A novel glycocluster molecule prevents timothy-induced allergic airway inflammation in mice.

BACKGROUND: Allergen-specific immunotherapy (SIT) effectively alleviates type I allergic diseases characterized by T helper (Th)2-type immunity. Our recent studies have shown that a synthetic trivalent glycocluster, triacedimannose (TADM), suppresses the Th2-type allergic inflammation. The aim of this study was to compare TADM with two well-known adjuvants, unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG) and monophosphoryl lipid A (MPLA) in a grass allergen-induced chronic allergic inflammation model in mice. METHODS: Female BALB/c mice were intranasally sensitized with 50 µL of timothy grass pollen extract (TE) twice a week for a period of 15 weeks. Therapeutic intranasal treatments were then performed once a week after the tenth intranasal TE instillation using TADM (10 or 25 µg/50 µL), CpG-ODN (20 µg/50 µL) or MPLA (2 µg/50 µL). Groups of 9-10 animals per treatment were killed 24 hours after the last timothy dosage. Blood, bronchoalveolar lavage (BAL) fluids and lung biopsies were taken for subsequent analysis. RESULTS: When mice were repeatedly exposed to TE for 15 weeks, the number of eosinophils and lymphocytes increased in the BAL fluids. The eosinophil and lymphocyte counts decreased dose-dependently and were practically abolished in the mice treated with TADM. Treatments with MPLA or CpG significantly increased the numbers of neutrophils, while CpG nonsignificantly decreased eosinophilia compared to timothy exposure. CONCLUSIONS: A novel synthetic glycocluster molecule inhibited the development of grass-induced eosinophilic pulmonary inflammation in mice when administrated in the airways. This compound could be a candidate to be used either as an adjuvant in SIT or as a topical anti-inflammatory treatment.

Blackcurrant seed oil for prevention of atopic dermatitis in newborns: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: The present increased incidence of atopic diseases has been associated with an altered intake of essential fatty acids (EFAs). The composition of blackcurrant seed oil (BCSO) corresponds to the recommended dietary intake of EFAs, and as a dietary supplement could, in small doses, modify the imbalance of EFAs in an efficient way. OBJECTIVE: To assess the effect of dietary supplementation with BCSO on the prevalence of atopy at 12 months of age. METHODS: Three hundred and thirteen pregnant mothers were randomly assigned to receive BCSO (151) or olive oil as placebo (162). The first doses were administered at 8th-16th weeks of pregnancy and were continued until the cessation of breastfeeding, followed by supplementation to the infants until the age of 2 years. Atopic dermatitis and its severity (SCORAD index) were evaluated, serum total IgE was measured and skin tests were performed at the age of 3, 12 and 24 months. RESULTS: Parental atopy was common (81.7%) among study subjects, making them infants with increased atopy risk. There was a significantly lower prevalence of atopic dermatitis in the BCSO group than in the olive oil group at the age of 12 months (33.0% vs. 47.3%, P=0.035). SCORAD was also lower in the BCSO group than in the olive oil group at 12 months of age (P=0.035). No significant differences in the prevalence of atopic dermatitis were observed between the groups at the age of 24 months (P=0.18). CONCLUSION: Dietary supplementation with BCSO was well tolerated and it transiently reduced the prevalence of atopic dermatitis. It could therefore be one potential tool in the prevention of atopic symptoms when used at an early stage of life. (Registration number SRCTN14869647, http://www.controlled-trials.com)

Allergen-induced in vitro expression of IL-18, SLAM and GATA-3 mRNA in PBMC during sublingual immunotherapy.

BACKGROUND: Signalling lymphocytic activation molecule (SLAM) and interleukin (IL)-18 induce interferon (IFN)-gamma production from Th1 cells. The allergen-induced SLAM and IL-18 mRNA expressions are increased during subcutaneous immunotherapy (SCIT), but nothing is known about their role during sublingual immunotherapy (SLIT). Transcription factor GATA-3 is associated with Th2 cells but its role in SCIT and SLIT is yet unexplored. This study was undertaken to analyse the allergen induced in vitro mRNA expression of IL-18, SLAM and GATA-3 in peripheral blood mononuclear cells (PBMC) of children with allergic rhinitis (AR) during SLIT. METHODS: Ten patients with AR undergoing pollen SLIT with a weekly dose of 200,000 SQ-U, 10 with 24,000 SQ-U of mixture of Betula verrucosa, Corylus avellana and Alnus glutinosa and 10 with placebo were included. Peripheral blood mononuclear cell were stimulated with birch extract prior to, after 1 and 2 years of the treatment. The mRNA expression was assessed using kinetic real-time RT-PCR (TaqMan); Applied Biosystems, Foster City, CA, USA). RESULTS: The expression of IL-18 mRNA was increased in the high-dose group in comparison to the placebo group after 1 year of therapy (P = 0.028) and had an inverse correlation with the late phase skin reaction after the second study year (r = -0.41, P = 0.041). SLAM mRNA expression increased in the high-dose group from baseline to 1 year (P = 0.028) and correlated with IL-10 (r = 0.96, P < 0.0001) and transforming growth factor-beta (r = 0.80, P = 0.0037) mRNA expression. No significant changes were seen in GATA-3 mRNA expression. CONCLUSIONS: During SLIT, IL-18 and SLAM are upregulated, suggesting that the Th2 type inflammatory response is downregulated during SLIT by increased Th1 type response.

Clinical efficacy and safety of sublingual immunotherapy with tree pollen extract in children.

BACKGROUND: Subcutaneous immunotherapy has been the principal approach of immunotherapy in the treatment of allergic diseases. Several clinical studies with birch, alder or hazel pollen extract conducted as subcutaneous immunotherapy have been published suggesting a well-tolerated and clinically effective treatment. Only a few clinical studies of sublingual immunotherapy (SLIT) with these allergens have been published. This study investigated the clinical efficacy, safety and dose-response relationship of SLIT in children suffering from rhinoconjunctivitis with/without asthma. METHODS: Eighty-eight children (5-15 years) with a history of tree pollen-induced allergic rhinoconjunctivitis with/without seasonal asthma for >or=2 years were included. Allergy to tree pollen was confirmed by positive skin-prick test, positive specific IgE and positive conjunctival provocation test. The extract used was a glycerinated mixture of Betula verrucosa, Corylus avellana and Alnus glutinosa 100,000 SQ-U/ml. Children were randomized into three groups receiving SLIT 5 days a week for up to 18 months; dose group 1: accumulated weekly dose of 24,000 SQ-U; dose group 2: accumulated weekly dose of 200,000 SQ-U; and placebo. RESULTS: In the birch pollen season, dose group 2 showed a significant reduction of symptom (P = 0.01) and medication scores (P = 0.04) compared with placebo. Dose group 1 showed a significant reduction of symptom scores (P = 0.03). There were no statistical differences between dose groups 1 and 2. All children tolerated the treatment well. CONCLUSION: SLIT with tree pollen extract provided dose-dependent benefits in tree pollen-allergic children in terms of significantly reduced symptoms and medication use. The treatment was well tolerated.

Sublingual immunotherapy in children modulates allergen-induced in vitro expression of cytokine mRNA in PBMC.

BACKGROUND: During subcutaneous immunotherapy (SCIT), there is a local mucosal shift from Th2 to Th1 type cytokine predominance and downregulation of interleukin (IL)-5 and eosinophilia. According to recent studies IL-10- and transforming growth factor (TGF)-beta-induced tolerance is another key phenomenon in SCIT. Few data to date is available on mechanisms and roles of these cytokines in sublingual immunotherapy (SLIT). SCOPE: This study was undertaken to analyse the allergen-induced in vitro mRNA expression of IL-4, IL-5, IL-10, TGF-beta and interferon (IFN)-gamma during SLIT in peripheral blood mononuclear cells (PBMC) of children with allergic rhinitis (AR). METHODS: Ten patients with AR undergoing pollen SLIT with a weekly dose of 200,000 SQ-U, 10 with a weekly dose of 24,000 SQ-U of glycerinated mixture of Betula verrucosa, Corylus avellana and Alnus glutinosa and 10 with placebo were included in the study. Peripheral blood mononuclear cell samples were collected and stimulated with pollen allergen extract prior to the treatment, after 1 and 2 years of the treatment. The cytokine mRNA expression was assessed using kinetic real time reverse transcription polymerase chain reaction (RT-PCR; TaqMan). RESULTS: The in vitro allergen-induced mRNA expression of IL-5 by PBMC in the placebo group at 1 (P = 0.0065) and 2 (P = 0.013) years of therapy were increased in comparison with the highest dose. The expression of IL-10 mRNA was increased in the highest dose group (P = 0.0016) and the lower dose group (P = 0.034) at 2 years of therapy when compared with placebo. The change in the expression of allergen-induced TGF-beta had an inversed correlation with the change of IL-5 (r = -0.38, P = 0.036) and positive correlation with the change of IL-10 (r = 0.58, P = 0.0019). CONCLUSIONS: Sublingual immunotherapy induced a dose-dependent systemic allergen-specific immunological response in children with AR. During high-dose SLIT, there was activation of regulatory cytokine IL-10 and an inhibitory effect on IL-5 expression increase that was associated with TGF-beta.

Increased expression of allergen-induced in vitro interleukin-10 and interleukin-18 mRNA in peripheral blood mononuclear cells of allergic rhinitis patients after specific immunotherapy.

BACKGROUND: During specific pollen immunotherapy (SIT) there is a local mucosal shift from Th2- to Th1- type cytokine predominance, with IL-12 having a major role in this shift. IL-10-induced tolerance is supposed to be a key phenomenon in venom immunotherapy (VIT). However, the role of Th1-promoting cytokines, on the one hand, and the role of regulatory cytokines, on the other hand, have not been studied in parallel during SIT. OBJECTIVE: This study was undertaken to analyse the allergen-induced in vitro mRNA expression of Th1-type effector cytokine IL-18 and regulatory cytokines IL-10 and TGF-beta during SIT in peripheral blood mononuclear cells (PBMC) of allergic rhinitis (AR) patients. METHODS: Thirty patients with AR undergoing pollen SIT and 10 patients with AR who were not treated with SIT were included in the study. The symptoms and medications were registered post-seasonally before the beginning of SIT and after 1 year of therapy. PBMC samples were collected and stimulated with pollen allergen extract prior to the treatment, at the maintenance phase in 12 patients and after 1 year of the treatment. The cytokine mRNA expression was assessed using kinetic real-time RT-PCR (TaqMan). RESULTS: There was a clear increase in the treated AR patients, in comparison with untreated AR patients, in the expression of both IL-10 (mean change from baseline (SEM): 3.1 (0.8) vs. -0.3 (0.3), P<0.002, Mann-Whitney U-test) and IL-18 (2.7 (0.9) vs. -0.2 (0.6), P<0.03) mRNA after 1 year. The clearest increase in IL-10 mRNA expression was seen in patients who did not benefit at all (6.0 (2.3), P<0.001 vs. untreated) and the least increase in patients that had the greatest reduction of symptoms (0.8 (0.6), n.s. vs. untreated) at 1 year. The clearest increase in IL-18 mRNA expression was seen in patients with moderate outcome (3.4 (1.6), P<0.04 vs. untreated). In intermediate samples, taken when the maintenance dose was reached, the peak expression of allergen-induced IL-10 mRNA was associated with the most favourable outcome of SIT (P=0.01, Fisher exact test). A similar trend was seen in IL-18 mRNA expression. CONCLUSIONS: The results suggest that an early and transient increase in allergen-specific IL-10 and IL-18 mRNA expression in PBMC is essential for the therapeutic outcome after 1 year of SIT.

In vitro kinetics of allergen- and microbe-induced IL-4 and IFN-gamma mRNA expression in PBMC of pollen-allergic patients.

BACKGROUND: According to a hypothesis allergens induce Th2 responses in allergic patients, and microbes induce Th1 responses. We studied the kinetics of in vitro allergen-, tuberculin (PPD)- and tetanus toxin (TT)-induced IFN-gamma and IL-4 mRNA expression in peripheral blood mononuclear cell (PBMC) cultures of pollen-allergic patients and healthy controls. METHODS: PBMC of 10 birch or timothy pollen-allergic patients and of 13 healthy controls were stimulated in vitro with allergen (birch or timothy), PPD or TT. Pellets and supernatants were collected at 24, 48, 72 and 96 h after stimulation. IFN-gamma and IL-4 production was measured by enzyme linked immunosorbent assay and mRNA expression using RT-PCR and time-resolved fluorometry. RESULTS: Allergen induced IFN-gamma production and mRNA expression in PBMC more in allergic patients than in healthy controls. Also allergen induced IL-4 mRNA expression more in allergic patients than in healthy controls. PPD induced IFN-gamma mRNA expression both in allergic patients and healthy controls, whereas IFN-gamma production was induced only in healthy controls and IL-4 was not induced at all. TT induced IFN-gamma mRNA expression in both groups, IFN-gamma production in allergic patients, and IL-4 mRNA expression in both allergic patients and healthy controls. CONCLUSIONS: In vitro stimulation with allergen induced both IFN-gamma and IL-4 mRNA expression of PBMC in allergic patients. These observations challenge the clearcut division of microbe-specific Th1 and allergen-specific Th2 responses in peripheral blood.

Electromyographic and kinematic analysis of therapeutic knee exercises under water.

OBJECTIVE: This study aimed to evaluate muscle function and kinematics during commonly used knee rehabilitation exercises performed in water. DESIGN: Maximal effort single extension and flexion trials in still water and repeated extension-flexion trials in flowing water in barefoot condition were analysed from 18 healthy participants (8 men, 10 women). BACKGROUND: Despite the fact that water exercises are widely used, there are only few studies involving biomechanical and hydrodynamical analysis of aquatic exercises in rehabilitation. METHODS: Electromyography of the quadriceps (vastus medialis, vastus lateralis) and hamstring muscles (biceps femoris, semitendinosus) and angular velocities of the movements were recorded under water. RESULTS: In the repeated extension-flexion exercises the early reduction of agonist activity occurred concurrently with a high level of activity of the antagonists. In the single trial exercises the level of antagonistic activity was low throughout the range of motion, whereas the level of agonist activity was higher during the final phase of the range of motion as compared with the repeated exercises. Angular velocity patterns and values were similar between the two types of exercises. CONCLUSIONS: The present data demonstrated that the flowing properties of water modified the neuromuscular function of the quadriceps and hamstring muscles acting as agonists and antagonists in the knee flexion-extension exercises.

Human isometric force production and electromyogram activity of knee extensor muscles in water and on dry land.

This study was designed to determine trial-to-trial and day-to-day reproducibility of isometric force and electromyogram activity (EMG) of the knee extensor muscles in water and on dry land as well as to make comparisons between the two training conditions in muscle activity and force production. A group of 20 healthy subjects (12 women and 8 men) were tested three times over 2 weeks. A measurement session consisted of recordings of maximal and submaximal isometric knee extension force with simultaneous recording of surface EMG from the vastus medialis, vastus lateralis and biceps femoris muscles. To ensure identical measurement conditions the same patient elevator chair was used in both the dry and the wet environment. Intraclass correlation coefficients (ICC) and coefficients of variation (CV) showed high trial-to-trial (ICC = 0.95-0.99, CV = 3.5%-11%) and day-to-day reproducibility (ICC=0.85-0.98, CV=11%-19%) for underwater and dry land measurements of force and EMG in each muscle during maximal contractions. The day-to-day reproducibility for submaximal contractions was similar. The interesting finding was that underwater EMG amplitude decreased significantly in each muscle during maximal (P < 0.01-P < 0.001) and submaximal contractions (P < 0.05-P < 0.001). However, the isometric force measurements showed similar values in both wet and dry conditions. The water had no disturbing effect on the electrodes as shown by slightly lowered interelectrode resistance values, the absence of artefacts and low noise levels of the EMG signals. It was concluded that underwater force and EMG measurements are highly reproducible. The significant decrease of underwater EMG could have electromechanical and/or neurophysiological explanations.

Genetic and environmental factors affecting the allergenicity of birch (Betula pubescens ssp. czerepanovii [Orl.] Hämet-ahti) pollen.

BACKGROUND: Environmental variation, such as an increase of mean temperature due to the greenhouse effect, as well as the genetic factors may affect the allergenicity of pollen and thus, the prevalence of allergies. The connection between these factors and the allergen content of pollen is poorly understood. OBJECTIVES: To evaluate the role of environmental and genetic factors on the allergenicity of birch pollen. METHODS: Mountain birch (Betula pubescens ssp. czerepanovii (Orl.) Hämet-Ahti) pollen was studied using SDS-PAGE and IgE-immunoblotting. Pollen samples were collected from the trees of 10 half-sib families. The study trees from each family were reared in two tree line gardens where the daily mean temperatures were different during the growing season. RESULTS: The quantitative analysis of band intensities suggested that the responses of the major birch pollen allergen, Bet v 1, were stronger in the samples collected from the garden with higher daily mean temperature. Half-sib families and individual trees differed in their Bet v 1 content. CONCLUSIONS: Our results show that both genetic and environmental factors have an effect on the amount of Bet v 1. This suggests that breeding for trees low in allergen content may be possible.

Effects of air pollution and other environmental factors on birch pollen allergens.

To determine the effects of anthropogenic pollution on water-soluble proteins and specifically allergens in birch (Betula pendula and B. pubescens) pollen, we analyzed extracts of pollen from the pollution gradient around a factory complex (emitting sulfur oxides and heavy metals) by sodium dodecyl sulfate (SDS)--polyacrylamide gel electrophoresis (PAGE) and IgE immunoblotting. In addition, tree density-associated shading of the tree habitat, and quantity and quality of proteins and allergens in pollen of the two birch species were studied. The two studied birch species gave identical allergen profiles even though their protein profiles differed. Distance from the factory did not affect the amount of birch pollen major allergen, Bet v 1 (17 kDa), or of two other strong allergens (23 and 36 kDa). Trees growing in shaded places had significantly stronger responses to Bet v 1 and to the 23-kDa allergen than trees growing in open or half-open environments. Thus, we propose that combined heavy metal and sulfur dioxide pollution does not have an important effect on birch pollen allergens. Instead, other factors, e.g., shading and soil properties of the tree habitat, as well as the genetic background of the tree, may have a stronger influence on the quantity and relative composition of allergens.

Candida albicans and atopic dermatitis.

The role of sensitization and exposure to Candida albicans in atopic dermatitis (AD) was studied with skin-prick tests, yeast cultures and immunoblotting in 156 young adults with AD attending the Department of Dermatology, University of Turku, during 1983-89. Eighteen patients with allergic rhinitis without eczema and 39 non-atopics were included as controls. Parameters associated with severe AD were simultaneous anti-C. albicans IgE and saprophytic C. albicans growth. A statistically significant correlation between C. albicans sensitization (specific IgE antibodies) and AD symptoms was observed only in patients with saprophytic C. albicans exposure. No correlation between C. albicans-specific IgE and AD severity was shown in patients without gastrointestinal growth. Furthermore, severe eczema was seldom seen in patients without saprophytic C. albicans growth. The most important IgE-binding components of C. albicans in immunoblotting were 27 and 46 kD proteins and mannan, a polysaccharide. IgG and IgA antibodies to C. albicans, mainly towards C. albicans mannan, were found in practically all 70 sera studied. These results suggest a continuous exposure and induction of IgE antibodies by C. albicans in AD patients. Severe phases of AD in colonized patients are associated with IgE synthesis against C. albicans. These findings suggest a role for C. albicans in the exacerbations of AD but the clarification of this subject needs double-blind placebo-controlled treatment trials.

Stability of Candida albicans allergens during storage.

Stability of Candida albicans allergens was studied under various storage conditions. Lyophilized extract was reconstituted with human serum albumin (NSA) diluent, glycerol-free and in the presence of 10% or 50% glycerol and stored at various temperatures for different time periods. All extracts were tested at the same time with immunoblotting using C. albicans allergic patient sera and galactosidase-labelled anti-IgE. The highest number of detected allergens in the immunoblotting pattern was found in the presence of 50% glycerol at +6 degrees C. The most important allergen of C. albicans, the 46 kD protein allergen was stable up to 10 weeks at +6 degrees C in the presence of 50% glycerol but thereafter began to lose its IgE-binding capacity. After 30 weeks more than 50% of the IgE binding had disappeared. The 27 kD protein, another important allergen, was also labile but retained the allergenicity better than the 46 kD one. The 29 kD protein allergen was stable at all storage conditions, except +37 degrees C tested even after one year. More than 6 months storage at +6 degrees C or higher temperature is, however, unacceptable even in the presence of the 50% glycerol. These findings have particular importance in the diagnosis and treatment of allergic diseases.

Acid and alkaline proteolytic activities of cast-immobilized rat hind-limb muscles after electric stimulation.

The activities of cathepsin D (CD), beta-glucuronidase (beta-GU), alkaline protease (AKP), and the rate of acid autolysis (AA) were measured in gastrocnemius, soleus, and tibialis anterior muscles of the rat after bilateral cast immobilization in the middle position for one and three weeks. The effects of daily indirect electric stimulation on the proteolytic activities of immobilized muscles were also studied. In gastrocnemius and tibialis anterior, electric stimulation partially prevented the loss of muscle weight after immobilization for one week. Activities of CD and beta-GU were highest in the soleus, but the activities did not change therein after immobilization or electric stimulation. In gastrocnemius, the activities of CD and AKP and the AA rate increased significantly after immobilization. Electric stimulation during immobilization seemed to prevent the increase of CD and AKP activities significantly after one (CD) and three (AKP) weeks immobilization. In tibialis anterior, the activities of CD and AKP and the AA rate increased significantly in the stimulated muscles after immobilization for three weeks. Both in the gastrocnemius and tibialis anterior the beta-GU activity increased significantly in the stimulated muscles. The results suggest that during the disuse atrophy process and electric stimulation of immobilized muscles, rat hind-limb muscles differ in acid and alkaline proteolytic responses.