Efficacy and safety of a novel nasal steroid, S0597, in patients with seasonal allergic rhinitis.

BACKGROUND: Allergic rhinitis (AR) poses a significant global burden with increasing prevalence. Although intranasal glucocorticosteroids are effective, older agents can have limiting side effects. S0597, a novel intranasal glucocorticosteroid, has demonstrated good safety and tolerability during preclinical and phase 1 studies. OBJECTIVE: To assess the clinical efficacy, safety, and tolerability of different doses of S0597 nasal spray vs placebo in patients with seasonal AR. METHODS: This phase 2, randomized, double-blinded, placebo-controlled, parallel-group, single-center study randomized 159 patients 18 to 65 years old (mean age 37.8 years) with a positive skin prick test reaction for Dactylis glomerata to receive S0597 at 200, 400, or 800 µg/d or placebo for 15 days. On days 1 (baseline), 15, and 16, patients underwent a 4-hour pollen challenge to evaluate treatment efficacy measured by the change in total nasal symptom score (TNSS) from baseline to days 15 and 16 and changes in TNSS subscales and nasal secretion. RESULTS: Statistically significant improvements in TNSS from baseline to days 15 and 16 were observed with all S0597 doses vs placebo (P = .0005 overall), with the greatest improvements observed in the highest-dose group (P < .0001). Significant decreases were observed in each S0597 dose group vs placebo for TNSS subscales and nasal secretion. Improvements in nasal secretion were related to dose, with the greatest decreases from baseline in the 800-µg/d group on days 15 and 16 (P < .0001). CONCLUSION: Treatment with S0597 at 200, 400, and 800 µg/d by 2 divided doses for 2 weeks was safe and significantly more effective than placebo for improving nasal symptoms associated with grass pollen-induced seasonal AR in adults. TRIAL REGISTRATION: ClinicalTrials.gov, identifier NCT01614691.

Allergen immunotherapy decreases LPS-induced NF-κB activation in neutrophils from allergic patients.

BACKGROUND: Allergen-specific immunotherapy (IT) is widely used to treat allergic diseases. The molecular mechanisms have not been clarified yet completely. The present work was undertaken to analyze the effect of IT in the activation of NF-κB. METHODS: Neutrophils from 15 pollen-allergic IT-treated patients, 10 untreated pollen-allergic patients, and 10 healthy donors were in vitro stimulated with LPS. NF-κB activation (p65/p52) was measured in their nuclear extracts by enzyme-linked immunosorbent assay (ELISA). IκBα phosphorylation, NF-κB-repressing factor (NRF) activation, and thromboxane A2 (TXA2 ) and Interleukin-8 (IL-8) release were measured by ELISA. RESULTS: There was a positive correlation between the score of symptoms and NF-κB activation in human neutrophils. IT significantly decreased NF-κB activation levels in neutrophils compared with neutrophils from untreated patients. IκBα phosphorylation and NRF activation levels were, respectively, significantly lower and higher in neutrophils from IT-treated patients than from untreated patients. IL-8 and TXA2 release were significantly lower in neutrophils from IT-treated patients than from untreated patients. CONCLUSIONS: IT positive effects are at least in part mediated by the negative regulation of NF-κB activation in human neutrophils. These observations represent a novel view of neutrophils as possible cell target to treat IgE-dependent diseases through NF-κB downmodulation.

Efficacy of epinastine hydrochloride for antigen-provoked nasal symptoms in subjects with orchard grass pollinosis.

BACKGROUND: Among the gramineae species, orchard grass is a typical causative pollen that provokes seasonal rhinitis. The purpose of this study was to examine the protective efficacy of epinastine hydrochloride for signs and symptoms caused by repeated nasal provocation with discs containing orchard grass pollen. METHODS: A single-dose, placebo-controlled, double-blind, crossover clinical study was conducted in subjects with orchard grass pollinosis. The pollen challenge was conducted with the use of provocation discs containing orchard grass pollen. RESULTS: Epinastine hydrochloride suppressed nasal symptoms caused by nasal provocation tests using orchard grass pollen discs. Among the nasal symptoms, the number of sneezing was significantly inhibited 30 minutes and 60 minutes after the administration of epinastine hydrochloride, as compared with placebo. There were no adverse reactions to the study drugs. CONCLUSIONS: Our results suggest that nasal provocation tests with discs containing orchard grass pollen is a useful method for evaluating the onset of action of antiallergic drugs. As compared with placebo, epinastine hydrochloride decreased early-phase sneezing and the total nasal symptom score after repeated nasal provocations with orchard grass pollen discs.

Generation of recombinant antibodies against orchardgrass Acidic nsLTP-like proteins.

Embryogenic and non-embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.) secreted into the culture medium a set of proteins, among which low molecular mass (11/12 kDa) proteins were found. However, only the 11/12 kDa proteins from the embryogenic suspension cultures reacted specifically with an antiserum raised against the carrot EP2 non-specific lipid transfer protein (nsLTP). Two-dimensional (2-D) electrophoretic analysis revealed that the extracellular nsLTP-like proteins from the embryogenic lines were acidic proteins, with pI values ranging between 4.3 and 6.4, and the 11/12 kDa proteins of the non-embryogenic lines were basic ones (pI 8-9.3). This is only the second case to report on the accumulation of extracellular acidic nsLTP-like proteins in the culture medium during somatic embryogenesis. A naive phage display Griffin1. library was used to select single-chain phage antibodies, which specifically bind to acidic nsLTP-like proteins. Nine phage clones were selected after four rounds of biopanning of the target proteins blotted on a nitrocellulose membrane. Three soluble monoclonal single-chain phage antibodies, expressed in the non-suppressor E. coli strain HB2151, were purified by metal affinity chromatography and found to be highly specific for the acidic nsLTP-like proteins from the embryogenic suspension cultures. The application of the selected monoclonal antibodies for localization and elucidation of the role of the acidic nsLTP-like proteins in vivo is discussed.

A modified enzyme-linked immunosorbent assay adapted for immunodetection of low amounts of water-insoluble proteins.

A mixture of thiourea, urea and CHAPS (TUC) is an excellent solvent compatible with isoelectrofocusing (IEF) separation of water-insoluble protein extracts, and their subsequent two-dimensional gel electrophoresis is an important step in proteomic studies. The main aim of this work was to quantify extremely low amounts of water-insoluble proteins contained, for instance, in samples collected in bio-aerosol samplers. High CHAPS concentrations solubilize many proteins. However, enzyme-linked immunosorbent assay (ELISA), which is the most popular immunodetection method of quantifying antigens, is unfortunately not compatible with these high CHAPS concentrations and with the low protein concentrations of TUC extracts. The most common mixture used to solubilize these proteins contains 2 mol l(-1) thiourea, 7 mol l(-1) urea and 5% w/v CHAPS. This paper shows that these components inhibit the adsorption and/or recognition of proteins on microtitration plates, preventing antigen quantification under classic ELISA conditions. We have tried several solvents (ethanol, isopropanol, acetonitrile and trichloroacetic acid) to make the TUC-soluble proteins stick to the ELISA plates, and ethanol was shown to be the most appropriate. In this study, we have defined a new ELISA protocol allowing rapid and sensitive detection of low concentrations (60-500 ng ml(-1)) of water-insoluble proteins extracted with high concentrations of TUC.

Effects of rupatadine vs placebo on allergen-induced symptoms in patients exposed to aeroallergens in the Vienna Challenge Chamber.

BACKGROUND: Rupatadine is a novel compound with potent dual antihistamine and platelet-activating factor antagonist activities and no sedative effects. OBJECTIVE: To evaluate the efficacy of rupatadine, 10 mg once daily, and placebo on allergen-induced symptoms (including nasal congestion), nasal airflow, nasal secretion, and subjective tolerability in response to grass pollen in a controlled allergen-exposure chamber. METHODS: In a randomized, double-blind, placebo-controlled, crossover trial, 45 patients with a history of seasonal allergic rhinitis received rupatadine or placebo every morning for 8 days in 2 different periods separated by a 14-day washout interval. On day 8 of each crossover period, patients underwent a 6-hour allergen exposure in the Vienna Challenge Chamber, where a constant and homogeneous concentration of aeroallergens was maintained. Subjective and objective assessments were performed online during the exposure. RESULTS: Subjective single and composite nasal and nonnasal symptoms were consistently less severe with rupatadine use than with placebo use starting from the first evaluation at 15 minutes to the end of the 6-hour Vienna Challenge Chamber challenge, with the most significant effects seen for nasal rhinorrhea, nasal itching, sneezing attacks, and total nasal symptoms (P < .001 for all). All the other symptoms (including nasal congestion, P < or = .005) were also significantly reduced with active treatment compared with placebo use. Mean secretion weights and overall feeling of complaint were significantly lower with rupatadine therapy than with placebo use (P < or = .001). Overall, rupatadine treatment was well tolerated. CONCLUSION: Rupatadine treatment is effective and well tolerated in patients with allergen-induced symptoms exposed to aeroallergens in a controlled exposure chamber.

Monoclonal antibody against a cell wall marker protein for embryogenic potential of Dactylis glomerata L. suspension cultures.

We identified and isolated a monoclonal antibody (MAb 3G2) raised against extracellular proteins from microcluster cells of orchard grass (Dactylis glomerata L.) embryogenic suspension culture. MAb 3G2 recognized with high specificity an antigen ionically bound within the primary cell wall and in the culture medium of microcluster cells. Two-dimensional polyacrylamide gel analysis and blotting of proteins on PVDF membrane showed that MAb 3G2 detected a single polypeptide of apparent molecular mass of 48 kDa and an isoelectric point (pI) of 5.2, designated EP48. A transient expression during somatic embryogenesis was observed for EP48. Indirect immunofluorescence showed that this protein highly accumulated in the cell walls of some single cells, microclusters and partly in proembryogenic masses (PEMs), but not in globular embryos of the embryogenic cell line and microclusters from the non-embryogenic cell line. Signal intensity varied between individual cells of the same population and in successive stages of somatic embryo development. Screening of several D. glomerata L. embryogenic and non-embryogenic cell lines with MAb 3G2 indicated the presence of ECP48 in only embryogenic suspension cultures at early stages of embryo development long before morphological changes have taken place and thus it could serve as an early marker for embryogenic potential in D. glomerata L. suspension cultures.

The major grass pollen group 5 allergen from Dactylis glomerata and its C-terminal split product both behave as dimers: implications for allergen standardization.

BACKGROUND: On SDS-PAGE grass pollen group-5 allergens migrate as a doublet with an apparent molecular mass (M(r)) of 25 kDa. Immunoblot analysis revealed additional group 5 reactivity at double and half this M(r). The aim of this study was to investigate these group 5 molecular entities and to compare their allergenicity and behavior in quantitative immunoassays. METHODS: Group-5-specific monoclonal antibodies were produced and used for the development of a group-5-specific sandwich ELISA. Affinity-purified Dac g 5 was separated by SDS-PAGE/Western blotting; individual bands were analyzed by N-terminal sequencing. Size exclusion chromatography (SEC) in conjunction with group-5-specific ELISA, competitive RIA and RAST inhibition were used to analyze the size distribution of Dac g 5. Basophil histamine release assays were used to assess biological activity. RESULTS: The lower band of the typical group 5 doublet was identified as a truncated form lacking the typical group 5 N-terminus AD(L)/(A)GY, observed in the upper band. The 12-kDa peptide was shown to be the C-terminal half of Dac g 5 (amino acid 127 onwards). SEC in conjunction with competitive RIA revealed that around 45% of Dac g 5 is represented by the 12-kDa peptide. Both the C-terminal half and the whole allergen dimerize under nondenaturing conditions. In competitive RIA and RAST inhibition both forms are equally well detected. In contrast, the half molecule is poorly recognized in sandwich ELISA and displays negligible biological activity in basophil histamine release tests with purified IgE. CONCLUSIONS: These observations stress the need to evaluate the performance of allergen standardization protocols in detail, with special attention to allergen size distribution.

Surfactant protein D increases phagocytosis and aggregation of pollen-allergen starch granules.

Recent studies have shown that surfactant components, in particular the collectins surfactant protein (SP)-A and -D, modulate the phagocytosis of various pathogens by alveolar macrophages. This interaction might be important not only for the elimination of pathogens but also for the elimination of inhaled allergens and might explain anti-inflammatory effects of SP-A and SP-D in allergic airway inflammation. We investigated the effect of surfactant components on the phagocytosis of allergen-containing pollen starch granules (PSG) by alveolar macrophages. PSG were isolated from Dactylis glomerata or Phleum pratense, two common grass pollen allergens, and incubated with either rat or human alveolar macrophages in the presence of recombinant human SP-A, SP-A purified from patients suffering from alveolar proteinosis, a recombinant fragment of human SP-D, dodecameric recombinant rat SP-D, or the commercially available surfactant preparations Curosurf and Alveofact. Dodecameric rat recombinant SP-D enhanced binding and phagocytosis of the PSG by alveolar macrophages, whereas the recombinant fragment of human SP-D, SP-A, or the surfactant lipid preparations had no effect. In addition, recombinant rat SP-D bound to the surface of the PSG and induced aggregation. Binding, aggregation, and enhancement of phagocytosis by recombinant rat SP-D was completely blocked by EDTA and inhibited by d-maltose and to a lesser extent by d-galactose, indicating the involvement of the carbohydrate recognition domain of SP-D in these functions. The modulation of allergen phagocytosis by SP-D might play an important role in allergen clearance from the lung and thereby modulate the allergic inflammation of asthma.

Human serum IgE-mediated mast cell degranulation shows poor correlation to allergen-specific IgE content.

BACKGROUND: Although allergen-specific IgE content in serum can be determined immunochemically, little is known about the relationship between this parameter and the strength of the degranulation response upon allergen triggering. OBJECTIVES: Analyse the degranulation capacity of immunochemically defined purified and serum IgE after challenge with anti-IgE or allergen using a rat mast cell line (RBL) transfected with the alpha-chain of the human high-affinity IgE receptor (FcepsilonRI). METHODS: Purified IgE specific for 4-hydroxy-3nitrophenylacetyl, purified IgE of unknown specificity, and sera from allergic patients sensitive to Dermatophagoides pteronyssinus and Dactylis glomerata were assessed. Degranulation was measured by a beta-hexosaminidase release assay after anti-IgE or allergen-specific challenge. RESULTS: For purified monoclonal IgE a significant correlation (r = 0.97) was found between the proportion of bound allergen-specific IgE and the strength of the degranulation response. In contrast, no correlation (r = 0.27) was detected after sensitization with serum IgE. CONCLUSION: Our studies demonstrate that mast cell activation mediated through IgE from allergic patients is a result of complex relationships that are not only dependent on allergen-specific IgE content but also relate to the capacity to efficiently sensitize and trigger the signalling responses that lead to degranulation.

Specific allergens enhance elastase release in stimulated neutrophils from asthmatic patients.

BACKGROUND: The presence of the three forms of IgE receptor - the heterotrimeric high-affinity receptor for IgE (Fc(epsilon)RI), the low-affinity receptor for IgE (Fc(epsilon)RII/CD23) and the Mac-2/IgE-binding protein (epsilonBP) - has been demonstrated on human neutrophils. We have previously shown that specific allergens are able to activate functional responses by neutrophils from allergic patients sensitized to those allergens. Neutrophils are present at the sites of allergic inflammation. The primary (azurophilic) granules of neutrophils contain a variety of enzymes, such as elastase, that might potentiate inflammation. It is not known whether specific allergens are able to elicit elastase release by neutrophils from allergic patients. In addition, we attempted to evaluate the relationship between neutrophil degranulation and lung function of the patients, measured as FEV(1). METHODS: Neutrophils were challenged in vitro with the specific allergens that produced clinical symptoms in asthmatic patients. The cells were also challenged with allergen to which the patients were not sensitive. Neutrophils from normal subjects were challenged with allergens as control. RESULTS: The in vitro challenge of neutrophils with allergens to which the patients were sensitive elicited a release of elastase by these cells. The in vitro activation of neutrophils was highly allergen specific; allergens other than those accounting for clinical symptoms did not evoke elastase release, and allergens were ineffective on neutrophils from healthy donors. A significant inverse correlation was observed between elastase release and patients' lung function, measured as FEV(1). CONCLUSION: An IgE-dependent mechanism might promote elastase release by neutrophils at allergic sites. There is a significant inverse relationship between levels of elastase released by neutrophils from allergic patients and lung function, as assessed by FEV(1).

Hypersensitivity to molds in New York City in adults who have asthma.

Molds have been linked epidemiologically to asthma as a key aeroallergen in several studies. Other allergens such as cockroach have been linked to asthma in New York City (NYC). To our knowledge, however, the pattern of mold hypersensitivity has never been examined systematically in the NYC area. Thus, we sought to determine the association between mold hypersensitivity and asthma in a large group of ambulatory patients evaluated for allergic disease for the years 1993 through 2001 at a single medical center. Serological testing for mold-specific immunoglobulin E (IgE) as well as IgE specific for other aeroallergens was performed and the associations between allergen-specific IgE and the presence of asthma were examined using bivariate and multivariate analysis. Factor analysis showed that three distinct groupings of aeroallergen-specific IgE existed within the panel of allergens used. Group 1 consisted of cat dander and dust mites (Dermatophagoides farinae). Group 2 consisted of tree, grass, and ragweed pollen. Group 3 consisted of the Deuteromycetes molds, Alternaria tenuis, Aspergillus fumigatus, and Cladosporium herbarum. Patients with asthma had a highly significant increase in the incidence of hypersensitivity to cat/dust mites and to the molds. Multivariate analysis showed that the presence of hypersensitivity to either A. tenuis or C. herbarum had a significant independent association with asthma after adjustment for cat/dust mite hypersensitivity and after adjustment for other clinical factors. On the other hand, pollen hypersensitivity was not associated independently with asthma. Mold hypersensitivity was strongly correlated with hypersensitivity to cat or dust mites in patients who did not have asthma but not in patients who did have asthma. In the NYC area, recent pollen and spore counts show that mold spores are measurable in at least 75% of the year. Thus it is conceivable that mold hypersensitivity plays a contributing and independent role in initiating or perpetuating the allergic response in patients with asthma in the New York area.