Rafal Pawliczak

Aspirin-triggered 15-HETE generation in peripheral blood leukocytes is a specific and sensitive Aspirin-Sensitive Patients Identification Test (ASPITest)*

Background:  We have previously demonstrated that aspirin triggers specific generation of 15‐hydroxyeicosateraenoic acid (15‐HETE) from nasal polyp epithelial cells and peripheral blood leukocytes (PBL) from aspirin‐sensitive (AS) but not aspirin‐tolerant (AT) patients with asthma/rhinosinusitis. The goal of this study was to assess the diagnostic value of ASA‐induced 15‐HETE generation measurement to identify AS patients.

Differential effects of aspirin and misoprostol on 15-hydroxyeicosatetraenoic acid generation by leukocytes from aspirin-sensitive asthmatic patients

BACKGROUND Although the mechanisms of aspirin-induced rhinosinusitis-asthma appear to be related to arachidonic acid abnormalities, only recently has a specific aspirin-triggered enhancement of 15-hydroxyeicosatetraenoic acid (15-HETE) generation in nasal polyp epithelial cells from aspirin-sensitive patients been demonstrated. OBJECTIVE The aim of this study was to assess generation of 15-HETE and other eicosanoids by peripheral blood leukocytes (PBLs) from aspirin-sensitive and aspirin-tolerant asthmatic patients and modulation of 15-HETE generation by a prostaglandin (PG) E(1) analogue (misoprostol). METHODS Twenty-four aspirin-sensitive patients with asthma-rhinosinusitis and 18 aspirin-tolerant asthmatic patients were studied, and eicosanoids released from PBLs were assessed by means of enzyme immunoassays. RESULTS Unstimulated PBLs from aspirin-sensitive and aspirin-tolerant patients generated similar amounts of PGE(2), leukotriene C(4), and 15-HETE, but lipoxin A(4) release was significantly less in aspirin-sensitive patients (300 +/- 70 pg/mL) in comparison with that seen in aspirin-tolerant patients (690 +/- 100 pg/mL, P <.05). Cell incubation with 2, 20, or 200 micromol/L aspirin resulted in a dose-dependent increase in 15-HETE generation (mean change of +85%, +189%, and +284% at each aspirin concentration, respectively) only in aspirin-sensitive asthmatic patients. Naproxen stimulated 15-HETE generation in aspirin-sensitive asthmatic patients, but indomethacin or specific COX-2 inhibitors (NS-398 and celecoxib) did not affect 15-HETE release. A synthetic PGE(1) analogue (misoprostol) inhibited aspirin-induced 15-HETE release but enhanced 15-HETE generation by aspirin in leukocytes from aspirin-tolerant patients. After preincubation with misoprostol, aspirin induced a dose-dependent production of lipoxin A(4) in both groups. CONCLUSION PBLs from patients with aspirin-sensitive rhinosinusitis-asthma might be specifically triggered by aspirin to generate 15-HETE. Metabolism of 15-HETE is differentially regulated by misoprostol in aspirin-tolerant and aspirin-sensitive asthmatic patients.

Decreased apoptosis and distinct profile of infiltrating cells in the nasal polyps of patients with aspirin hypersensitivity

Background:  Patients with aspirin‐hypersensitive rhinosinusitis/asthma suffer from a severe form of hyperplastic rhinosinusitis with recurrent polyposis. We aimed to assess the presence of apoptotic cells in nasal polyps from aspirin‐hypersensitive (AH) and aspirin‐tolerant (AT) patients with rhinosinusitis as related to the characteristics of local inflammation.

Association of stem cell factor expression in nasal polyp epithelial cells with aspirin sensitivity and asthma

Mast cells constitute a significant proportion of cells infiltrating nasal polyp tissue, and epithelial cells may release stem cell factor (SCF), a cytokine with chemotactic and survival activity for mast cells. We aimed to assess the expression of SCF in human nasal polyp epithelial cells (NPECs) as related to patients’ clinical phenotypes. Nasal polyp tissues were obtained from 29 patients [including nine with aspirin (ASA)‐hypersensitivity and 12 with bronchial asthma] undergoing polypectomy for nasal obstruction. Epithelial cells were obtained following 6‐week culture of nasal polyps explants. The SCF released into the culture supernatant was assessed by enzyme‐linked immunosorbent assay (ELISA) and total SCF mRNA in the polyp tissue was determined by semiquantitative reverse transcriptase polymerase chain reaction (RT‐PCR). For the whole group of patients, the number of polypectomies correlated with expression of SCF mRNA (r = 0.62; P < 0.005), SCF protein in the NPECs supernatants (r = 0.39; P < 0.05) and with density of mast cells in epithelial layer (r = 0.37; P < 0.05) and stromal layer (r = 0.5; P < 0.01) of nasal polyps. The SCF/β‐actin mRNA ratios were significantly higher in ASA‐hypersensitive (AH) asthmatics (median 0.97, range: 0.8–1.5) when compared with ASA‐tolerant (AT) patients (median 0.5, range: 0.1–0.7; P < 0.001). The SCF protein concentration in NPEC supernatants was also significantly higher in AH asthmatics (median 1.10 pg/μg DNA, range: 0.4–1.9) when compared with AT patients (median 0.1 pg/μg DNA, range: 0.02–1.2; P < 0.001). In the subpopulation of ASA‐sensitive asthmatics the number of poypectomies correlated also with the density of mast cells and eosinophils in the polyp tissue.

The Active Metabolite of Vitamin D3 as a Potential Immunomodulator.

In the past, vitamin D was known for its classical, skeletal action as a regulator of calcium and bone homoeostasis. Currently, vitamin D was found to have a role in numerous physiological processes in the human body; thus, vitamin D has pleiotropic activity. The studies carried out in the past two decades showed the role of vitamin D in the regulation of immune system functions. Basically, these effects may be mediated not only via endocrine mechanism of circulating calcitriol but also via paracrine one (based on cell–cell communication that leads to production of signal inducing the changes in nearby/adjacent cells and modulating their differentiation or behaviour) and intracrine mechanism (the action of vitamin D inside a cell) of 1,25‐dihydroxycholecalciferol (1,25(OH)2D3) synthetized from its precursor 25‐hydroxyvitamin D3 (25(OH)D3). Both vitamin D receptor (VDR) and 25‐hydroxyvitamin D3 1‐α‐hydroxylase (CYP27B1) are expressed in several types of immune cells (i.e. antigen presenting cells, T and B cells), and thus, they are able to synthetize the bioactive form of vitamin D that modulates both the innate and adaptive immune system. This review discusses the role of vitamin D as regulator of immune system, and our understanding of how vitamin D regulates both adaptive and innate immunity as well as inflammatory cascade on the cellular level.

Apocynin reduces reactive oxygen species concentrations in exhaled breath condensate in asthmatics

ABSTRACT Asthma is an inflammatory airway disease, and oxidative stress was proven to be involved in its pathogenesis. Apocynin effectively inhibits the main source of reactive oxygen species (ROS)—nicotinamide adenine dinucleotide phosphate (NADPH) oxidase—by blocking its activation. The aim of this study was to investigate the effect of inhaled apocynin on ROS and RNS (reactive nitrogen species) concentration in 14 nonsmoking mild asthmatics. Effects of nebulized apocynin (0.5 mg/mL) were assessed in exhaled breath condensate (EBC) after 30, 60, and 120 minutes, and safety parameters have been analyzed. Apocynin significantly decreased H2O2 concentration in EBC in comparison with placebo after 60 and 120 minutes. Moreover, apocynin significantly reduced NO−2 concentration 30 and 60 minutes after nebulization and caused a significant decrease of NO−3 concentration in EBC 60 and 120 minutes after administration, comparing with placebo. No adverse events have been observed throughout the study. This research confirmed anti-inflammatory properties of nebulized apocynin, which might be an effective and safe drug in bronchial asthma.

Expression of arachidonate metabolism enzymes and receptors in nasal polyps of aspirin-hypersensitive asthmatics.

Background: The pathogenesis of rhinosinusitis in aspirin-exacerbated airway disease is closely linked to the disequilibrium in arachidonic acid metabolism. Although considerable amounts of data concerning impaired eicosanoid production are available, the precise mechanism and pathogenesis of the disease are still unknown. The aim of the present study was to assess the expression of enzymes belonging to the arachidonic acid cascade and receptors for arachidonate derivative metabolites in nasal polyps from aspirin- hypersensitive (AH) and aspirin-tolerant (AT) patients with rhinosinusitis. Methods: Cells expressing cysteinyl leukotriene (CysLT) receptors (CysLT1 and CysLT2), arachidonate 5-lipoxygenase, leukotriene B4 receptor type 1, E-prostanoid receptors (EP2 and EP4), cyclooxygenase (COX)-1 and COX-2 were detected by immunocytochemistry in nasal polyps obtained from 10 AH patients and 18 AT patients. Results: There was a significantly higher density of cells expressing CysLT1 and CysLT2 receptors in nasal polyps from AH patients than from AT patients (p < 0.001). In contrast, the density of cells expressing EP2 receptor and COX-2 was significantly lower in AH patients than in AT patients (p < 0.02). The number of COX-2-positive epithelial cells was significantly reduced in AH polyps (p < 0.04). Conclusions: The elevated number of nasal polyp cells expressing CysLT receptors and lack of cells expressing EP2 receptor and COX-2 may be related to a more severe course of hyperplastic rhinosinusitis in aspirin hypersensitivity.

An enhanced risk of basal cell carcinoma is associated with particular polymorphisms in the VDR and MTHFR genes

Background:  Vitamin D and folate are influenced by ultraviolet radiation (UVR), and both are implicated in skin carcinogenesis. Polymorphisms in the genes involved in the metabolism of these two compounds may alter the risk of basal cell carcinoma (BCC).

Apocynin decreases hydrogen peroxide and nirtate concentrations in exhaled breath in healthy subjects

The imbalance between reactive oxygen species (ROS) synthesis and antioxidants might be involved in the pathogenesis of many inflammatory diseases. NADPH oxidase, an enzyme responsible for ROS production, may represent an attractive therapeutic target to inhibit, for the treatment of these diseases. Apocynin is an inhibitor of activation of NADPH oxidase complex present in the inflammatory cells. In double blind, placebo-controlled, cross-over study, we investigated the effect of nebulized apocynin on ROS synthesis in 10 nonsmoking healthy volunteers. Apocynin (6ml of 0.5mg/ml) was administered by nebulization and its effects on H(2)O(2), NO(2)(-) and NO(3)(-) generation were assessed after 30, 60 and 120min by collecting exhaled breath condensate (EBC) samples using an EcoScreen analyzer. Additionally, respiratory parameters have been evaluated, utilizing spirometry and DLCO. We also analyzed peripheral blood differential counts and NO(2)(-) serum level, cough scale control and blood pressure as safety parameters. Apocynin caused reduction of H(2)O(2) concentration in EBC as compared to placebo, after 60min. of inhalation (0.18microM vs. 0.31microM, p<0.05) as well as after 120min. (0.2microM vs. 0.31microM, p<0.05). Similarly, apocynin significantly decreased concentration of NO(3)(-) as compared to placebo, after 60 and 120min. (6.8microM vs. 14.4microM and 6.5microM vs. 14.9microM respectively, p<0.05). Apocynin was well tolerated and no adverse events have been observed throughout the study. Thus, as apocynin significantly influence ROS concentration, it might have also antiinflammatory properties. As it is safe, it may have a potential to become a drug in airway inflammatory diseases treatment.

β2-ADR haplotypes/polymorphisms associate with bronchodilator response and total IgE in grass allergy

Association and linkage studies of β2‐adrenergic receptor (β2‐ADR) polymorphisms in relation to the expression of asthmatic phenotypes and immune regulatory mechanisms have shown inconsistent results. In order to analyse the relevance of particular combinations of single nucleotide polymorphisms (SNPs) or haplotypes of β2‐ADR gene to bronchial asthma, bronchodilator response and total immunoglobulin E (IgE) we determined by direct DNA sequencing five SNPs (in positions: −47, −20, 46, 79, 252) in a group of 180 Caucasian subjects (110 patients with grass allergy and 70 nonatopic controls). The eight different β2‐ADR haplotypes were identified, with three the most common of them representing 92% of the studied cohort. Significantly higher (pcor = 0.0045) bronchodilator response was observed in patients with homozygotic genotype 46A/A in comparison with respective homo‐ and hetero‐zygotes. There was no significant difference in bronchodilator response when β2‐ADR haplotypes were analysed. Significantly higher (pcor = 0.0005) total IgE levels were found in patients with β2‐ADR haplotype −47T/−20T/46A/79C/252G and homozygotic carriers of 46A (pcor = 0.0015) and 79C (pcor = 0.003) genotypes. No significant associations were found in regards to asthmatic phenotype and atopy. These results indicate that depending on phenotype studied, either an individual β2‐ADR SNP or β2‐ADR haplotype might affect disease manifestation.