Molecular allergy diagnosis using pollen marker allergens and pollen panallergens: Five patterns seen in multiple test reactions to pollen extracts.

Polysensitizations to tree, grass, and weed pollen are found in ~ 20% of pollen-allergic individuals. They are often based on broad IgE cross-reactivities to pollen panallergens belonging to highly conserved protein families: 1. profilins, 2. polcalcins (calcium-binding proteins in pollen), 3. cyclophilins. They represent highly conserved cross-reactive minor allergens present in all pollen species, but also in plant foods and other organisms. Despite being rarely clinically relevant they can hamper allergy diagnostic tests with extracts. In this situation, molecular allergy diagnosis is able to distinguish broad cross-reactivity due to allergen-specific IgE to pollen panallergens (i.e. profilins Bet v 2 or Phl p 12; polcalcins Bet v 4 or Phl p 7; and, in the future, cyclophilins Bet v 7 or Ole e 15) from primary IgE sensitizations to so-called marker allergens represented by important pollen major allergens: Bet v 1 for the birch and beech family (Fagales), Ole e 1 for olive and ash (Oleaceae), Phl p 1 for temperate climate grasses (Poaceae), Art v 1 for mugwort (Artemisia), Amb a 1 for Ambrosia species (Ambrosia). Five typical cases (A - E) with positive skin prick test results to tree, grass, and weed pollen extracts demonstrate typical patterns of IgE sensitization with a variable impact of pollen panallergens: A - profilins, B - polcalcins, C - profilins and polcalcins, D - presumably cyclophilins, E - primary polysensitization to tree, grass, and weed pollen without interference from profilins or polcalcins. Differences between pollen extract-based skin prick test diagnosis and molecular allergen-specific IgE testing are explained using the presented concept. This approach allows to reduce the number of allergen extracts - presuming they are also clinically relevant - for allergen immunotherapy (i.e., only tree and/or grass pollen extracts), particularly in pollen-polysensitized patients.

Basophil histamine release decreases during omalizumab therapy in allergic asthmatics.

BACKGROUND: Omalizumab is an established add-on therapy efficacious in allergic diseases with additional anti-inflammatory activity in the treatment of asthma. The evaluation of responders to anti-IgE treatment is critical to maximize benefit/risk/cost ratio. The aim of the study was to monitor the efficacy of anti-IgE treatment by ex vivo basophil histamine release. METHODS: Seventeen patients with allergic asthma were enrolled and received omalizumab at a dose of > or =0.016 mg/kg/IgE every 4 weeks. Histamine release from basophils was evaluated fluorometrically after dose-dependent allergen challenge at baseline and after 16 weeks of treatment. Maximal histamine release and cellular sensitivity to the allergen were calculated. Clinical measurements consisted of body plethysmography, skin prick test, beta2-agonist usage, serum free IgE levels, peripheral eosinophils and investigator ratings of global evaluation of treatment effectiveness. RESULTS: Maximal histamine release and cellular sensitivity to the allergen were significantly decreased in the omalizumab group compared to placebo. These changes were accompanied by significant changes in the clinical markers airway resistance, beta-agonist usage, skin prick test wheal area and investigator ratings of global evaluation of treatment effectiveness. CONCLUSIONS: Omalizumab therapy decreases basophil histamine release and cellular sensitivity with high effectivity of 95.8% (median). The decline of ex vivo basophil responses does not always parallel individual clinical improvement. Basophil-based stimulation tests should be further evaluated before being regarded as useful parameters monitoring omalizumab therapy.

Monocytes of allergics and non-allergics produce, store and release the neurotrophins NGF, BDNF and NT-3.

INTRODUCTION: Recent studies have shown that neurotrophins (NTs) are involved in inflammatory processes. Elevated plasma levels of NTs were found allergic diseases with the highest levels in allergic asthma. However, the exact cellular sources involved in the regulation and release of neurotrophins in allergic inflammation are still not well defined. OBJECTIVE: The aim of this study was to assess whether monocytes of allergic and non-allergic subjects produce, store and release the neurotrophins NGF, BDNF and NT-3. METHODS: Monocytes of allergic and non-allergic donors were purified by immunomagnetic selection. APAAP-staining for the presence of NTs and their receptors was performed. RT-PCR and Western blot evaluated the production and storage of NTs. Monocytes were incubated and supernatants were collected for measurement of neurotrophic factors after stimulation with lipopolysaccharide (LPS) as inflammatory stimulus. The neurotrophin content in lysates and cell culture supernatants was determined by ELISA. RESULTS: Human monocytes express the neurotrophins NGF, BDNF and NT-3 but also their specific receptors TrkA, TrkB and TrkC. RT-PCR amplification of isolated mRNA demonstrated expression of the examined neurotrophins. Proteins were detectable by Western blot. NTs were found in the monocyte lysates and supernatants at different levels in allergic and non-allergic donors. Cell stimulation with LPS leads to release of NGF and NT3. CONCLUSIONS: Monocytes, produce, store and release NGF, BDNF and NT-3. They are a possible source of elevated neurotrophin levels found in allergy and asthma.

Regulation of NGF and BDNF by dexamethasone and theophylline in human peripheral eosinophils in allergics and non-allergics.

BACKGROUND: Recent studies have shown that the neurotrophins NGF and BDNF are produced by eosinophils. The influence of neurotrophins in allergic diseases including asthma has been described. The regulation by pharmacological substance remains unclear. OBJECTIVES: The aim of this study was to assess whether approved pharmacological substances in the treatment of asthma such as corticosteroids or theophylline regulate neurotrophins on a cellular level. METHODS: Eosinophils were purified by negative immunoselection from allergics and non-allergic donors. Eosinophils were incubated with dexamethasone and theophylline and supernatants were collected for measurement of neurotrophic factors. The content of neurotrophins in eosinophil lysates was determined by ELISA. Regulation of stored NGF and BDNF was demonstrated by Western-blotting and flow cytometry while influence on transcription level was demonstrated by RT-PCR. RESULTS: Eosinophils produce and release the neurotrophins NGF and BDNF at different levels in allergics and non-allergics. Dexamethason lead to a significant downregulation of NGF in eosinophils of allergics. The levels of BDNF were not significantly reduced. Theophylline did not influence the levels of NGF nor BDNF significantly. CONCLUSIONS: The production of the neurotrophin NGF was downregulated by an established substance such as dexamethasone. This might further contribute to the pharmacological potential of corticosteroids in allergic asthma.

Effect of omalizumab treatment on peripheral eosinophil and T-lymphocyte function in patients with allergic asthma.

BACKGROUND: Omalizumab is a recombinant monoclonal anti-IgE antibody with proven efficacy in allergic diseases and further anti-inflammatory potency in the treatment of asthma. OBJECTIVES: To explore the anti-inflammatory mechanism of omalizumab, we investigated the induction of immunologic changes leading to eosinophil apoptosis and examined T-lymphocyte cytokine profiles in patients with allergic asthma. METHODS: Nineteen patients with allergic asthma were enrolled and received omalizumab at a dose of at least 0.016 mg/kg/IgE (IU/mL) every 4 weeks. Peripheral eosinophils and T-lymphocyte cytokine profiles were evaluated by fluorescence-activated cell sorting before treatment (baseline), at 12 weeks of treatment, and 12 weeks after discontinuation of treatment with omalizumab or placebo. RESULTS: Markers of eosinophil apoptosis (Annexin V) were significantly increased in omalizumab recipients compared with placebo, whereas no changes in markers of necrosis (7-amino-actinomycin) or eosinophil activation CD69 or Fas receptor (CD95) were detected. GM-CSF+ lymphocytes were reduced in omalizumab recipients compared with placebo. Fewer IL-2+ and IL-13+ lymphocytes were evident in omalizumab recipients than in the placebo group. There were no significant differences in IL-5, IFN-gamma, or TNF-alpha between the omalizumab and placebo groups. CONCLUSION: These findings provide further evidence that omalizumab has additional anti-inflammatory activity demonstrated by induction of eosinophil apoptosis and downregulation of the inflammatory cytokines IL-2 and IL-13. Further studies are needed to determine the underlying mechanisms. CLINICAL IMPLICATIONS: These findings support the critical role of IgE in the regulation of inflammation in allergic asthma: influencing the inflammation is the key to control the more severe type of asthma.

Immunological and clinical changes in allergic asthmatics following treatment with omalizumab.

IgE plays a key role in allergic asthma. We investigated whether omalizumab treatment of patients with moderate to severe allergic asthma leads to changes in inflammatory mediators and clinical symptoms. This sub-study was conducted on 35 patients with a positive skin prick test (SPT) requiring daily administration of beclomethasone dipropionate (500-1,000 microg), who participated in a multicentre, randomised, double-blind, placebo-controlled study. Omalizumab or placebo was administered at 0.016 mg/kg/IgE every 4 weeks. Patients recorded peak expiratory flow, asthma symptom score and beta(2)-agonist use in daily diaries and spirometry was performed at each visit. beta(2)-Agonist use and SPT wheal reaction decreased significantly (p < 0.05). Circulating levels of IL-5, IL-6, IL-8, IL-10, IL-13 and s-ICAM were measured before and after 16 weeks of treatment. IL-13 and s-ICAM were measured before and after 16 weeks of treatment. IL-13 decreased significantly (p < 0.01). IL-5 and IL-8 decreased in the omalizumab group compared to baseline. The other circulating mediators did not demonstrate any changes. Histamine release was significantly reduced (p < 0.01). Airway resistance (p < 0.05) and the provocative concentration inducing a 20% decrease in FEV(1) (p < 0.05) were measured before, after 16 weeks, and 3 months after completion of treatment. Both parameters decreased significantly (p < 0.05). Peripheral eosinophil count decreased significantly compared to placebo (p < 0.01). These findings suggest that omalizumab has potential as a novel treatment for allergic asthma.

Ozone-induced release of neuropeptides from human nasal mucosa cells.

Studies examining the effect of pharmacological agents on respiratory responses to ozone support the concept that the effects are mediated, at least in part, by neural mechanisms, including neuropeptide release. Using a special tissue culture system the influence of ozone (0.1 ppm/24 h) on nasal mucosa from allergic and nonallergic patients undergoing surgery for chronic nasal obstruction was examined. Substance P (SP)-immunoreactive nerves were found in air-exposed as well as in ozone-exposed tissue samples. The content of neurokinin A (NKA) and SP in the culture supernatants was significantly increased following ozone exposure compared to controls. Tissue of allergic patients showed an ozone-induced increase in the release of NKA and SP compared to tissue of nonallergic patients. These results suggest that the mode of action of ozone results in an increased activity of sensory nerves in the upper airways with a subsequent increased release of neuropeptides. In addition to the known ozone-induced release of proinflammatory mediators, these mechanisms may explain the increased responsiveness of patients with hypersensitive airways.