Sambucus nigra

The selected area for tick monitoring is located in the southern outskirts of Prague (49°58′43″N; 14°24′52″E; altitude 325 MSL), 4 km from the main observatory station of the Czech Hydrometeorological Institute (CHMI) in Prague-Libuš, one of the best equipped observatories in the Czech Republic (Fig. 1). An adequate size (eg. 600 m²) was required to provide a large enough sample size for registering the spring start and autumn cessation of questing activity when numbers of active ticks are comparatively low. The site comprises eutrophicated, acid oak wood (Quercus robur, 70–100 year-old trees), with patches of younger hornbeam (Carpinus betulus). Most of the stand is evenly dense, with a few small openings. The tree canopy cover is 70 % and the shrub canopy cover (Rubus fruticosa, Sambucus nigra) is <10 %. The herb canopy cover is ~25 %, with prevailing low (<20 cm) sparse grass (mostly Poanemoralis) and patches completely covered with dead leaves or moss. In this vegetation community, considered typical habitat for I. ricinus in Central Europe, three adjacent plots were established each of 200 m² having all the features described, which were consistent year-on-year. Low vegetation of the undergrowth (remaining <20 cm in height throughout the year) in the monitored plots limited the possible bias from seasonal growth of the vegetation on the monitoring of ticks and microclimate. The habitat under study is situated on flat ground completely exposed to sunlight giving homogenous conditions over the whole site. Although the study area is under forestry management, with a controlled population of game (including roe deer), there was no forestry activity on the site or in the surrounding area; the absence of fencing allows free movement of all animals (including humans).Fig. 1

Location and habitat of the monitoring site. Circle indicates location of the Czech Hydrometeorological Institute in Prague-Libuš; circle and arrow indicate location of the monitoring site. Insert shows typical habitat of the monitoring site

This is a prospective study. Approval was received on May 5, 2013 from the Ethical Board for Clinical Research, Faculty of Medicine, Celal Bayar University, Turkey, and the Decision Reference Number 2013/153 was assigned. Written informed consent of all patients was obtained.
Diagnosis of AR was based on history, physical examination, and laboratory findings. The patients having applied for the adult allergy-immunology clinic in Tepecik Training Research Hospital and after medical history and physical examination, who were predicted to have AR, were administered skin prick test.
Allergic sensitization was demonstrated by the skin prick test. Skin prick tests were performed according to the EAACI guidelines for the most common inhalant allergens in Turkey, including house dust mites (Dermatophagoides pteronyssinus and Dermatophagoides farinae), fungi (Aspergillus fumigatus, Alternaria alternata, Cladosporium herbarum, and Penicillium notatum), grasses (Lolium perenne, Fectuca pratensis, Phleum pratense, Poa pratensis, and Dactylis glomerata), weeds (Plantago lanceolata, Artemisia vulgaris, Rumex acetosa, Taraxacum vulgare, and Parietaria offıcinalis), and trees (Sambucus nigra, Populus alba, Ulmus scabra, Salix caprea, Fagus sylvatica, Carpinus betulus, Quercus robur, Fraxinus excelsior, and Olea europaea) with histamine and diluent control (Allergopharma Ltd, Reinbek, Germany) [18 (link)].
Allergic rhinitis clinic severity was demonstrated by the nasal symptom scores (NSS). NSS (sneezing, nasal obstruction, nasal itching, and watery nasal discharge; graded as 0 = none, 1 = mild, 2 = moderate, and 3 = severe, up to a maximum of 12 points) were recorded for all patients during the pollen season, when their symptoms were at their worst.
In skin prick test, out of the patients detected to have sensitivity for only mite and olive tree pollen, those having symptoms for 2 years were included in the study. Patients known to have had any immunosuppressive treatment within the last 1 month, to have had antihistamine treatment within the last 10 days, to have smoked, to have any active infection, and to have another chronic disease such as chronic rhinosinusitis, nasal polyposis, and cancer were excluded from the study.
AR patients were divided into two groups according to allergen sensitivities as seasonal allergic rhinitis (SAR) and perennial allergic rhinitis (PAR). Patients only with olive tree sensitivity and having seasonal symptoms were evaluated for SAR. Patients only with mite sensitivity and having year-long symptoms were included in the study for PAR.
Peripheral blood samples were taken for evaluation of serum cytokine level in the period with the highest symptom scores for both patient groups, and cytokine measurement was performed with ELISA method.
The control group consisted of volunteer people with similar age and gender range with patients, with no allergic and systemic disease history and with no drug use within the last one month. Skin prick tests of the control group were negative.

The expression of NA protein on MDCK-II cells was quantified by flow cytometry using FACS analysis as previously described with few modifications [65 (link)]. Briefly, MDCK-II cells in 24-well plates were infected with MOI 1 of different viruses in three replicates. At 8 and 24 h, the supernatant (0.5mL) were firstly collected then cell sheets were dissociated by trypsin (0.250 mL) and anti-N1 polyclonal antibodies were added at a ratio 1:1000 for 1 h to ~5x10⁶ non-permeabilized single cells. After washing, secondary anti-rabbit antibodies were added 1:20000 and the cells were subjected to immuno-staining for flow cytometry analysis using a BD FACSCanto flow cytometer (BD Biosciences). The mean fluorescence intensity (MFI) of non-infected cells was subtracted from infected cells and the results were shown as ΔMFI and standard deviation of all replicates. For the detection of SA moieties, MDCK-II and MDCK-SIAT cells were infected at an MOI of 1 with rg-AL or rg-HL-16. After 2 hour incubation at 37°C cells were detached with trypsin and washed once with staining buffer. Fluorescein-labelled Sambucus nigra lectin (SNA; Vector Laboratories) and biotin-labelled Maackia amurensis lectin II (MAL II; Vector Laboratories) were used for the detection of (α-2,6) or (α-2,3) linked sialic acids, respectively, in infected and non-infected cells. After washing, MAL II binding was detected using PE-Cy7-labelled streptavidin (Biolegend). All incubation steps were carried out at 4°C in the dark. Stained infected and non-infected cells were treated with fixation buffer according to the manufacturer’s protocol (Biolegend). Reading was done using a BD FACSCanto flow cytometer (BD Biosciences). The results are the average and standard deviation of a triplicate experiment.

An ELLA was performed to analyze the influence of glycoprotein coupling conditions on terminal sialic acid residues of fetuin. 200 µL of a 5 µg x mL⁻¹ fetuin solution (in PBS) was immobilized on 96-well adsorptive microtiter plates (Nunc Immuno Maxisorp, VWR, Langenfeld, Germany) overnight at 4 °C, blocked with BSA, and washed with PBS-Tween (0.05% v/v), as described above. The adsorbed fetuin was then subjected to 50 µL of buffers used for Sepharose coupling (see Section 4.3) with PBS pH 7.5 as a control for one hour at room temperature. After washing three times, samples were incubated with 50 µL of 2 µg x mL⁻¹ biotinylated Maackia amurensis lectin II (MALII, Roche, Mannheim, Germany) and Sambucus nigra elderberry bark lectin (EBL, Roche, Mannheim, Germany) in lectin buffer (10 mM HEPES pH 7.5, 150 mM NaCl, 0.1 mM CaCl₂ ·2H₂O) for one hour. Afterward, for one hour, lectin binding was detected with streptavidin-peroxidase (1:5000 in PBS). The absorption was measured with TMB as described.