Development and evaluation of a sublingual tablet based on recombinant Bet v 1 in birch pollen-allergic patients.

BACKGROUND: Sublingual immunotherapy (SLIT) applied to type I respiratory allergies is commonly performed with natural allergen extracts. Herein, we developed a sublingual tablet made of pharmaceutical-grade recombinant Bet v 1.0101 (rBet v 1) and investigated its clinical safety and efficacy in birch pollen (BP)-allergic patients. METHODS: Following expression in Escherichia coli and purification, rBet v 1 was characterized using chromatography, capillary electrophoresis, circular dichroism, mass spectrometry and crystallography. Safety and efficacy of rBet v 1 formulated as a sublingual tablet were assessed in a multicentre, double-blind, placebo-controlled study conducted in 483 patients with BP-induced rhinoconjunctivitis. RESULTS: In-depth characterization confirmed the intact product structure and high purity of GMP-grade rBet v 1. The crystal structure resolved at 1.2 Å documented the natural conformation of the molecule. Native or oxidized forms of rBet v 1 did not induce the production of any proinflammatory cytokine by blood dendritic cells or mononuclear cells. Bet v 1 tablets were well tolerated by patients, consistent with the known safety profile of SLIT. The average adjusted symptom scores were significantly decreased relative to placebo in patients receiving once daily for 5 months rBet v 1 tablets, with a mean difference of 17.0-17.7% relative to the group treated with placebo (P < 0.025), without any influence of the dose in the range (12.5-50 µg) tested. CONCLUSION: Recombinant Bet v 1 has been produced as a well-characterized pharmaceutical-grade biological drug. Sublingual administration of rBet v 1 tablets is safe and efficacious in patients with BP allergic rhinoconjunctivitis.

Characterization of oral immune cells in birch pollen-allergic patients: impact of the oral allergy syndrome and sublingual allergen immunotherapy on antigen-presenting cells.

BACKGROUND: A detailed characterization of human oral immune cells is needed to better understand local mechanisms associated with allergen capture following oral exposure. METHODS: Oral immune cells were characterized by immunohistology and immunofluorescence in biopsies obtained from three healthy individuals and 23 birch pollen-allergic patients with/without oral allergy syndrome (OAS), at baseline and after 5 months of sublingual allergen immunotherapy (AIT). RESULTS: Similar cell subsets (i.e., dendritic cells, mast cells, and T lymphocytes) were detected in oral tissues from healthy and birch pollen-allergic individuals. CD207+ Langerhans cells (LCs) and CD11c+ myeloid dendritic cells (DCs) were found in both the epithelium and the papillary layer of the Lamina propria (LP), whereas CD68+ macrophages, CD117+ mast cells, and CD4+ /CD8+ T cells were rather located in both the papillary and reticular layers of the LP. Patterns of oral immune cells were identical in patients with/without OAS, except lower numbers of CD207+ LCs found in oral tissues from patients with OAS, when compared to OAS- patients (P < 0.05). A 5-month sublingual AIT had a limited impact on oral immune cells, with only a significant increase in IgE+ cells in patients from the active group. Colocalization experiments confirmed that such IgE-expressing cells mostly encompass CD68+ macrophages located in the LP, and to a lesser extent CD207+ LCs in the epithelium. CONCLUSION: Two cell subsets contribute to antigen/allergen uptake in human oral tissues, including (i) CD207+ LCs possibly involved in the physiopathology of OAS and (ii) CD68+ macrophages likely critical in allergen capture via IgE-facilitated mechanisms during sublingual AIT.

Recommendations for the standardization of clinical outcomes used in allergen immunotherapy trials for allergic rhinoconjunctivitis: an EAACI Position Paper.

BACKGROUND: Allergen immunotherapy (AIT) has been thoroughly documented in randomized controlled trials (RCTs). It is the only immune-modifying and causal treatment available for patients suffering from IgE-mediated diseases such as allergic rhinoconjunctivitis, allergic asthma and insect sting allergy. However, there is a high degree of clinical and methodological heterogeneity among the endpoints in clinical studies on AIT, for both subcutaneous and sublingual immunotherapy (SCIT and SLIT). At present, there are no commonly accepted standards for defining the optimal outcome parameters to be used for both primary and secondary endpoints. METHODS: As elaborated by a Task Force (TF) of the European Academy of Allergy and Clinical Immunology (EAACI) Immunotherapy Interest Group, this Position Paper evaluates the currently used outcome parameters in different RCTs and also aims to provide recommendations for the optimal endpoints in future AIT trials for allergic rhinoconjunctivitis. RESULTS: Based on a thorough literature review, the TF members have outlined recommendations for nine domains of clinical outcome measures. As the primary outcome, the TF recommends a homogeneous combined symptom and medication score (CSMS) as a simple and standardized method that balances both symptoms and the need for antiallergic medication in an equally weighted manner. All outcomes, grouped into nine domains, are reviewed. CONCLUSION: A standardized and globally harmonized method for analysing the clinical efficacy of AIT products in RCTs is required. The EAACI TF highlights the CSMS as the primary endpoint for future RCTs in AIT for allergic rhinoconjunctivitis.

Efficacy, safety, and immunological effects of a 2-year immunotherapy with Depigoid birch pollen extract: a randomized, double-blind, placebo-controlled study.

BACKGROUND: Rhinoconjunctivitis due to birch pollen sensitization is common in Northern Europe. A depigmented polymerized birch pollen extract - Depigoid has been developed for immunotherapy. OBJECTIVE: To evaluate its clinical efficacy, safety, and effects on immunological parameters. METHODS: Sixty-one patients aged 7-69 years were included in a randomized, double-blind, placebo-controlled trial of subcutaneous immunotherapy (SCIT) using depigmented polymerized birch pollen extract. SCIT consisted of four increasing doses at 7-day intervals, followed by maintenance injections of 500 DPP (corresponding to 30 microg Bet v1 before depigmentation) at 6-week intervals for 18 months. The primary outcome was the combined symptom and medication score during the 2006 birch pollen season. The frequency of peripheral blood mononuclear cells (PBMC)producing IL-4, IL-10, IL-12, and IL-13 was assessed in a subgroup of patients by ELISPOT assay. RESULTS: After 18 months of treatment, the median combined symptom and medication score (upper/lower quartile) of treated patients was significantly lower than those on placebo: 8.0 (5.8-10.3) and 12.6 (8.6-16.2), respectively (P=0.004). Systemic reactions occurred in 29 patients (12 active, 17 placebo), were grades 1 or 2, and none required specific treatment. After 18 months of treatment, mean serum concentrations of specific IgE increased significantly in both groups (P<0.0001) whereas serum concentrations of both specific IgG1 and IgG4 only increased significantly in the SCIT group (P=0.002) and not in the placebo group. The seasonal increase in numbers of IL-4- and IL-13-producing PBMC was blunted by immunotherapy. CONCLUSIONS: SCIT with depigmented polymerized birch pollen extract significantly reduced symptom and medication scores when compared with the placebo, was well tolerated, and resulted in immunological changes comparable with those of native pollen extracts.

A follow-up study of immunotherapy-treated birch-allergic patients: effect on the expression of chemokines in the nasal mucosa.

BACKGROUND: Specific immunotherapy (SIT) is the only treatment producing lasting clinical improvement in patients with allergy. We investigated the long-term effect of SIT treatment on the expression of chemokines: eotaxin, RANTES (regulated upon activation, normal T cell expressed and secreted) and thymus and activation-regulated chemokine (TARC), and their receptors CCR3 and CCR4 in biopsies of nasal mucosa from birch-allergic individuals. METHODS: Sixteen patients who completed a 3-year treatment programme 3-5 years ago, and 12 untreated, matched controls were included in the study. Patients recorded symptoms and use of rescue medication before and during the pollen season. Nasal mucosa samples obtained before and during the season were stained for eosinophil and mast cell markers and for eotaxin, RANTES, TARC, CCR3 and CCR4. RESULTS: During the pollen season, rhinoconjunctivitis symptoms increased in both SIT and control groups (P=0.001 and 0.002, respectively). However, SIT patients had 37% fewer symptoms than controls. Medication use increased in both groups (P=0.002) during the season but the SIT group used 28% less than the controls (P=0.02). The number of eosinophils in the nasal mucosa increased in the control group (P=0.01) and the difference between the groups was significant during the season (P=0.01). No seasonal increase in the numbers of mast cells was seen, but during the pollen season, more (P=0.02) AA(+) cells were found in the controls than in the SIT group. The number of eotaxin(+) and RANTES(+) cells increased in the control group (P=0.01 and 0.03, respectively) and the difference between groups during the season was significant (P=0.01 and 0.01, respectively). The TARC(+) cell numbers were lower in the SIT group during the season (P=0.003). The CCR3(+) cells increased only in the control group during the pollen season and remained unchanged in SIT patients, while CCR4(+) cell numbers increased in both the control (P=0.03) and SIT (P=0.02) groups. CONCLUSION: This study confirmed that decreased numbers of eosinophils in the nasal mucosa is a long-lasting effect of birch SIT. SIT also prevented seasonal rises in the number of cells expressing the chemokines eotaxin and RANTES.

A double-blind placebo-controlled birch allergy vaccination study II: correlation between inhibition of IgE binding, histamine release and facilitated allergen presentation.

BACKGROUND: The pathogenesis of IgE-mediated allergic disease is closely related to the production of T-helper type 2 (Th2) cytokines, which lead to IgE production pivotal for activation of mast cells and basophils. Proliferating T cells along with eosinophils expanded and attracted by Th2 cytokines are major contributors to the late-phase reaction. The activation of these Th2 cells is strongly enhanced by CD23-mediated IgE facilitated allergen presentation (FAP). OBJECTIVE: The present study aims to investigate the effect of specific immunotherapy (SIT)-induced allergen-specific non-IgE antibodies (blocking antibodies) on IgE binding to allergen, histamine release (HR) and CD23-mediated allergen uptake in antigen-presenting cells. METHODS: Competition between IgE and non-IgE for allergen binding was studied by Advia Centaur antibody measurements, passively sensitized basophils were used to study HR and IgE-facilitated binding of allergen to B cells (FAP) was studied by flow cytometry. FAP measurements were performed both with and without the addition of a reference IgE serum, which was included to obtain optimal complex formation. The serum samples were obtained from birch pollen immunotherapy (n=21) or placebo control patients (n=21) before and after 1 and 2 years of treatment. RESULTS: Statistically significant reduction of all parameters investigated was observed after 1 year of treatment and the effect was maintained during the second year of treatment. There was a clear correlation between the two FAP measurements and between each of them and the level of T cell activation reported upon previously. Moreover, strong correlations were found between changes in FAP, IgE binding and HR. CONCLUSION: The present study clearly demonstrates that SIT induces changes in the composition of serum antibodies that inhibit IgE binding, HR and FAP to a similar extent. This suggests that these measurements, individually or in combination, may be used to monitor the immunological effect of SIT, even though direct correlations to changes in clinical parameters could not be demonstrated.

The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification.

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

Early and late phase asthmatic response in lower airways of cat-allergic asthmatic patients--a comparison between experimental and environmental allergen challenge.

BACKGROUND: Standardized experimental allergen challenges are usually adopted to investigate the effect of allergen exposure on the lower airways. Environmental (natural) allergen challenges are used less often, mainly because of difficulties in standardizing the method, safety reasons and costs. The aim of this study was to investigate the relationship between an experimental and an environmental bronchial challenge. For this reason a natural challenge model was developed. METHODS: Sixty-two patients with a history of cat allergen-induced symptoms involving the lower airways, positive skin prick test, positive in vitro specific IgE to cat allergen and bronchial hyper-responsiveness were included. All 62 patients underwent an experimental challenge in the laboratory followed by an environmental allergen challenge. RESULTS: All 62 patients developed an early asthmatic response [>or=20% fall in forced expiratory volume in 1 s (FEV1)] in the experimental challenge and 60% (37/62) during the environmental challenge. A late asthmatic response (>or=15% fall in FEV1 within 3-24 h) was seen in 56% (35/62) of the patients after the experimental challenge. Following the environmental challenge 47% (29/62) of the patients developed a late response. Thirty-four per cent (21/62) of the patients developed a late response in both challenge models and 31% (19/62) did not develop a late response in any model. Thus, there was consistency in 65% (40/62) of the patients in both challenge models. CONCLUSION: We found consistency in the pattern of response to inhaled allergen between the two challenge models and we believe that experimental bronchial challenge is likely to reflect the development of relevant inflammation in the lower airways after low-dose allergen exposure in the environment.

[Comparison of in vitro and in vivo methods of evaluation of the efficacy of influenza virus hemagglutinin inhibitors].

One of the problem in the selection of the most effective antiviral preparations with a broad spectrum of antiviral protective activity, is the "continuity" of assays of different level of complexity so, that the most effective antiviral therapeutic, selected by in vitro assays would be the most effective in vivo. Comparative study of the efficacy of the influenza virus inhibitor in the assays of inhibition of virus binding with fetuin, inhibition of infectious focus forming units in MDCK cells, inhibition of virus yield in infected MDCK cells, and inhibition of influenza virus infectivity in mice infected by viral aerosol are presented. The value of 50% inhibiting concentration IC50 for the pare "influenza virus strain A/NIB/23/89-MA-inhibitor tetra-Aca6-6'SLN" corresponded to 6-10 microM and was invariant for three different tests--in vitro assay of inhibition of virus binding with fetuin, inhibition of yield in infected MDCK cell culture, and inhibition of virus infectivity in mice, but not for the assay of inhibition of infectious focus forming units in cell culture.

Increased expression of lipoxygenase enzymes during pollen season in nasal biopsies of pollen-allergic patients.

BACKGROUND: Exposure of patients sensitized to pollen triggers development of seasonal allergic rhinitis symptoms (SAR). Eicosanoids are a group of arachidonic acid metabolites contributing to the symptoms of SAR. The aim of this study was to investigate seasonal changes in the expression of enzymes of the eicosanoid pathway in the nasal mucosa of patients with SAR. METHODS: Twenty SAR patients allergic to birch or grass and eight healthy subjects were included in the study. Patients registered rhinoconjunctivitis symptoms and use of rescue medication before and during the pollen season. Nasal biopsies were obtained before and around the peak of the season, sectioned and stained using markers for eosinophils, mast cells, T cells and neutrophils. Antibodies against the following enzymes were also used: cyclo-oxygenase (COX-1, COX-2), 5-lipoxygenase (5-LO), 5-lipoxygenase-activating factor (FLAP), LTA4 hydrolase (LTA4h) and LTC4 synthase (LTC4s). RESULTS: During the pollen season symptoms of rhinoconjunctivitis and medication score increased significantly (P=0.001; P=0.001 respectively). During the pollen season numbers of eosinophils (P=0.02) and cell positive 5-LO (P=0.02), LTC4s (P=0.04) and LTA4h (P=0.02) increased significantly. During season number of mast cells and cells expressing 5-LO and LTA4h were higher in SAR than in healthy controls group (P=0.02; P=0.01; P=0.03 respectively). CONCLUSION: In sensitized patients exposure to pollen allergen results in increased expression of enzymes of the eicosanoid pathway.

Specific immunotherapy with SQ standardized grass allergen tablets in asthmatics with rhinoconjunctivitis.

BACKGROUND: The best way to prevent allergy symptoms is to treat the allergic condition. Specific immunotherapy with grass allergen tablets 75,000 SQ-T (Grazax, Phleum pratense, ALK-Abelló) is safe and efficacious in rhinoconjunctivitis patients. As rhinoconjunctivitis often co-exists with asthma, we aimed to confirm safety and efficacy in grass allergic subjects with asthma and rhinoconjunctivitis. METHODS: A randomized, double-blind, placebo-controlled, multicentre trial was performed 10-14 weeks prior to and during the grass pollen season 2004. About 114 subjects were randomized 2 : 1 to grass allergen tablets or placebo. The primary end points were average asthma medication and symptom scores during the grass pollen season, and secondary variables were average rhinoconjunctivitis symptom and medication scores during the grass pollen season. Additionally, number of well days was defined post hoc. RESULTS: Differences in asthma medication and symptom scores between the treatment groups were negligible. The mean difference in asthma medication score was below 0.1 and 0.3 for asthma symptom score [a single inhalation of salbutamol (200 microg) was scored 2]. No serious adverse events were reported. A reduction in rhinoconjunctivitis symptom score of 37% (P = 0.004) and a 41% (P = 0.036) reduction in medication score was found in the grass pollen season for subjects treated with the grass allergen tablet compared with placebo. Well days increased by 54% (P = 0.002). CONCLUSIONS: Self-administration of the grass allergen tablet was safe. The treatment did not impair asthma control and confirmed considerable symptom prevention and reduced medication use. It addresses the allergic condition and represents a baseline treatment for grass pollen allergy.

Circulating eosinophils in asthma, allergic rhinitis, and atopic dermatitis lack morphological signs of degranulation.

BACKGROUND: In allergic diseases, eosinophils in affected tissues release granule proteins with cytotoxic, immunoregulatory, and remodelling-promoting properties. From recent observations, it may be assumed that eosinophils degranulate already in circulating blood. If degranulation occurs in the circulation, this could contribute to widespread systemic effects and provide an important marker of disease. OBJECTIVE: To determine the degranulation status of circulating eosinophils in common allergic diseases. METHODS: Using a novel approach of whole blood fixation and leucocyte preparation, the granule morphology of blood eosinophils from healthy subjects, non-symptomatic patients, symptomatic patients with asthma, asthma and Churg-Strauss syndrome, allergic rhinitis, and atopic dermatitis was evaluated by transmission electron microscopy (TEM) and eosinophil peroxidase (TEM) histochemistry. Plasma and serum levels of eosinophil cationic protein were measured by fluoroenzymeimmunoassay. Selected tissue biopsies were examined by TEM. RESULTS: Regardless of symptoms, circulating eosinophils from allergic patients showed the same granule morphology as cells from healthy subjects. The majority of eosinophil-specific granules had preserved intact electron-density (96%; range: 89-98%), while the remaining granules typically exhibited marginal coarsening or mild lucency of the matrix structure. Abnormalities of the crystalline granule core were rarely detected. Furthermore, granule matrix alterations were not associated with any re-localization of intracellular EPO or increase in plasma eosinophil cationic protein. By contrast, eosinophils in diseased tissues exhibited cytolysis (granule release through membrane rupture) and piecemeal degranulation (loss of granule matrix and core structures). CONCLUSION: In symptomatic eosinophilic diseases, circulating blood eosinophils retain their granule contents until they have reached their target organ.

Comparison of nasal immunohistology in patients with seasonal rhinoconjunctivitis treated with topical steroids or specific allergen immunotherapy.

BACKGROUND: Specific allergen immunotherapy (SIT) and nasal steroids (NS) are considered effective anti-inflammatory treatments for allergic rhinitis, although their mechanism of action differs. OBJECTIVE: The aim of this study was to examine the effect of treatment with NS and SIT on different populations of inflammatory cells in the nasal mucosa and to compare cell numbers before and during the birch pollen season in patients with seasonal allergic rhinitis. METHODS: In a randomized, double-blind, double dummy comparative study, 41 patients with seasonal rhinoconjunctivitis were treated with birch SIT or NS (budesonide 400 microg daily). Treatment with NS started before the birch pollen season and at the same time SIT-treated patients reached the maintenance dose. Nasal biopsies for immunohistochemistry were obtained before the season and start of the treatments and at the peak of the pollen season during treatment. RESULTS: Symptoms of rhinoconjunctivitis increased significantly in both groups during the pollen season but less in the NS-treated group and the difference between the treatment groups was significant at the end of the season (P = 0.03). Immunohistochemistry of nasal biopsies from NS-treated patients showed significantly fewer CD1a+, IgE+ and Fc epsilonRI+ cells during the season compared with preseason (P = 0.02, P = 0.001 and P = 0.0004, respectively) and with seasonal values of the SIT-treated group (P = 0.002, P = 0.002 and P = 0.0004 respectively). CONCLUSION: Treatment with NS but not SIT decreased the numbers of CD1a+, IgE+ and Fc epsilonRI+ cells during the birch pollen season. Our data indicate that treatment with NS has a broader anti-inflammatory range than SIT.

Defective suppression of Th2 cytokines by CD4CD25 regulatory T cells in birch allergics during birch pollen season.

BACKGROUND: CD4(+)CD25+ regulatory T cells suppress proliferation and cytokine production by human T cells both to self-antigens and exogenous antigens. Absence of these cells in human newborns leads to multiple autoimmune and inflammatory disorders together with elevated IgE levels. However, their role in human allergic disease is still unclear. OBJECTIVE: This study aimed to evaluate the capacity of CD4(+)CD25+ regulatory T cells to suppress proliferation and cytokine production outside and during birch-pollen season in birch-allergic patients relative to non-allergic controls. METHODS: CD4+ cells were obtained from blood of 13 birch-allergic patients and six non-allergic controls outside pollen season and from 10 birch-allergic patients and 10 non-allergic controls during birch-pollen season. CD25+ and CD25- fractions were purified with magnetic beads and cell fractions, alone or together in various ratios, were cultured with antigen-presenting cells and birch-pollen extract or anti-CD3 antibody. Proliferation and levels of IFN-gamma, IL-13, IL-5 and IL-10 were measured by thymidin incorporation and ELISA, respectively. Numbers of CD25+ cells were analysed by flow cytometry. RESULTS: CD4(+)CD25+ regulatory T cells from both allergics and non-allergics potently suppressed T cell proliferation to birch allergen both outside and during birch-pollen season. However, during season CD4(+)CD25+ regulatory T cells from allergic patients but not from non-allergic controls were defective in down-regulating birch pollen induced IL-13 and IL-5 production, while their capacity to suppress IFN-gamma production was retained. In contrast, outside pollen season the regulatory cells of both allergics and non-allergic controls were able to inhibit T-helper 2 cytokine production. CONCLUSION: This is the first study to show differential suppression of Th1 and Th2 cytokines, with CD4(+)CD25+ regulatory T cells from birch-pollen-allergic patients being unable to down-regulate Th2, but not Th1 responses during birch-pollen season.

Living & learning with allergy: a European perception study on respiratory allergic disorders.

BACKGROUND: Knowledge of allergy patients' perception of own disease is inadequate, and understanding of the impact of local environment, including family and health-care system, on patients' management of disease is insufficient. We examined the potential of telephone-based survey techniques for establishing this knowledge in 10 European countries. METHODS: A two-phased questionnaire developed by use of focus groups in seven countries was translated into 10 languages. To ensure that the true values of the populations were restored in randomly selected populations, 75,343 telephone numbers selected for screening represented balanced national distributions of households. RESULTS: Eight thousand two hundred and sixty-eight respiratory allergy sufferers were identified by the telephone screening process. 85.4% accepted to participate in the survey and 89.6% completed both phases comprising 34 questions and rating of 49 statements. Data for each country were weighted in terms of age, sex and the recorded allergy prevalence within age intervals. CONCLUSIONS: The telephone survey technique allowed for establishment of random representative samples, and application of mathematical weighting procedures assured that the true national values were restored in the data set. As all interviews were performed in a standardised manner we conclude that the telephone-based survey methodology enables national representative data set to be established and compared.

A double-blind, placebo-controlled birch allergy vaccination study: inhibition of CD23-mediated serum-immunoglobulin E-facilitated allergen presentation.

BACKGROUND: The clinical efficacy of specific allergy vaccination (SAV), previously called specific immunotherapy is well documented. The working mechanism of this treatment is not completely known at present. Allergen-specific CD4+ T lymphocytes are activated at extremely low allergen concentrations in vivo possibly as a result of serum IgE-facilitated allergen presentation (S-FAP). Previously, we have shown that this process can be inhibited by long-term birch SAV sera. METHODS: In the present study, we have analysed sera from birch-allergic patients in a randomized double-blind, placebo-controlled clinical trial for their ability to mediate S-FAP. Birch-specific IgE levels were not changed after SAV. Bet v 1-specific IgE levels, however, were significantly decreased (P<0.05) and Bet v 1-specific IgG4 levels were strongly increased after SAV (P<0.001). None of these changes were observed in the placebo group. When the sera were tested for their ability to induce S-FAP, a complete abrogation of this effect was noted in the sera from patients receiving active treatment (P<0.001), but not in the control group. This inhibition of S-FAP seemed to be associated with the reduction in the ratio between Bet v 1-specific IgE and IgG4 antibodies in serum, but a clear correlation could not be demonstrated. CONCLUSION: In conclusion, the present study clearly shows that SAV leads to an inhibition of the S-FAP needed to obtain optimal T cell activation at the low allergen concentrations present in vivo. This novel mechanism may explain the increased allergen threshold levels found in allergen provocation tests and the reduction of late-phase reactions observed after SAV.

Cytokine production in peripheral blood cells during and outside the pollen season in birch-allergic patients and non-allergic controls.

BACKGROUND: The naturally occurring pollen season permits observation of the kinetic changes in the process of allergic inflammation. We examined cytokine production in peripheral blood (PB) T cells and monocytes obtained from birch-allergic patients both during and outside the pollen season. METHODS: PB from 16 patients and six healthy controls was obtained during the alder pollen season, at the beginning and the peak of the birch pollen season and outside the pollen season. Mononuclear cells (MNC) were stimulated with allergen and polyclonal activators. For flow cytometric analysis, MNC were stained with monoclonal antibodies (MoAbs) against the cell surface markers CD3, CD8, CD14 and the intracellular cytokines IL-4, IL-5, IL-10, IL-12, IL-13, granulocyte macrophage-colony stimulating factor (GM-CSF) and IFN-gamma. RESULTS: In allergic patients, significant increases in clinical symptoms, use of medication, eosinophil numbers and birch-specific IgE were found during the pollen season. In vitro allergen stimulation increased the number of GM-CSF+ monocytes (P<0.01) and this increase was dependent on allergen exposure. The IL-4/IFN-gamma ratio rose (P<0.001) at the peak of birch pollen season and the ratio correlated with symptom scores during the birch season. In the CD4+ cell population, the numbers of GM-CSF+ cells were higher throughout the alder and birch seasons compared with outside the pollen season (P<0.05). No such changes were seen in the healthy controls. CONCLUSIONS: The main finding of our study was the increased percentage of GM-CSF+ monocytes in atopic subjects compared with healthy controls. In allergic patients, natural seasonal pollen exposure resulted in increased numbers of GM-CSF+ cells among both monocytes and CD4+ T cells. We have also shown that a seasonal change in Th2/Th1 cytokine ratio requires an adequate and prolonged allergen stimulation that is seen late in the pollen season.

Allergen specific immunotherapy attenuates early and late phase reactions in lower airways of birch pollen asthmatic patients: a double blind placebo-controlled study.

BACKGROUND: Few placebo-controlled studies have examined the effect of allergen specific immunotherapy (SIT) on early and late phase asthmatic reactions. In this placebo-controlled study we have investigated the effect of 1 year of SIT with standardized birch pollen extract on early and late phase asthmatic reactions in adult asthmatic patients. METHODS: Nineteen patients with a history of birch-pollen-induced seasonal symptoms from upper and lower airways, positive skin prick test and in vitro specific immunoglobulin E to birch pollen extract were included. Allergen and methacholine bronchial challenges were performed and blood samples obtained for analyses of total eosinophil count and eosinophil cationic protein (ECP) in serum, before and after 1 year of immunotherapy treatment. RESULTS: All patients developed early and 16 of 19 both early and late phase asthmatic reactions. A significant increase in allergen dose was required to evoke early asthmatic reaction in the immunotherapy group (P < 0.01) after 1 year of treatment. The difference between the groups was significant (P < 0.01). Also the size of late asthmatic reaction was significantly reduced in the SIT group compared with placebo treated patients (P < 0.01). Twenty-four hours after allergen challenge methacholine sensitivity, number of total eosinophils and ECP increased significantly in the placebo (P < 0.02, P < 0.05 and P < 0.05 respectively), but not in the SIT group. CONCLUSION: Allergen SIT with standardized birch pollen extract decreased early and late asthmatic responses following bronchial challenge in pollen allergic patients, thus confirming anti-inflammatory effect of the treatment.

The effect of specific immunotherapy on the expression of costimulatory molecules in late phase reaction of the skin in allergic patients.

BACKGROUND: Specific immunotherapy (SIT) modulates immune responses to allergens resulting in improvement of allergic symptoms. However, the mechanisms behind the clinical changes are not clear. Participation of costimulatory molecules on antigen-presenting cells and T cells in the process of antigen recognition is suggested to be of essential importance. The SIT effect on expression of costimulatory molecules has not been earlier examined. METHODS: Forty-one birch-allergic patients were treated with SIT or placebo. After 1 year of treatment skin biopsies were obtained 24 h following allergen challenge. Sections were stained with antibodies against: EG2 (eosinophils), CD4 (T cells), CD68 (macrophages), CD1a (Langerhans cells), CD28 (on T cells) and costimulatory molecules (CD80, CD86). RESULTS: Following allergen challenge number of the CD4(+) and CD68(+) cells increased significantly (P=0.002, 0.0001, respectively) in the placebo, but not in the SIT-treated patients. The difference between groups was significant (P=0.003, 0.01, respectively). The numbers of EG2(+) cells increased significantly in both groups. CD80(+) cell numbers increased in the placebo (P=0.01) but not in the SIT group. The number of CD86(+) cells increased in both groups (placebo, P=0.001; SIT, P=0.01) but significantly less in the SIT group (P=0.05). The numbers of CD28(+) cells increased in the placebo (P=0.001) but remained unchanged in the SIT group. The difference between the groups was significant (P=0.05). CONCLUSION: There were lower numbers of cells expressing costimulatory molecules in SIT-treated than in placebo-treated patients. Decreased costimulation may lead to diminished immune response following allergen exposure. This could be an important factor contributing to the clinical improvement after SIT.

Chromosomal assignment of 20 candidate genes for canine congenital sensorineural deafness by FISH and RH mapping.

The analysis of inherited diseases in the domestic dog (Canis familiaris) provides a resource for the continued use of this species as a model system for human diseases. Many different dog breeds are affected by congenital sensorineural deafness. Since mutations in various genes have already been found causative for sensorineural hearing impairment in humans or mice, 20 of these genes were considered as candidates for deafness in dogs. For each of the candidate genes a canine BAC clone was isolated by screening with heterologous human or murine cDNA probes. The gene-containing BAC clones were physically assigned to the canine genome by FISH and the BAC-derived STS-markers were positioned with the RHDF5000 panel on the canine RH map. The mapping data, which confirm the established conservation of synteny between canine and human chromosomes, provide a resource for further association studies in segregating canine populations and the basis for new insights into this common canine and human disease.