Diurnal pattern of Poaceae and Betula pollen flight in Central Europe.

In Central Europe the most common allergies are provoked by grass or birch pollen allergens. We determined the intra-daily behavior of airborne pollen grains of grasses (Poaceae) and birch (Betula ssp.) in Central Europe, based on data obtained from a network of automatic pollen monitors over Europe (www.pollenscience.eu). Our aim was to determine the time of day when the lowest concentrations occur, to provide allergic individuals the optimal time to ventilate their homes. The study was carried out in three Central European capitals, Berlin (Germany), Paris-Saclay (France), and Luxembourg (Luxembourg), as well as in eight stations in Germany (Altötting, Feucht, Garmisch-Partenkirchen, Hof, Marktheidenfeld, Mindelheim, Munich and Viechtach). The diurnal rhythm of these eleven locations was analyzed for either the complete, first week, peak week, peak day and last week of the pollen season. The data studied were reported as pollen/m3 measured in 3 h periods. Stations were classified as city, semi-populated or countryside areas using land-use and population density criteria. Grass pollen has a more pronounced diurnal rhythm than birch pollen concentrations. A significant difference was observed when comparing day (6-21 h) versus night (21-6 h) for all stations. No difference was detected between city and countryside for both pollen types, although for Poaceae a longer period of maximum concentrations was observed in big cities and higher day/night-time differences were registered in the countryside (6.4) than in cities (3.0). The highest pollen concentrations were observed between 9 and 18 h for grass, but the rhythm was less pronounced for birch pollen. For allergic individuals who want to bring in fresh air in their homes, we recommend opening windows after 21 h, but even better early in the morning between 6 and 9 h before pollinations (re)starts.

Component-resolved diagnosis using guinea-pig allergens elucidates allergen sensitization profiles in allergy to furry animals.

BACKGROUND: Furry animals are an important source of indoor allergens. Diagnosis of allergy to small pets such as guinea-pigs still relies on animal dander extracts which do not allow to define the primary sensitization source. OBJECTIVE: To identify major guinea-pig allergens and to evaluate their potential as marker allergens for in vitro IgE-diagnosis in comparison with dander extracts. METHODS: A group of patients allergic to guinea-pig (n = 29) and a group of patients allergic to cat and dog (n = 30) were recruited for the study. A panel of four guinea-pig lipocalin allergens was expressed as recombinant proteins in E. coli. Specific IgE were quantified by ImmunoCAP and ELISA. RESULTS: The combination of 4 guinea-pig lipocalin allergens, including 2 new lipocalins, Cav p 1.0201 and Cav p 6.0101, and the previously characterized lipocalins Cav p 2 and Cav p 3, enabled the identification of 90% of all patients allergic to guinea-pig. The vast majority had specific IgE to Cav p 1 (83%). Cav p 6 shares 54% sequence identity with Fel d 4 and Can f 6 and was found to be IgE-cross-reactive with these allergens. In the group of cat- and dog-allergic patients, 73% had also specific IgE to guinea-pig dander. However, only 27% of the cat /dog-allergic patients had specific IgE to any of the non-cross-reactive guinea-pig allergens Cav p 1, Cav p 2 or Cav p 3. The high prevalence of IgE to guinea-pig dander could be explained by IgE-cross-reactivity among serum albumins and certain lipocalins. CONCLUSIONS AND CLINICAL RELEVANCE: The availability of specific allergen markers is essential for the assessment of primary sensitization, especially in polysensitized patients. The proposed panel of guinea-pig allergens Cav p 1, Cav p 2 and Cav p 3 is a first step to component-resolved IgE-diagnosis of allergy to small furry pets.

Comprehensive mapping of immune tolerance yields a regulatory TNF receptor 2 signature in a murine model of successful Fel d 1-specific immunotherapy using high-dose CpG adjuvant.

BACKGROUND: The prevalence of allergy to cat is expanding worldwide. Allergen-specific immunotherapy (AIT) has advantages over symptomatic pharmacotherapy and promises long-lasting disease control in allergic patients. However, there is still a need to improve cat AIT regarding efficacy, safety, and adherence to the treatment. Here, we aim to boost immune tolerance to the major cat allergen Fel d 1 by increasing the anti-inflammatory activity of AIT with the established immunomodulatory adjuvant CpG, but at a higher dose than previously used in AIT. METHODS: Together with CpG, we used endotoxin-free Fel d 1 as therapeutic allergen throughout the study in a BALB/c model of allergy to Fel d 1, thus mimicking the conditions of human AIT trials. Multidimensional immune phenotyping including mass cytometry (CyTOF) was applied to analyze AIT-specific immune signatures. RESULTS: We show that AIT with high-dose CpG in combination with endotoxin-free Fel d 1 reverts all major hallmarks of allergy. High-dimensional CyTOF analysis of the immune cell signatures initiating and sustaining the AIT effect indicates the simultaneous engagement of both, the pDC-Treg and B-cell axis, with the emergence of a systemic GATA3+ FoxP3hi biTreg population. The regulatory immune signature also suggests the involvement of the anti-inflammatory TNF/TNFR2 signaling cascade in NK and B cells at an early stage and in Tregs later during AIT. CONCLUSION: Our results highlight the potential of CpG adjuvant in a novel formulation to be further exploited for inducing allergen-specific tolerance in patients with cat allergy or other allergic diseases.

Long-Term Pollen Monitoring in the Benelux: Evaluation of Allergenic Pollen Levels and Temporal Variations of Pollen Seasons.

Airborne pollen is a major cause of allergic rhinitis, affecting between 10 and 30% of the population in Belgium, the Netherlands, and Luxembourg (Benelux). Allergenic pollen is produced by wind pollinating plants and released in relatively low to massive amounts. Current climate changes, in combination with increasing urbanization, are likely to affect the presence of airborne allergenic pollen with respect to exposure intensity, timing as well as duration. Detailed analysis of long-term temporal trends at supranational scale may provide more comprehensive insight into these phenomena. To this end, the Spearman correlation was used to statistically compare the temporal trends in airborne pollen concentration monitored at the aerobiological stations which gathered the longest time-series (30-44 years) in the Benelux with a focus on the allergenic pollen taxa: Alnus, Corylus, Betula, Fraxinus, Quercus, Platanus, Poaceae, and Artemisia. Most arboreal species showed an overall trend toward an increase in the annual pollen integral and peak values and an overall trend toward an earlier start and end of the pollen season, which for Betula resulted in a significant decrease in season length. For the herbaceous species (Poaceae and Artemisia), the annual pollen integral and peak values showed a decreasing trend. The season timing of Poaceae showed a trend toward earlier starts and longer seasons in all locations. In all, these results show that temporal variations in pollen levels almost always follow a common trend in the Benelux, suggesting a similar force of climate change-driven factors, especially for Betula where a clear positive correlation was found between changes in temperature and pollen release over time. However, some trends were more local-specific indicating the influence of other environmental factors, e.g., the increasing urbanization in the surroundings of these monitoring locations. The dynamics in the observed trends can impact allergic patients by increasing the severity of symptoms, upsetting the habit of timing of the season, complicating diagnosis due to overlapping pollen seasons and the emergence of new symptoms due allergens that were weak at first.

Male-specific submaxillary gland protein, a lipocalin allergen of the golden hamster, differs from the lipocalin allergens of Siberian and Roborovski dwarf hamsters.

BACKGROUND: An increasing number of asthma cases upon exposure to hamsters and anaphylactic reactions following hamster bites are being reported, but the allergens responsible are still poorly characterized. In the Golden hamster, male-specific submaxillary gland protein (MSP), a lipocalin expressed in a sex- and tissue-specific manner in the submaxillary and lacrimal glands, is secreted in the saliva, tears and urine. The purpose of this study was to determine if MSP is an allergen, to identify IgE-reactive proteins of different hamster species and to analyse potential cross-reactivities. METHODS: Fur extracts were prepared from four hamster species. Hamster-allergic patients were selected based on a history of positive IgE-test to hamster epithelium. The IgE-reactivity of patients' sera was investigated by means of immunoblot and ELISA. IgE-reactive proteins in fur extracts and the submaxillary gland were identified using anti-MSP antibodies, Edman sequencing or mass spectrometry. MSP was purified from Golden hamster and recombinant MSP was expressed in E. coli. RESULTS: Four patients had IgE-antibodies against 20.5-kDa and 24-kDa proteins of Golden hamster fur extract, which were identified as MSP. IgE-reactive MSP-like proteins were detected in European hamster fur extract. Three patient sera showed IgE-reactive bands at 17-21 kDa in Siberian and Roborovski hamster fur extracts. These proteins were identified as two closely related lipocalins. Immunoblot inhibition experiments showed that they are cross-reactive and are different from MSP. CONCLUSION: MSP lipocalin of the Golden hamster was identified as an allergen, and it is different from the cross-reactive lipocalin allergens of Siberian and Roborovski hamsters. Our findings highlight the need for specific tools for the in vitro and in vivo diagnosis of allergy to different hamster species.

Neurturin influences inflammatory responses and airway remodeling in different mouse asthma models.

Neurturin (NTN) was previously described for its neuronal activities, but recently, we have shown that this factor is also involved in asthma physiopathology. However, the underlying mechanisms of NTN are unclear. The aim of this study was to investigate NTN involvement in acute bronchial Th2 responses, to analyze its interaction with airway structural cells, and to study its implication in remodeling during acute and chronic bronchial inflammation in C57BL/6 mice. We analyzed the features of allergic airway inflammation in wild-type and NTN(-/-) mice after sensitization with two different allergens, OVA and house dust mite. We showed that NTN(-/-) dendritic cells and T cells had a stronger tendency to activate the Th2 pathway in vitro than similar wild-type cells. Furthermore, NTN(-/-) mice had significantly increased markers of airway remodeling like collagen deposition. NTN(-/-) lung tissues showed higher levels of neutrophils, cytokine-induced neutrophil chemoattractant, matrix metalloproteinase 9, TNF-α, and IL-6. Finally, NTN had the capacity to decrease IL-6 and TNF-α production by immune and epithelial cells, showing a direct anti-inflammatory activity on these cells. Our findings support the hypothesis that NTN could modulate the allergic inflammation in different mouse asthma models.

Immune responses to inhalant Mammalian allergens.

In Europe and the USA, at least one person in four is exposed every day to inhalant allergens of mammalian origin, a considerable number is regularly exposed for professional reasons and almost everyone is occasionally exposed to inhalant allergens from pets or domestic animals. The production of IgE to these inhalant allergens, often complicated by asthma and rhinitis, defines the atopic status. However, the immune response to these allergens largely imprints the cellular immune compartment and also drives non-IgE humoral immune responses in the allergic and non-allergic population. During the recent years, it has become clear that IgE antibodies recognize mammalian allergens that belong to three protein or glycoprotein families: the secretoglobins, the lipocalins, and the serum albumins. In this article, we review the humoral and cellular immune responses to the major members of these families and try to define common characteristics and also distinctive features.

Fish allergens at a glance: variable allergenicity of parvalbumins, the major fish allergens.

Fish is a common trigger of severe, food-allergic reactions. Only a limited number of proteins induce specific IgE-mediated immune reactions. The major fish allergens are the parvalbumins. They are members of the calcium-binding EF-hand protein family characterized by a conserved protein structure. They represent highly cross-reactive allergens for patients with specific IgE to conserved epitopes. These patients might experience clinical reactions with various fish species. On the other hand, some individuals have IgE antibodies directed against unique, species-specific parvalbumin epitopes, and these patients show clinical symptoms only with certain fish species. Furthermore, different parvalbumin isoforms and isoallergens are present in the same fish and might display variable allergenicity. This was shown for salmon homologs, where only a single parvalbumin (beta-1) isoform was identified as allergen in specific patients. In addition to the parvalbumins, several other fish proteins, enolases, aldolases, and fish gelatin, seem to be important allergens. New clinical and molecular insights advanced the knowledge and understanding of fish allergy in the last years. These findings were useful for the advancement of the IgE-based diagnosis and also for the management of fish allergies consisting of advice and treatment of fish-allergic patients.

NK cell killer Ig-like receptor repertoire acquisition and maturation are strongly modulated by HLA class I molecules.

The interaction between clonally distributed inhibitory receptors and their activating counterparts on NK cells and HLA class I molecules defines NK cell functions, but the role of HLA class I ligands in the acquisition of their receptors during NK development is still unclear. Although some studies demonstrated that HLA-C affects the expression of killer Ig-like receptors (KIR), other studies showed that NK cells acquire their KIR repertoire in a stochastic manner. Only when infected with human CMV is an expansion of self-specific KIR(+) NKG2C(+) NK cells detected. To gain more insight into this question, we compared the coexpression of different KIR molecules, NKG2A, CD8, and CD57, on NK cells in healthy donors and seven patients with deficient HLA class I expression due to mutations in one of the TAP genes. Our results show a correlation between the presence/absence of HLA class I molecules and the coexpression of their receptors. In an HLA class I low-expression context, an increase in KIR molecules' coexpression is detected on the NKG2A(+) CD8(+) subset. In functional assays, hyporesponsiveness was observed for TAP-deficient NK cells derived from four patients. In contrast, NK cells from patient five were functional, whereas CD107a(+) and IFN-γ(+) CD56(dim) NK cells presented a different pattern of HLA class I receptors compared with healthy donors. Taken together, our results provide strong evidence for the role of HLA class I molecules in NK cell maturation and KIR repertoire acquisition.

Identification and isolation of a Fel d 1-like molecule as a major rabbit allergen.

BACKGROUND: Rabbits are increasingly kept as domestic pets. Several rabbit allergens have been characterized. However, their sequences are still elusive, and none of these molecules are available for diagnosis. OBJECTIVE: We sought to isolate major allergens from the rabbit Oryctolagus cuniculus and to investigate their importance in sensitized patients. METHODS: Proteins were extracted from rabbit hair, and IgE-reactive proteins were purified by using sequential chromatography. Allergens were characterized by means of N-terminal sequencing and mass spectrometry. IgE reactivity to a new allergen was analyzed in sera of 35 patients sensitized to rabbits in a domestic setting. A model of the crystal structure of the isolated proteins was constructed. RESULTS: A new IgE-reactive allergen, Ory c 3, was identified as rabbit lipophilin. The molecule that belongs to the secretoglobin family is a heterodimer of 18 to 19 kDa composed of 2 polypeptide chains, CL2 and AL. CL2 has a predicted N-linked glycosylation site confirmed by using mass spectrometry. Of the 35 patients with rabbit allergy studied, 27 (77%) had IgE to both the glycosylated and deglycosylated Ory c 3 heterodimer. Allergenicity of Ory c 3 was confirmed by using skin prick tests and the basophil activation assay. Modeling of the structure revealed a marked homology to Fel d 1, the major cat allergen. However, no IgE cross-reactivity was detected between Fel d 1 and Ory c 3. CONCLUSION: The rabbit lipophilin heterodimer AL-CL2 has been identified as a major rabbit allergen. After Fel d 1, Ory c 3 is the second mammalian secretoglobin shown to be a major allergen.

Targeting glioblastoma with NK cells and mAb against NG2/CSPG4 prolongs animal survival.

Glioblastoma (GBM) is the most malignant brain tumor where patients' survival is only 14.6 months, despite multimodal therapy with debulking surgery, concurrent chemotherapy and radiotherapy. There is an urgent, unmet need for novel, effective therapeutic strategies for this devastating disease. Although several immunotherapies are under development for the treatment of GBM patients, the use of natural killer (NK) cells is still marginal despite this being a promising approach to treat cancer. In regard of our knowledge on the role of NG2/CSPG4 in promoting GBM aggressiveness we investigated the potential of an innovative immunotherapeutic strategy combining mAb9.2.27 against NG2/CSPG4 and NK cells in preclinical animal models of GBM. Multiple immune escape mechanisms maintain the tumor microenvironment in an anti-inflammatory state to promote tumor growth, however, the distinct roles of resident microglia versus recruited macrophages is not elucidated. We hypothesized that exploiting the cytokine release capabilities of activated (NK) cells to reverse the anti-inflammatory axis combined with mAb9.2.27 targeting the NG2/CSPG4 may favor tumor destruction by editing pro-GBM immune responses. Combination treatment with NK+mAb9.2.27 diminished tumor growth that was associated with reduced tumor proliferation, increased cellular apoptosis and prolonged survival compared to vehicle and monotherapy controls. The therapeutic efficacy was mediated by recruitment of CCR2low macrophages into the tumor microenvironment, increased ED1 and MHC class II expression on microglia that might render them competent for GBM antigen presentation, as well as elevated IFN-γ and TNF-α levels in the cerebrospinal fluid compared to controls. Depletion of systemic macrophages by liposome-encapsulated clodronate decreased the CCR2low macrophages recruited to the brain and abolished the beneficial outcomes. Moreover, mAb9.2.27 reversed tumor-promoting effects of patient-derived tumor-associated macrophage/microglia(TAM) ex vivo.Taken together, these findings indicate thatNK+mAb9.2.27 treatment may be an amenable therapeutic strategy to treat NG2/CSPG4 expressing GBMs. We provide a novel conceptual approach of combination immunotherapy for glioblastoma. The results traverse beyond the elucidation of NG2/CSPG4 as a therapeutic target, but demonstrate a proof of concept that this antibody may hold potential for the treatment of GBM by activation of tumor infiltrated microglia/macrophages.

NK cells in central nervous system disorders.

NK cells are important players in immunity against pathogens and neoplasms. As a component of the innate immune system, they are one of the first effectors on sites of inflammation. Through their cytokine production capacities, NK cells participate in the development of a potent adaptive immune response. Furthermore, NK cells were found to have regulatory functions to limit and prevent autoimmunity via killing of autologous immune cells. These paradoxical functions of NK cells are reflected in CNS disorders. In this review, we discuss the phenotypes and functional features of peripheral and brain NK cells in brain tumors and infections, neurodegenerative diseases, acute vascular and traumatic damage, as well as mental disorders. We also discuss the implication of NK cells in neurotoxicity and neuroprotection following CNS pathology, as well as the crosstalk between NK cells and brain-resident immune cells.

Mouse lung and spleen natural killer cells have phenotypic and functional differences, in part influenced by macrophages.

NK cells are lymphocytes of the innate immune system which are a first line of defense against infections and tumor cells, in bone marrow and peripheral organs like lung and spleen. The lung is an organ in contact with respiratory pathogens and the site of inflammatory disorders triggered by the respiratory environment. In contrast, spleen is a lymphatic organ connected to the blood system which regulates the systemic immune response. Here we compare NK cell maturation and expansion as well as expression of NK cell receptors in spleen and lung compartments. We show that spleen and lung NK cells differ in phenotypic and functional characteristics due to a difference of maturity and cellular microenvironment. Indeed we observe that spleen and lung macrophages have the capacity to influence the cytotoxicity of NK cells by cell-to-cell contact. This suggests that the differences of NK cell subsets are in part due to a modulation by the organ environment.

Animal lipocalin allergens.

Lipocalins represent the most important group of inhalant animal allergens. For some of them, three-dimensional protein structures have been resolved, but their functions are still elusive. Lipocalins generally display a low sequence identity between family members. The characterization of new lipocalin allergens has revealed however that some of them display a high sequence identity to lipocalins from another species. They constitute a new group of potentially cross-reactive molecules which, in addition to serum albumins, may contribute to allergic cross-reactions between animal dander of different species. However, the clinical relevance of cross-reactivity needs to be assessed. Further studies are needed to understand which of these animal lipocalins are the primary allergens and which are cross-reacting molecules. The use of single, well characterized allergens for diagnosis will allow the identification of the sensitizing animal, which is a prerequisite for specific immunotherapy.

Consequences of the crosstalk between monocytes/macrophages and natural killer cells.

The interaction between natural killer (NK) cells and different other immune cells like T cells and dendritic cells is well-described, but the crosstalk with monocytes or macrophages and the nature of ligands/receptors implicated are just emerging. The macrophage-NK interaction is a major first-line defense against pathogens (bacteria, viruses, fungi, and parasites). The recruitment and the activation of NK cells to perform cytotoxicity or produce cytokines at the sites of inflammation are important to fight infections. The two main mechanisms by which macrophages can prime NK cells are (1) activation through soluble mediators such as IL-12, IL-18, and (2) stimulation through direct cell-to-cell contact. We will discuss the progress in matters of modulation of NK cell functions by monocytes and macrophages, in the steady state and during diseases.