseases are frequent in patients with chronic obstructive disorders, particularly in COPD patients. The strongest predictors of CVD are age and airway obstruction. COPD patients have higher prevalence of ischemic heart disease and pulmonary hypertension. In the elderly the prevalence of PO and VO in asthma and COPD patients is similar.

Functional imbalance in Th1/Th2 cell response toward allergens is a recognized hallmark of allergic patients and a major role of dendritic cells (DCs) in redirecting T-cell phenotypes after specific immunotherapy has been suggested. This study investigates the proliferative and cytokine responses of T cells cocultured with monocyte-derived DCs (MoDCs) after allergen stimulation in birch-allergic patients compared with controls and investigates whether sublingual immunotherapy (SLIT) could change the DC-driven immune response. T cells were stimulated with the major birch pollen allergen (nBet v1) and MoDCs from eight birch-allergic patients with seasonal allergic rhinitis and eight nonallergic controls. Proliferation and cytokine production were measured before and after one course of SLIT with birch allergoid. Significantly lower levels of proinflammatory (IL-1beta, p = 0.027; IL-6, p = 0.030; TNF-alpha, p = 0.019) and Th1 (interferon gamma, p = 0.032; IL-12, p = 0.05) cytokines were measured in supernatants of T cells and MoDCs cultures from allergic patients compared with nonallergic controls. After SLIT, significant increase in IL-12 (p = 0.039), IL-1beta (p = 0.040), IL-6 (p = 0.041), TNF-α (p = 0.048), and IL-10 (p = 0.048) and significant decrease in IL-13 (p = 0.001) were observed. MoDCs/T-cell cocultures, pulsed with the specific allergen, produced lower quantities of proinflammatory and Th1 cytokines in allergic patients compared with healthy subjects, suggesting an allergen-specific impairment of natural immunity and Th1 immune response. A single course of SLIT was able to enhance allergen-specific innate immunity and to modify lymphocyte response, promoting Th1 and T-cell regulatory activity.

Objectives: Churg-Strauss syndrome (CSS) is a necrotising vasculitis of small vessels in which oligoclonally expanded TCR Vβ CD8+ effector memory T cells populations (TEM) may be involved in vasculitic damage. The aim of this study was to assess the functional role of CD8+ T cells in CSS patients by flow cytometry analysis of membrane expression of cytotoxic markers NKG2D and CD107a., Methods: Immunostaining of peripheral T cells and effector memory lymphocytes (TEM) from CSS patients and controls was performed by gating CD28 and CD45RA in the CD8+NKG2D+ and CD4+NKG2D+ populations. CD107a expression was evaluated in both whole CD8+ and CD4+ and the TEM cells by gating CD62 and CD45RA following polyclonal stimulation., Results: NKG2D expression was shifted toward the CD8+CD28- fraction of T cells in CSS patients compared to healthy controls (56.1±25.8% versus 17.2±7.3%, respectively, p=0.002). CD8+Vβ+ expanded T cells showed a significantly increased expression of NKG2D compared to the whole CD8+ T cell population (91.4±1.9% versus 79.7±3.8%, respectively, p=0.015). Moreover the CD8+ population from CSS upregulates CD107a on its surface upon polyclonal stimulation in a significantly higher proportion than healthy subjects (26.2±10.8% versus 8.2±2.9%, p=0.0031) and the majority CD8+ CD107+ cells from CSS patients showed a TEM phenotype compared to controls (64.8±4.9% vs. 19.8±2.9, respectively, p<0.001)., Conclusions: In CSS, CD8+ TEM lymphocytes show markers of cytotoxic activity, which suggests a role for these cells in vasculitic damage.

Churg Strauss Syndrome (CSS) is a systemic vasculitis in which oligoclonal T cell expansions might be involved in the pathogenesis. Combined analysis of TCR-Vbeta expression profile by flow cytometry and of TCR gene rearrangement by heteroduplex PCR was used to detect and characterize T cell expansions in 8 CSS patients, 10 asthmatics and 42 healthy subjects. In all CSS patients one or two Vbeta families were expanded among CD8+ cells, with an effector memory phenotype apt to populate tissues and inflammatory sites. Heteroduplex PCR showed the presence of one or more clonal TCR rearrangements, which reveals monoclonal or oligoclonal T cells subpopulations. After purification with a Vbeta specific monoclonal antibody, each CD8+/Vbeta+ expanded family showed a single TCR rearrangement, clearly suggestive of monoclonality. All CD8+ expansions were detectable throughout the disease course. TCR-Vbeta expanded or deleted populations were not observed in asthmatic patients. Clonal CD8+/Vbeta+ T cell expansions might be useful as a disease marker.