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.     

Increased apoptosis of peripheral blood mononuclear cells in patients with perennial allergic asthma/rhinitis: relation to serum markers of apoptosis

BACKGROUND: The goal of our study was to examine spontaneous and stimulated apoptosis of peripheral blood MNC from allergic patients, sensitized to Der p I antigen as compared to cells from non-atopic subjects. Furthermore we aimed to investigate which populations of mononuclear cells (lymphocytes, monocytes) undergo the apoptosis and to determine relations between apoptosis and serum levels of sFas/APO-1, ICE/caspase-1 or TNF-alpha. METHODS: The study included 17 patients with perennial, allergic asthma and/or allergic rhinitis [6 male and 11 female; mean age 29,5 years; (range 15-49)]. Apoptosis was assessed by fluorescence technique and confirmed by flow-cytometric method and DNA ladder. Serum levels of sFas, ICE/caspase-1 or TNF-alpha were determined by immunoassays (ELISA). RESULTS: Apoptotic index of unfractionated mononuclear cells (MNC) and lymphocytes (but not monocytes) were significantly higher in allergic patients as compared to non-allergic subjects after 48 and 72 hours of culture (p<0.05). Incubation of cells with ConA (10 microg/ml) resulted in a significant increase in the proportion of apoptotic cells in all populations once the apoptotic index for MNC and lymphocytes (but not monocytes) was again significantly higher in allergic as compared to non-allergic subjects after 24, 48 and 72 hour of culture. In allergic patients, mean serum sFas level, was significantly lower then in non-allergic group (mean value 624.8 pg/ml +/- 25.67 versus 802.0 pg/ml +/- 31.91; p = 0.003) and in both groups sFas level correlated inversely with apoptosis of MNC. The mean ICE/caspase-1 concentration was significantly higher in sera of allergic patients as compared to non-allergic group (mean value 27.71 pg/ml +/- 3.79 vs 23.54 pg/ml respectively; p<0.01). ICE/caspase-1 levels in allergic patients correlated with apoptotic index of mononuclear cells (r = 0.57; p<0.001). CONCLUSIONS: An increased spontaneous and mitogen-induced apoptosis of MNC from peripheral blood of atopic patients as well as different serum levels of sFas and ICE/caspase-1 correlating with apoptosis, suggest different regulation of apoptotic process in peripheral blood mononuclear cells of patients with allergic asthma and/or rhinitis.     

Gender differences in inflammatory proteins and pathways in seasonal allergic rhinitis

In model organisms, thousands of genes differ in expression between females and males. It is not known if differences on a similar scale are found in humans nor how this relates to disease. However, in allergic disease gender differences in the levels of both inflammatory cells and proteins have been shown. In this study, we found lower nasal fluid allergen-specific IgE in women than men with seasonal allergic rhinitis (SAR). This led to genome-wide analyses of gene expression in allergen-challenged CD4⁺ cells from patients with SAR before and after treatment with cortisone. Before treatment, 975 genes differed in expression between women and men: 337 were higher in women. After treatment only 428 genes and one pathway differed in expression. The genes that differed in expression between women and men were over-represented in 10 pathways. Five of the pathways regulated chemotaxis. All five were less active in women. One of the pathways was induced by the eosinophilic chemokine CCL4. Analysis of nasal fluid CCL4 protein confirmed lower levels in women with seasonal allergic rhinitis, before and during the pollen season. By contrast, nasal fluid CCL3 levels did not differ between the genders. In summary, this study shows gender differences in specific inflammatory pathways and proteins in patients with seasonal allergic rhinitis. Further studies are warranted to examine if such differences have diagnostic and therapeutic implications in allergic diseases.     

Pharmacoeconomics of sublingual immunotherapy with the 5-grass pollen tablets for seasonal allergic rhinitis

Allergic rhinitis has a very high burden regarding both direct and indirect costs. This makes essential in the management of AR to reduce the clinical severity of the disease and thus to lessen its costs. This particularly concerns allergen immunotherapy (AIT), that, based on its immunological action on the causes of allergy, extends its benefit also after discontinuation of the treatment. From the pharmacoeconomic point of view, any treatment must be evaluated according to its cost-effectiveness, that is, the ratio between the cost of the intervention and its effect. A favorable cost-benefit ratio for AIT was defined, starting from the first studies in the 1990s on subcutaneous immunotherapy (SCIT) in AR patients, that highlighted a clear advantage on costs over the treatment with symptomatic drugs. Such outcome was confirmed also for sublingual immunotherapy (SLIT), that has also the advantage on SCIT to be free of the cost of the injections. Here we review the available literature on pharmacoeconomic data for SLIT with the 5-grass pollen tablets.     

Randomised controlled trial of butterbur and cetirizine for treating seasonal allergic rhinitis

ObjectivesTo compare the efficacy and tolerability of butterbur (Petasites hybridus) with cetirizine in patients with seasonal allergic rhinitis (hay fever).DesignRandomised, double blind, parallel group comparison.SettingFour outpatient general medicine and allergy clinics in Switzerland and Germany.Participants131 patients were screened for seasonal allergic rhinitis and 125 patients were randomised (butterbur 61; cetirizine 64).InterventionsButterbur (carbon dioxide extract tablets, ZE 339) one tablet, four times daily, or cetirizine, one tablet in the evening, both given for two consecutive weeks.ResultsImprovement in SF-36 score was similar in the two treatment groups for all items tested hierarchically. Butterbur and cetirizine were also similarly effective with regard to global improvement scores on the clinical global impression scale (median score 3 in both groups). Both treatments were well tolerated. In the cetirizine group, two thirds (8/12) of reported adverse events were associated with sedative effects (drowsiness and fatigue) despite the drug being considered a non-sedating antihistamine.ConclusionsThe effects of butterbur are similar to those of cetirizine in patients with seasonal allergic rhinitis when evaluated blindly by patients and doctors. Butterbur should be considered for treating seasonal allergic rhinitis when the sedative effects of antihistamines need to be avoided.

What is already known on this topic

Seasonal allergic rhinitis (hay fever) is common in countries with temperate climates.Most patients have their symptoms treated for short periods, particularly during peaks in atmospheric pollen count

What this study adds

After two weeks, the effects of butterbur and cetirizine were comparable in patients with hay feverButterbur produced fewer sedating effects than cetirizineButterbur should be considered when the sedating effects of antihistamines must be avoided     

Prevalence of arthropod antibodies in Korean patients with allergic rhinitis.

Arthropod antigens are main causative agents which induce allergic responses in humans. However, little information is known about the prevalence of specific arthropod allergens in Koreans with allergic diseases. The current study was designed to determine the positive rates of arthropod antibodies by the Korean inhalant panel of MAST-CLA. One hundred sixty patients, who were diagnosed with allergic rhinitis from an out-patient center at the Soonchunhyang University Chunan Hospital, were studied between August 1998 to July 2000. The overall positive rate, at least more than one specific antibody of arthropods such as Dermatophagoides farinae (Df), Dermatophagoides pteronyssinus (Dp), and cockroach mix (Cm), was 46.9%. Each positive rate of Df, Dp, and Cm was 45.0%, 43.1%, and 8.8%, respectively. A significant agreement among arthropod allergens was observed (Df and Dp: 95.6%, Kappa = 0.911, P < 0.001). Our data supported the fact that arthropods were the most common allergens in Korean patients with allergic rhinitis; however, the MAST-CLA should be modified to increase specificity of arthropod allergens.     

Effects of Citrus unshiu powder on the cytokine balance in peripheral blood mononuclear cells of patients with seasonal allergic rhinitis to pollen

We evaluated the effects of a 50% methanol extract of Citrus unshiu powder (MEC) on cytokines in peripheral blood mononuclear cells (PBMCs) obtained from patients with seasonal allergic rhinitis to cedar pollen. The levels of cytokines, such as TNF-alpha, IFN-gamma, IL-2, IL-4, IL-5, IL-10, IL-12 (p70), IL-13, and GM-CSF, produced by pollen-stimulated PBMC were measured. We found that MEC suppressed pollen-induced TNF-alpha release and increased IFN-gamma release from PBMCs. The results suggest that Citrus unshiu powder has an immunomodulatory effect in vitro and that its use could improve seasonal allergic rhinitis symptoms.     

Survey on the impact of comorbid allergic rhinitis in patients with asthma

Background

Allergic rhinitis (AR) and asthma are inflammatory conditions of the airways that often occur concomitantly. This global survey was undertaken to understand patient perspectives regarding symptoms, treatments, and the impact on their well-being of comorbid AR and asthma.

Methods

Survey participants were adults with asthma (n = 813) and parents of children with asthma (n = 806) from four countries each in the Asia-Pacific region and Europe. Patients included in the survey also had self-reported, concomitant AR symptoms. Patients and parents were recruited by telephone interview or by direct interview.

Results

Most patients (73%) had pre-existing symptoms of AR when their asthma was first diagnosed. Shortness of breath (21%) was the most troublesome symptom for adults, and wheezing (17%) and coughing (17%) the most troublesome for children. Patients used different medications for treating asthma (most commonly short-acting β-agonists and inhaled corticosteroids) and for treating AR (most commonly oral antihistamines). The concomitant presence of AR and asthma disrupted the ability to get a good night's sleep (79%), to participate in leisure and sports activities (75%), to concentrate at work or school (69% of adults, 73% of children), and to enjoy social activities (57% of adults, 51% of children). Most patients (79%) reported worsening asthma symptoms when AR symptoms flared up. Many (56%) avoided the outdoors during the allergy season because of worsening asthma symptoms. Many (60%) indicated difficulty in effectively treating both conditions, and 72% were concerned about using excessive medication. In general, respondents from the Asia-Pacific region reported more disruption of activities caused by symptoms and more concerns and difficulties with medications than did those from Europe. Differences between the two regions in medication use included more common use of inhaled corticosteroids in Europe and more common use of Chinese herbal remedies in the Asia-Pacific region.

Conclusion

Results of this survey suggest that comorbid asthma and AR substantially impact patient well-being and that the worsening of AR symptoms in patients with asthma can be associated with worsening asthma symptoms. These findings underscore the need for physicians who treat patients with asthma to evaluate treatment options for improving symptoms of both AR and asthma when present concomitantly.

     

In vitro suppression of lymphocyte activation in patients with seasonal allergic rhinitis and pollen-related asthma by cetirizine or azelastine in combination with ginkgolide B or astaxanthin

Novel strategies are evaluated for management of allergic rhinitis and asthma in patients co-afflicted with both disorders. It is hypothesized that the platelet activating factor receptor antagonist ginkgolide B (GB) and the carotenoid antioxidant astaxanthin (ASX) interact with antihistamines cetirizine dihydrochloride (CTZ) and azelastine (AZE) to potentiate their ability to downregulate potentially pathological immune activation. Peripheral blood mononuclear cells from asthmatics and healthy subjects, cultured 24 hours with 50 μg/ml phytohemaglutinin (PHA) or PHA plus each drug are analyzed by flow cytometry for expression of CD25+ or HLA-DR+ by CD3+ (T cells). Results are reported as stimulation indices for CD3+CD25+ (SICD3+CD25+) and CD3+HLA-DR+ (SICD3+HLADR+) cells in cultures treated with PHA alone, versus cultures treated with both PHA and drugs. Optimal suppression of activated cells was observed in cultures stimulated with ASX 10-6 M + CTZ 10-6 M (SICD3+CD25+, p = 0.016; SICD3+HLADR, p = 0.012); ASX 10-6 M + AZE 10-6 M (SICD3+CD25+, p = 0.012; SICD3+HLADR, p = 0.015); GB 10-6 M + CTZ 10-6 M (SICD3+CD25+, p = 0.024, SICD3+HLADR+, p = 0.019). Results demonstrate improved activity of antihistamines by 2 phytochemicals, suggesting dosing strategies for animal trials of ASX- or GB-augmented formulations for seasonal allergic rhinitis and asthma.     

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.     

Development and pilot evaluation of a novel probiotic mixture for the management of seasonal allergic rhinitis

Microbial exposure may direct the immune system away from allergic-type responses, but until now probiotic interventions have had limited success in the prevention and treatment of allergic diseases. In this study, a novel probiotic mixture was specifically created based on preliminary in vitro investigations on pollen-induced immune responses. A mixture with Lactobacillus rhamnosus GR-1 and a novel fecal Bifidobacterium adolescentis isolate was formulated into a yogurt and tested for its effects in 36 subjects with allergic rhinitis over 2 pollen seasons in a double-blind, placebo-controlled trial. The new formulation was well tolerated, but did not have significant effects on the quality of life scores, use of antihistamines, or eosinophil cationic protein concentration in nasal lavage. However, at the end of the grass pollen season, serum IL-10 and IL-12 levels were increased in the probiotic group compared to the controls. During the ragweed season, the serum TGF-β levels were significantly higher in the probiotic group than in the controls. In conclusion, the novel probiotic formulation had potentially desirable effects on the cytokine profile of patients with allergic rhinitis, but provided few clinical benefits. The study highlights the challenges in designing efficient immunomodulatory probiotic therapies based upon in vitro findings.     

Gene expression profiling and functional analysis of angiogenic markers in murine collagen-induced arthritis

Introduction

Dysregulated angiogenesis is implicated in the pathogenesis of rheumatoid arthritis (RA). To provide a more profound understanding of arthritis-associated angiogenesis, we evaluated the expression of angiogenesis-modulating genes at onset, peak and declining phases of collagen-induced arthritis (CIA), a well-established mouse model for RA.

Methods

CIA was induced in DBA/1 mice with type II collagen. Functional capillary density in synovial tissue of knee joints was determined by intravital fluorescence microscopy. To assess the ability of arthritic joint homogenates to induce angiogenesis, an endothelial chemotaxis assay and an in vivo matrigel plug assay were employed. The temporal expression profile of angiogenesis-related genes in arthritic paws was analysed by quantitative real-time RT-PCR using an angiogenesis focused array as well as gene specific PCR. Finally, we investigated the therapeutic effect of a monoclonal antibody specifically blocking the binding of VEGF to neuropilin (NRP)-1.

Results

Although arthritic paw homogenates displayed angiogenic activity in vitro and in vivo, and synovia of arthritic paws appeared highly vascularised on histological examination, the functional capillary density in arthritic knee synovia was significantly decreased, whereas capillary diameter was increased. Of the 84 genes analysed, 41 displayed a differential expression in arthritic paws as compared to control paws. Most significant alterations were seen at the peak of clinical arthritis. Increased mRNA expression could be observed for VEGF receptors (Flt-1, Flk-1, Nrp-1, Nrp-2), as well as for midkine, hepatocyte growth factor, insulin-like growth factor-1 and angiopoietin-1. Signalling through NRP-1 accounted in part for the chemotactic activity for endothelial cells observed in arthritic paw homogenates. Importantly, therapeutic administration of anti-NRP1^B antibody significantly reduced disease severity and progression in CIA mice.

Conclusions

Our findings confirm that the arthritic synovium in murine CIA is a site of active angiogenesis, but an altered balance in the expression of angiogenic factors seems to favour the formation of non-functional and dilated capillaries. Furthermore, our results validate NRP-1 as a key player in the pathogenesis of CIA, and support the VEGF/VEGF receptor pathway as a potential therapeutic target in RA.     

变应性鼻炎患者外周血中IL-27和Treg细胞的相关性分析

目的探讨外周血IL-27和CD4^＋CD25^＋调节性T细胞（Treg）在变应性鼻炎（AR）发病机制中的作用。方法2012年3月至7月,收集AR患者32例（AR组）和20例健康志愿者（对照组）外周血,采用流式细胞术（FCM）检测外周血中Treg细胞比例;ELISA检测血清中IL-27、IL-10和TGF-β1的水平。结果AR组Treg细胞百分率[（1．75±0．56）％]明显低于对照组[（4．76±1．75）％],两组比较的差异有统计学意义（P〈0．01）。AR组IL-27、IL-10和TGF-β1的水平分别为（24．43±16．36）pg／ml、（14．29±6．16）pg／ml、（0．34±0．04）pg／ml,均低于对照组（44．09±13．12）pg／ml、（31．32±21．20）pg／ml、（O．49±0．06）pg／ml,两组之间的差异有统计学意义（P〈0．01）。AR患者外周血中IL-27和Treg细胞百分率、IL-10、TGF-β1存在正相关（r分别为0．825,0．646,0．517,P〈0．01）,Treg细胞百分率和IL-10、TGF-β1存在正相关（r=0.622,0．738,P〈0．01）,IL-10和TGF-β1无相关性（r=0．304,P〉0．05）结论AR患者外周血中IL-27水平降低,Treg细匏百分率降低及其主要分泌因子IL-10、TGF-β1水平降低,且IL-27与Treg细胞百分率、IL-10、TGF-β1水平呈正相关,提示在AR发病中IL-27对Treg细胞可能具有免疫调节作用。