World Allergy Organization Journal / Clinical efficacy of sublingual immunotherapy is associated with restoration of steady-state serum lipocalin 2 after SLIT : a pilot study

Background: So far, only a few biomarkers in allergen immunotherapy exist that are associated with a clinical benefit. We thus investigated in a pilot study whether innate molecules such as the molecule lipocalin-2 (LCN2), with implications in immune tolerance demonstrated in other fields, may discriminate A) between allergic and non-allergic individuals, and B) between patients clinically responding or non-responding to sublingual allergen immunotherapy (SLIT) with house dust mite (HDM) extract. Moreover, we assessed haematological changes potentially correlating with allergic symptoms. Methods: LCN2-concentrations were assessed in sera of healthy and allergic subjects (n = 126) as well as of house dust mite (HDM) allergics before and during HDM- sublingual immunotherapy (SLIT) in a randomized, double-blind, placebo-controlled trial for 24 weeks. Sera pre-SLIT (week 0), post-SLIT (week 24) and 9 months after SLIT were assessed for LCN2 levels and correlated with total nasal symptom scores (TNSS) obtained during chamber challenge at week 24 in patients receiving HDM- (n = 31) or placebo-SLIT (n = 10). Results: Allergic individuals had significantly (p < 0.0001) lower LCN2-levels than healthy controls. HDM-allergic patients who received HDM-SLIT showed a significant increase in LCN2 9 months after termination of HDM-SLIT (p < 0.001), whereas in subjects receiving placebo no increase in LCN2 was observed. Among blood parameters a lower absolute rise in the lymphocyte population (p < 0.05) negatively correlated with symptom improvement (Pearson r 0.3395), and a lower relative increase in the neutrophils were associated with improvement in TNSS (p < 0.05). LCN2 levels 9 months after immunotherapy showed a low positive correlation with the relative improvement of symptoms (Pearson r 0.3293). LCN2-levels 9 months off-SLIT were significantly higher in patients whose symptoms improved during chamber challenge than in those whose symptoms aggravated (p < 0.01). Conclusion: Serum LCN2 concentrations 9 months off-SLIT correlated with clinical reactivity in allergic patients. An increase in the LCN2 levels 9 months after HDM-SLIT was associated with a clinical benefit. Serum LCN2 may thus contribute to assess clinical reactivity in allergic patients. Trial registration: Part of the data were generated from clinicaltrials.gov Identifier NCT01644617.(VLID)468365

Scientific Reports / Sublingual house dust mite immunotherapy has no impact on decrease of circulating erythrocytes upon airway allergen challenge in allergic rhinitis

House dust mite (HDM) allergy is a predominant cause for perennial allergic rhinitis (AR) in Europe. We recently reported that circulating erythrocyte numbers decrease after airway allergen challenge in a murine asthma model and in grass-pollen sensitized AR subjects. Consequently, we aimed to evaluate these findings in HDM sensitized AR subjects and the influence of preceding allergen immunotherapy. Seventy-seven (age 26.87.3 years; 54.5% female) HDM-allergic rhinitis subjects previously enrolled in a randomized, monocentric sublingual immunotherapy (SLIT) trial at the Vienna Challenge Chamber (VCC) were included. Subjects had either received placebo (n=22), low-dose HDM (n=29) or high-dose HDM specific sublingual immunotherapy (n=26) daily for 24 weeks. Blood sampling was performed before and after 6hours of HDM allergen exposure. Overall, specific airway allergen challenge resulted in a significant decrease in circulating erythrocytes and hematocrit (p<0.001), and elevation of leukocytes (p<0.001), particularly segmented neutrophils (p<0.001). Gender had no significant effect on the observed changes in circulating blood cells. Erythrocytes decreased and neutrophil counts increased significantly after airway allergen challenge regardless of preceding immunotherapy. These findings imply a rapid systemic mobilization of neutrophils occurring within immediate type hypersensitivity response upon a specific allergen challenge, which is possibly inversely linked with the erythrocyte numbers.(VLID)460635

Mechanisms, safety and efficacy of a B cell epitope-based vaccine for immunotherapy of grass pollen allergy

Background: We have developed a recombinant B cell epitope-based vaccine (BM32) for allergen-specific immunotherapy (AIT) of grass pollen allergy. The vaccine contains recombinant fusion proteins consisting of allergen-derived peptides and the hepatitis B surface protein domain preS as immunological carrier. Methods: We conducted a randomized, double-blind, placebo-controlled AIT study to determine safety, clinical efficacy and immunological mechanism of three subcutaneous injections of three BM32 doses adsorbed to aluminum hydroxide versus aluminum hydroxide (placebo) applied monthly to grass pollen allergic patients (n = 70). Primary efficacy endpoint was the difference in total nasal symptom score (TNSS) through grass pollen chamber exposure before treatment and 4 weeks after the last injection. Secondary clinical endpoints were total ocular symptom score (TOSS) and allergen-specific skin response evaluated by titrated skin prick testing (SPT) at the same time points. Treatment-related side effects were evaluated as safety endpoints. Changes in allergen-specific antibody, cellular and cytokine responses were measured in patients before and after treatment. Results: Sixty-eight patients completed the trial. TNSS significantly decreased with mean changes of −1.41 (BM32/20 μg) (P = 0.03) and −1.34 (BM32/40 μg) (P = 0.003) whereas mean changes in the BM32/10 μg and placebo group were not significant. TOSS and SPT reactions showed a dose-dependent decrease. No systemic immediate type side effects were observed. Only few grade 1 systemic late phase reactions occurred in BM32 treated patients. The number of local injection site reactions was similar in actively and placebo-treated patients. BM32 induced highly significant allergen-specific IgG responses (P < 0.0001) but no allergen-specific IgE. Allergen-induced basophil activation was reduced in BM32 treated patients and addition of therapy-induced IgG significantly suppressed T cell activation (P = 0.0063). Conclusion: The B cell epitope-based recombinant grass pollen allergy vaccine BM32 is well tolerated and few doses are sufficient to suppress immediate allergic reactions as well as allergen-specific T cell responses via a selective induction of allergen-specific IgG antibodies. (ClinicalTrials.gov number, NCT01445002.

Molecular, structural and immunological characterization of Der p 18, a chitinase-like house dust mite allergen

The house dust mite (HDM) allergen Der p 18 belongs to the glycoside hydrolase family 18 chitinases. The relevance of Der p 18 for house dust mite allergic patients has only been partly investigated.To perform a detailed characterization of Der p 18 on a molecular, structural and immunological level.Der p 18 was expressed in E. coli, purified to homogeneity, tested for chitin-binding activity and its secondary structure was analyzed by circular dichroism. Der p 18-specific IgG antibodies were produced in rabbits to localize the allergen in mites using immunogold electron microscopy and to search for cross-reactive allergens in other allergen sources (i.e. mites, crustacea, mollusca and insects). IgE reactivity of rDer p 18 was tested with sera from clinically well characterized HDM-allergic patients (n = 98) and its allergenic activity was analyzed in basophil activation experiments.Recombinant Der p 18 was expressed and purified as a folded, biologically active protein. It shows weak chitin-binding activity and partial cross-reactivity with Der f 18 from D. farinae but not with proteins from the other tested allergen sources. The allergen was mainly localized in the peritrophic matrix of the HDM gut and to a lower extent in fecal pellets. Der p 18 reacted with IgE from 10% of mite allergic patients from Austria and showed allergenic activity when tested for basophil activation in Der p 18-sensitized patients.Der p 18 is a rather genus-specific minor allergen with weak chitin-binding activity but exhibits allergenic activity and therefore should be included in diagnostic test panels for HDM allergy

Peripheral Erythrocytes Decrease upon Specific Respiratory Challenge with Grass Pollen Allergen in Sensitized Mice and in Human Subjects

<div><p>Background and Aims</p><p>Specific hyper-responsiveness towards an allergen and non-specific airway hyperreactivity both impair quality of life in patients with respiratory allergic diseases. We aimed to investigate cellular responses following specific and non-specific airway challenges locally and systemically in i) sensitized BALB/c mice challenged with grass pollen allergen Phl p 5, and in ii) grass pollen sensitized allergic rhinitis subjects undergoing specific airway challenge in the Vienna Challenge Chamber (VCC).</p><p>Methods and Results</p><p>BALB/c mice (n = 20) were intraperitoneally immunized with grass pollen allergen Phl p 5 and afterwards aerosol challenged with either the specific allergen Phl p 5 (n = 10) or the non-specific antigen ovalbumin (OVA) (n = 10). A protocol for inducing allergic asthma as well as allergic rhinitis, according to the united airway concept, was used. Both groups of exposed mice showed significantly reduced physical activity after airway challenge. Specific airway challenge further resulted in goblet cell hyperplasia, enhanced mucous secretion, intrapulmonary leukocyte infiltration and lymphoid follicle formation, associated with significant expression of IL-4, IL-5 and IL-13 in splenocytes and also partially in lung tissue. Concerning circulating blood cell dynamics, we observed a significant drop of erythrocyte counts, hemoglobin and hematocrit levels in both mouse groups, challenged with allergen or OVA. A significant decrease in circulating erythrocytes and hematocrit levels after airway challenges with grass pollen allergen was also found in grass pollen sensitized human rhinitis subjects (n = 42) at the VCC. The effects on peripheral leukocyte counts in mice and humans however were opposed, possibly due to the different primary inflammation sites.</p><p>Conclusion</p><p>Our data revealed that, besides significant leukocyte dynamics, particularly erythrocytes are involved in acute hypersensitivity reactions to respiratory allergens. A rapid recruitment of erythrocytes to the lungs to compensate for hypoxia is a possible explanation for these findings.</p></div

Specific and nonspecific airway provocation of allergic mice: impact on blood cells.

<p>(<b>A</b>) RBC, (<b>B</b>) HGB and (<b>C</b>) HCT levels of naïve, non-sensitized versus sensitized and Phl p 5 nebulized (red squares) and OVA challenged mice (black points) after airway challenge. Data are mean ± SD and n = 5 mice/group. *, p<0.05, **, p<0.01 and n.s. means not significant. (<b>D</b>) White blood cell counts (WBC) from naïve versus sensitized and Phl p 5 challenged (red squares) or OVA aerosolized mice (black points).</p

Changes in number of peripheral blood cells before and after airway challenge with grass-pollen mix in subjects with allergic rhinitis, after 4<b> </b>h (<i>group 1</i>) or 2<b> </b>h (<i>group 2</i>) of allergen exposure; follow-up data shows the respective blood cell counts 6–21 days after airway allergen challenge.

<p>Data are median (minimum, maximum); *p<0.05, **p<0.01, ***p<0.001 compared to baseline values;</p>a<p>Significantly more intra-individual decreases than increases from baseline.</p

Demographic and clinical characteristics of study subjects.

<p>Data are mean±standard deviation, SPT = Skin-Prick-Test, RAST = Radioallergosorbent Test, FEV1 = forced expiratory volume in 1 second.</p