Comparative proteomics of nasal fluid in seasonal allergic rhinitis

A comparative proteomic approach was applied to examine nasal lavage fluid (NLF) from patients with seasonal allergic rhinitis (SAR, n = 6) and healthy subjects (controls, n = 5). NLF samples were taken both before allergy (pollen) season and during season, and proteins were analyzed by two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) after tryptic cleavage. Twenty proteins were selected and quantified. During allergy season, the levels of six sialylated isoforms of PLUNC (palate lung nasal epithelial clone) were lower in SAR patients than controls, as were the levels of six isoforms of von Ebner's gland protein (VEGP), including a previously undescribed form with N-linked glycosylation, and of cystatin S. PLUNC is a new innate immunity protein and VEGP and cystatin S are two endogenous proteinase inhibitors. By contrast, the levels of an acidic form of alpha-1-antitrypsin were higher in SAR patients than controls. One previously unidentified NLF protein was found in all samples from the SAR patients during allergy season but not in any sample before allergy season: this protein was identified as eosinophil lysophospholipase (Charcot-Leyden crystal protein/galactin 10). MS/MS analysis of the N-terminus of the protein showed removal of Met and acetylation of Ser. Altogether, these findings illustrate the potential use of proteomics for identifying protein changes associated with allergic rhinitis and for revealing post-translational modifications of such new potential markers of allergic inflammation.     

Clara cell protein (CC16) in serum and bronchoalveolar lavage fluid of subjects exposed to asbestos

The Clara cell protein (CC16) is a small and readily diffusible protein of 16kDa secreted by bronchiolar Clara cells in the distal airspaces. These epithelial cells are altered in several pulmonary pathological processes induced by various lung toxicants. In the search for a new biomarker of asbestos-induced lung impairment, we used a sensitive immunoassay to determine the levels of CC16 in bronchoalveolar fluid (BALF) and serum of subjects exposed to asbestos compared with a group of healthy controls. In the BALF of asbestos-exposed subjects there was an insignificant trend towards CC16 elevation compared with controls, with a (mean ±SD of 0.81 ±0.65mg l^-1 for asbestos-exposed subjects (n = 23) versus 0.39 ±0.19mg l^-1 for controls (n = 11) (p = 0.09). In serum, CC16 concentration was significantly increased among asbestos-exposed subjects, with values of 27.2 ±24.0 µg l^-1 for asbestos-exposed subjects (n = 34) versus 16.1 ±7.6 µg l^-1 for controls (n = 34) (p = 0.01). Regarding the effects of smoking, there were significant differences between generally lower CC16 levels in serum and BALF (p = 0.05 and 0.001, respectively) of smokers compared with the higher levels in non-smokers. Serum CC16 levels positively correlated with those in BALF, which is consistent with a diffusional transfer of CC16 from the bronchoalveolar space into the serum. No association, however, emerged between the levels of CC16 in serum or BALF and either the duration of asbestos exposure or the severity of the lung impairment as assessed by chest X-ray. These findings suggest that exposure to asbestos elicits early changes in the local and, importantly, also the systemic levels of CC16. This pneumoprotein therefore appears as a promising non-invasive biomarker of asbestos-induced lung injury and occupational disease in both smoking and non-smoking exposed subjects.     

Exhaled nitric oxide and nasal tryptase are associated with wheeze,rhinitis and nasal allergy in primary school children

Abstract

Rhinitis and asthma are the most common respiratory diseases in children. We assessed whether airway inflammation markers were associated with nasal allergies and self-reported symptoms of wheeze and rhinitis in 130 children 6–12 year old in an epidemiological context. Independent of sex and age, the fraction of exhaled nitric oxide (FeNO) and nasal mast cell (MC) activation (tryptase ≥?5?ng/mL) were positively associated with wheeze, rhinitis and with nasal allergy. Nasal eosinophil cationic protein (ECP) and exhaled breath condensate (EBC) markers (pH, 8-isoprostane, interleukin-1β) were not associated with symptoms or with nasal allergy. In conclusion, FeNO and nasal tryptase reflect allergic inflammation in the respiratory system.     

Microflora of the nasal mucosa in allergic perennial and infectious rhinitis

The microbiological study of 69 patients with allergic annual rhinitis (AAR) and infectious rhinitis (IR) was carried out. In AAR the isolated representatives of 15 genera and 40 species were distributed in 2 to 7 component; in IR the isolated representatives of 16 genera and 25 species were grouped in 2 to 4-component associations. In AAR Staphylococcus aureus was found to belong to the main species and in IR, S. aureus and S. epidermidis, while the number of species regarded as occasional in AAR was 7 (S. auricularis, S. cohnii, S. hominis, S. haemolyticus, S. warneri, S. apitis, S. schleiferi). Differences in the distribution of Neisseria, nonfermenting Gram negative bacteria, Streptococcus in associations in cases of AAR and IR were established. In AAR Corynebacterium pseudodiphthericum and in IR C. pseudotuberculosis were the dominant species.     

Identification of novel biomarkers in seasonal allergic rhinitis by combining proteomic, multivariate and pathway analysis

Background

Glucocorticoids (GCs) play a key role in the treatment of seasonal allergic rhinitis (SAR). However, some patients show a low response to GC treatment. We hypothesized that proteins that correlated to discrimination between symptomatic high and low responders (HR and LR) to GC treatment might be regulated by GCs and therefore suitable as biomarkers for GC treatment.

Methodology/Principal Findings

We identified 953 nasal fluid proteins in symptomatic HR and LR with a LC MS/MS based-quantitative proteomics analysis and performed multivariate analysis to identify a combination of proteins that best separated symptomatic HR and LR. Pathway analysis showed that those proteins were most enriched in the acute phase response pathway. We prioritized candidate biomarkers for GC treatment based on the multivariate and pathway analysis. Next, we tested if those candidate biomarkers differed before and after GC treatment in nasal fluids from 40 patients with SAR using ELISA. Several proteins including ORM (P<0.0001), APOH (P<0.0001), FGA (P<0.01), CTSD (P<0.05) and SERPINB3 (P<0.05) differed significantly before and after GC treatment. Particularly, ORM (P<0.01), FGA (P<0.05) and APOH (P<0.01) that belonged to the acute phase response pathway decreased significantly in HR but not LR before and after GC treatment.

Conclusions/Significance

We identified several novel biomarkers for GC treatment response in SAR with combined proteomics, multivariate and pathway analysis. The analytical principles may be generally applicable to identify biomarkers in clinical studies of complex diseases.     

Synthesis of novel 4-substituted-7-trifluoromethylquinoline derivatives with nitric oxide releasing properties and their evaluation as analgesic and anti-inflammatory agents

Six derivatives of the general formula 2- or 4-(7-trifluoromethylquinolin-4-ylamino) benzoic acid N'-(nitrooxyacetyl or propionyl) hydrazide and an oxime of the formula 1-[4-(7-trifluoromethylquinolin-4-ylamino)phenyl]ethanone oxime were synthesized and tested for their in vivo anti-inflammatory, analgesic, and ulcerogenic properties, as well as their in vitro nitric oxide release ability. Compound 2-(7-trifluoromethylquinolin-4-ylamino)benzoic acid N'-(2-nitrooxy propionyl)hydrazide 12 showed an anti-inflammatory activity comparable to that of indomethacin in the carrageenan-induced rat paw edema test, and equipotency to glafenine in the acetic acid mice induced writhing model at 100mg/kg p.o., respectively. All the final compounds showed no tendency to induce stomach ulceration in rats; nitric oxide seems to contribute to their excellent safety profile.     

Psoriasin,one of several new proteins identified in nasal lavage fluid from allergic and non-allergic individuals using 2-dimensional gel electrophoresis and mass spectrometry

Background

Extravasation and luminal entry of plasma occurs continuously in the nose. This process is markedly facilitated in patients with symptomatic allergic rhinitis, resulting in an increased secretion of proteins. Identification of these proteins is an important step in the understanding of the pathological mechanisms in allergic diseases. DNA microarrays have recently made it possible to compare mRNA profiles of lavage fluids from healthy and diseased patients, whereas information on the protein level is still lacking.

Methods

Nasal lavage fluid was collected from 11 patients with symptomatic allergic rhinitis and 11 healthy volunteers. 2-dimensional gel electrophoresis was used to separate proteins in the lavage fluids. Protein spots were picked from the gels and identified using mass spectrometry and database search. Selected proteins were confirmed with western blot.

Results

61 spots were identified, of which 21 were separate proteins. 6 of these proteins (psoriasin, galectin-3, alpha enolase, intersectin-2, Wnt-2B and hypothetical protein MGC33648) had not previously been described in nasal lavage fluids. The levels of psoriasin were markedly down-regulated in allergic individuals. Prolactin-inducible protein was also found to be down-regulated, whereas different fragments of albumin together with Ig gamma 2 chain c region, transthyretin and splice isoform 1 of Wnt-2B were up-regulated among the allergic patients.

Conclusion

The identification of proteins in nasal lavage fluid with 2-dimensional gelelectrophoresis in combination with mass spectrometry is a novel tool to profile protein expression in allergic rhinitis and it might prove useful in the hunt for new therapeutic targets or diagnostic markers for allergic diseases. Psoriasin is a potent chemotactic factor and its down-regulation during inflammation might be of importance for the outcome of the disease.     

Orally administrated Lactobacillus gasseri TMC0356 and Lactobacillus GG alleviated nasal blockage of guinea pig with allergic rhinitis

Lactobacillus GG (LGG) and L. gasseri TMC0356 (TMC0356) were investigated for their ability to alleviate nasal blockage associated with allergic rhinitis using a guinea pig model. The increases in sRaw at 10 min and 5 hr after the exposure of the nasal mucosa to OVA were significantly alleviated in the guinea pigs orally administrated with LGG and TMC0356 compared with those of the control (P<0.05 and P<0.01). The total numbers of leukocytes, particularly eosinophils and neutrophils from the nasal cavity lavage fluid, and the OVA-specific IgE concentration in the serum were also decreased in the guinea pigs orally administrated with LGG and TMC0356, although the decreases were not statistically significant. These results suggest that LGG and TMC0356 can alleviate antigen-induced nasal blockage in earlyphase and late-phase inflammatory responses associated with allergic rhinitis.     

Glycated collagen alters endothelial cell actin alignment and nitric oxide release in response to fluid shear stress

People with diabetes suffer from early accelerated atherosclerosis, which contributes to morbidity and mortality from myocardial infarction, stroke, and peripheral vascular disease. Atherosclerosis is thought to initiate at sites of endothelial cell injury. Hyperglycemia, a hallmark of diabetes, leads to non-enzymatic glycosylation (or glycation) of extracellular matrix proteins. Glycated collagen alters endothelial cell function and could be an important factor in atherosclerotic plaque development. This study examined the effect of collagen glycation on endothelial cell response to fluid shear stress. Porcine aortic endothelial cells were grown on native or glycated collagen and exposed to shear stress using an in vitro parallel plate system. Cells on native collagen elongated and aligned in the flow direction after 24 h of 20 dynes/cm(2) shear stress, as indicated by a 13% decrease in actin fiber angle distribution standard deviation. However, cells on glycated collagen did not align. Shear stress-mediated nitric oxide release by cells on glycated collagen was half that of cells on native collagen, which correlated with decreased endothelial nitric oxide synthase (eNOS) phosphorylation. Glycated collagen likely inhibited cell shear stress response through altered cell-matrix interactions, since glycated collagen attenuated focal adhesion kinase activation with shear stress. When focal adhesion kinase was pharmacologically blocked in cells on native collagen, eNOS phosphorylation with flow was reduced in a manner similar to that of glycated collagen. These detrimental effects of glycated collagen on endothelial cell response to shear stress may be an important contributor to accelerated atherosclerosis in people with diabetes.     

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery.

In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 microM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5'-dithiobis 2-nitrobenzoic acid (DTNB) (100 microM) or inhibiting PDI with bacitracin (5mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry). cGMP accumulation in response to GSNO (1 microM) was inhibited by MEG-01 treatment with bacitracin or DTNB, suggesting a role for PDI and surface thiols in NO delivery. PDI activity was present in MEG-01 conditioned medium, and was inhibited by high concentrations of GSNO (500 microM). A number of cell surface thiol-containing proteins were labelled using the impermeable thiol specific probe 3-(N-maleimido-propionyl) biocytin (MPB). Pretreatment of cells with GSNO resulted in a loss of thiol reactivity on some but not all proteins, suggesting selective cell surface thiol modification. Immunoprecipitation experiments showed that GSNO caused a concentration-dependent loss of thiol reactivity of PDI. Our data indicate that PDI is involved in both rapid metabolism of GSNO and intracellular NO delivery and that during this process PDI is itself altered by thiol modification. In contrast, the relevance of PDI-mediated albSNO metabolism to NO signalling is uncertain.     

Endothelial nitric oxide synthase and calcium production in arterial geometries: an integrated fluid mechanics/cell model

It is well known that atherosclerosis occurs at very specific locations throughout the human vasculature, such as arterial bifurcations and bends, all of which are subjected to low wall shear stress. A key player in the pathology of atherosclerosis is the endothelium, controlling the passage of material to and from the artery wall. Endothelial dysfunction refers to the condition where the normal regulation of processes by the endothelium is diminished. In this paper, the blood flow and transport of the low diffusion coefficient species adenosine triphosphate (ATP) are investigated in a variety of arterial geometries: a bifurcation with varying inner angle, and an artery bend. A mathematical model of endothelial calcium and endothelial nitric oxide synthase cellular dynamics is used to investigate spatial variations in the physiology of the endothelium. This model allows assessment of regions of the artery wall deficient in nitric oxide (NO). The models here aim to determine whether 3D flow fields are important in determining ATP concentration and endothelial function. For ATP transport, the effects of a coronary and carotid wave form on mass transport is investigated for low Womersley number. For the carotid, the Womersley number is then increased to determine whether this is an important factor. The results show that regions of low wall shear stress correspond with regions of impaired endothetial nitric oxide synthase signaling, therefore reduced availability of NO. However, experimental work is required to determine if this level is significant. The results also suggest that bifurcation angle is an important factor and acute angle bifurcations are more susceptible to disease than large angle bifurcations. It has been evidenced that complex 3D flow fields play an important role in determining signaling within endothelial cells. Furthermore, the distribution of ATP in blood is highly dependent on secondary flow features. The models here use ATP concentration simulated under steady conditions. This has been evidenced to reproduce essential features of time-averaged ATP concentration over a cardiac cycle for small Womersley numbers. However, when the Womersley number is increased, some differences are observed. Transient variations are overall insignificant, suggesting that spatial variation is more important than temporal. It has been determined that acute angle bifurcations are potentially more susceptible to atherogenesis and steady-state ATP transport reproduces essential features of time-averaged pulsatile transport for small Womersley number. Larger Womersley numbers appear to be an important factor in time-dependent mass transfer.     

Relationship between surfactant protein A polymorphisms and allergic rhinitis in a Chinese Han population

The development of allergic rhinitis is considered to be determined by the interaction between genetic and environmental factors. Surfactant protein A (SP-A), a member of the collectin family of proteins, plays an important role in immune regulation. The purpose of this study was to investigate the association between SP-A polymorphisms and allergic rhinitis. We conducted a case–control association study on a Chinese Han population, comprising 216 adult individuals with AR and 84 healthy controls. A total of 9 single-nucleotide polymorphisms (SNPs) mapped to the SP-A were genotyped using PCR-based molecular identification methods. The frequency of A allele at amino acid 223 in the patient group was significantly higher than that in the control group after correcting for multiple testing (P = 0.006). The 1A² allele haplotype in SFTPA2 was associated with decreased risk for allergic rhinitis, after applying Bonferroni corrections (P = 0.003). However, genetic variants of the SFTPA1 genes were not found to be associated with AR. In addition, no significant associations were established between any of the 9 SFTPA gene polymorphisms and the skin-prick test responses (P > 0.05). Further, no association was established between the 9 SNP loci and the levels of total serum immunoglobulin E (IgE) (P > 0.05). These results indicate that the gene polymorphism at the residue 223 in the carbohydrate recognition domain of SFTPA2 may be a genetic marker for the development of AR in the adult Chinese Han population.     

Interaction between nitric oxide and prostaglandin synthesis in the acute phase of allergic conjunctivitis

Both nitric oxide and prostaglandins induce vasodilatation which is an important feature of local inflammation. The purpose of the study described here was to investigate a possible interaction between these two types of mediators in an experimental model of allergic conjunctivitis. A conjunctival allergic reaction was induced with antigen in sensitized guinea pigs. Conjunctival vascular permeability changes were evaluated with the prophylactic use of an inhibitor of nitric oxide synthase (L-NAME) and a cycloxygenase inhibitor (indomethacin). To study a possible interaction between nitric oxide and prostaglandin synthesis in the acute phase of allergic conjunctivitis, the levels of nitrite and PGE₂ were determined in lavage fluid. The prophylactic use of L-NAME on the formation of conjunctival edema in response to topical PGD₂ administration was studied by measurement of albumin levels in lavage fluid. Both nitric oxide and PGE₂ are synthesized in response to antigen provocation and after histamine administration. Nitric oxide and PGE₂ are produced simultaneously in the conjunctiva and they showed identical synthesis profiles in response to antigen provocation. Pretreatment with L-NAME inhibited the synthesis of PGE₂ whereas exogenous administration of nitric oxide increased the level of PGE₂ in lavage fluid. Prophylactic treatment with L-NAME significantly inhibited the PGD₂ induced albumin extravasation. Nitric oxide seems to play an important role in the acute phase of allergic conjunctivitis it may stimulate PGE₂ production and acts as a secondary mediator in PGD₂ and histamine induced conjunctival edema.     

A flavo-diiron protein from Desulfovibrio vulgaris with oxidase and nitric oxide reductase activities. Evidence for an in vivo nitric oxide scavenging function

A few members of a widespread class of bacterial and archaeal flavo-diiron proteins, dubbed FprAs, have been shown to function as either oxidases (dioxygen reductases) or scavenging nitric oxide reductases, but the questions of which of these functions dominates in vivo for a given FprA and whether all FprAs function as oxidases or nitric oxide reductases remain to be clarified. To address these questions, an FprA has been characterized from the anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris. The gene encoding this D. vulgaris FprA lies downstream of an operon encoding superoxide reductase and rubredoxin, consistent with an O(2)-scavenging oxidase function for this FprA. The recombinant D. vulgaris FprA can indeed serve as the terminal component of an NADH oxidase. However, this oxidase turnover results in irreversible inactivation of the enzyme. On the other hand, the recombinant D. vulgaris FprA shows robust anaerobic nitric oxide reductase activity in vitro and also protects a nitric oxide-sensitive Escherichia coli strain against exposure to exogenous nitric oxide. It is, therefore, proposed that this D. vulgaris FprA functions as a scavenging nitric oxide reductase in vivo and that this activity protects D. vulgaris against anaerobic exposure to nitric oxide. The location of a gene encoding a second FprA homologue in the D. vulgaris genome also suggests its involvement in nitrogen oxide metabolism.     

Early and late allergic phase related cough response in sensitized guinea pigs with experimental allergic rhinitis

Cough is a common and important symptom of asthma and allergic rhinitis. Previous experimental evidence has shown enhanced cough sensitivity during early phase of experimental allergic rhinitis in guinea pigs. We hypothesized that airway inflammation during the late phase response after repeated nasal antigen challenge may affect the afferent sensory nerve endings in the larynx and tracheobronchial tree and may also modulate cough response. In the present study we evaluated the cough sensitivity during a period of early and late allergic response in sensitized guinea pigs after repeated nasal antigen challenges. Forty-five guinea pigs were sensitized with ovalbumin (OVA). Four weeks later 0.015 ml of 0.5 % OVA was intranasally instilled to develop a model of allergic rhinitis that was evaluated from the occurrence of typical clinical symptoms. Animals were repeatedly intranasally challenged either by OVA (experimental group) or by saline (controls) in 7-day intervals for nine weeks. Cough was elicited by inhalation of citric acid aerosols. Cough was evaluated at 1 or 3 h after the 6th nasal challenge and 17 or 24 h after the 9th nasal challenge. The cough reflex was significantly increased at 1 and 3 h after repeated nasal challenge in contrast to cough responses evoked at 17 and 24 h after repeated nasal challenge. In conclusion, enhanced cough sensitivity only corresponds to an early allergic response after repeated nasal challenges.     

PGE2 and PGF2 alpha in the nasal lavage fluid from healthy subjects: methodological aspects

Prostaglandins E2 and F2 alpha were estimated in nasal secretions from ten healthy volunteers by high performance liquid chromatography and radioimmunological assay. Prostaglandin concentrations determined in five consecutive nasal washes with saline at room temperature showed that the nasal mucosa was stimulated after instillation. A substantial increase of basal levels was associated with the second nasal lavage. In all volunteers aspirin treatment inhibited prostaglandin release.