Grass Pollen Extract Research Articles

Identification of cross-reactive IgE-binding proteins from Philippine allergenic grass pollen extracts.

Respiratory allergies are one of the most common allergic diseases that affect Filipinos. Grass pollen accounts for the majority of the outdoor allergens triggering these respiratory allergies. Cross-reactivity among the Philippine grass pollen grains has not been extensively studied. This study aims to investigate the cross-reactivity of our local grasses and identify the cross-reactive allergens. Grass pollen grains were collected and processed into crude allergenic extracts. The IgE-reactivity of these crude allergenic pollen extracts was studied using sera from patients who tested positive for the mentioned extracts. The proteins from the immunoblots of cross-reactive pollen allergen extracts were sequenced and identified. Allergenic pollen proteins were identified as cross-reactive among the grass pollen extracts. Four of these have not been listed yet as grass allergens in the World Health Organization/International Union of Immunological Societies allergen nomenclature database. Local grass pollen allergens are cross-reactive with probable new allergens identified.

Tablet allergen immunotherapy: the anaphylaxis issue.

For the first time 15 years ago, tablet allergen immunotherapy (T-AIT) formulations were approved by regulatory agencies for treating allergic rhinitis caused by grass pollen in adults and children aged >5 years. Extensive evidences existed about effectiveness and safety of AIT. However, the safety profile is particularly compelling in children. Generally, T-AIT causes local reactions, mostly in the oral cavity, that are usually mild-to-moderate and often self-resolving. However, systemic allergic reactions are also observed with T-AIT, anaphylaxis representing the most fearsome adverse event, considering that it occurs in subjects treated for allergic rhinitis. Therefore, we conducted a literature search of patients reporting anaphylaxis because of T-AIT. Nine cases of anaphylactic reactions were reported in literature. Notably, no death was reported using T-AIT. This outcome was very important as it underscored the substantial safety of T-AIT. However, T-AIT deserves careful attention, mainly in the pediatric population. In this regard, after the first report of anaphylactic reaction at the first administration of T-AIT, manufacturers recommended that the first dose should be administered in a medical facility in the presence of staff with experience in managing anaphylaxis and the patient should be observed for at least 30 min. Interestingly, reported anaphylactic reactions were due to grass pollen extracts, with no report concerning other allergen extracts. However, it is relevant to note that anaphylactic reactions because of T-AIT are not reported in recent years.

Pollen allergen products: current standardisation issues

Scientific relevance. Pollen allergen medicines are in high demand, and their therapeutic benefits directly correlate with their standardisation. Better diagnosis and treatment of allergic diseases require state-of-the-art procedures for assessing the allergenic activity of pollen allergen products using reference standards and physicochemical testing methods.Aim. The study aimed at developing methodological approaches to the standardisation of pollen allergen products in order to shift to measuring their potency in allergenic activity units (AAU) and bring their quality in line with the requirements of the State Pharmacopoeia of the Russian Federation.Materials and methods. The study used pollen allergen reference standards by Microgen, the WHO International Standard for timothy grass (Phleum pratense) pollen extract, a gel filtration standard kit of molecular weight markers ranging from 1.35 to 670 kDa, bovine serum albumin, serum samples with specific IgE obtained from donors sensitised to the study pollen allergens, labelled anti-human IgE antibodies, and reference standards for determining residual volatile solvents by gas chromatography. The identification of in-house reference standards for the potency of pollen allergens involved Western blotting (for allergenic components). The total protein content was determined by Bradford’s assay. In addition, the authors used high-performance liquid chromatography to study protein fractions and gas–liquid chromatography to determine the content of residual organic solvents.Results. To substitute the existing method of non-specific characterisation of allergenic activity in protein nitrogen units (PNU), the authors developed and tested a new method to control allergenic activity in allergenic activity units (AAU) based on an in vitro competitive enzyme-linked immunosorbent assay (ELISA) procedure developed and validated in this study. Furthermore, the authors developed and certified 15 primary in-house reference standards with allergenic activity established in AAU/mL using skin tests in vivo. The experimental data were analysed to standardise the allergenic activity of the pollen allergens manufactured by Microgen. The authors developed physicochemical methods for the certification of in-house reference standards and validated these methods in accordance with the State Pharmacopoeia of the Russian Federation. The study involved selecting chromatographic separation conditions for residual organic solvents (acetone and diethyl ether) and establishing system suitability criteria for the chromatographic system. The allergenic activity of secondary in-house reference standards was certified against that of primary in-house reference standards using competitive ELISA. Thus, the authors managed to shift to the standardisation of pollen allergen products in vitro.Conclusions. The authors developed their competitive ELISA-based method to standardise pollen allergen products by comparing the inhibition of immune responses to a product and a standard. The study demonstrated the feasibility of substituting allergenic activity quantification (in AAU) for protein nitrogen content determination (in PNU) and showed the first example of using AAU for the certification of in-house reference standards. Additionally, the authors developed and validated an analytical procedure for determining the content of residual organic solvents in pollen allergen products by gas–liquid chromatography.

Factors predicting the outcome of allergen-specific nasal provocation test in children with grass pollen allergic rhinitis.

Nasal provocation testing (NPT) is a reference methodology to identify the culprit allergen in patients with allergic rhinitis. Selecting the right allergen for NPT is particularly difficult in poly-sensitized patients with seasonal allergic rhinitis (SAR). Predictors of NPT outcomes may facilitate the proper use of this test or even substitute it. To identify predictors of grass pollen NPT outcome from an array of clinical data, e-diary outcomes, and allergy test results in poly-sensitized pediatric patients with SAR. Poly-sensitized, SAR patients with grass pollen allergy, participating in the @IT.2020 pilot project in Rome and Pordenone (Italy), participated in a baseline (T0) visit with questionnaires, skin prick testing (SPT), and blood sampling to measure total (ImmunoCAP, TFS, Sweden) and specific IgE antibodies to grass pollen extracts and their major allergenic molecules (ESEP, Euroimmun Labordiagnostika, Germany). During the pollen season, patients filled the AllergyMonitor® e-diary app measuring their symptoms, medication intake, and allergy-related well-being via the Visual Analogue Scale (VAS). After the pollen season (T1), patients answered clinical questionnaires and underwent a nasal provocation test (NPT) with grass pollen extract. We recruited 72 patients (age 14.3 ± 2.8 years, 46 males) sensitized to grass and/or other pollens, including olive (63; 87.5%) and pellitory (49; 68.1%). Patients positive to grass pollen NPT (61; 84.7%), compared to the negative ones, had worse VAS values in the e-diary, larger SPT wheal reactions, and higher IgE levels, as well as specific activity to timothy and Bermuda grass extracts, rPhl p 5 and nCyn d 1. A positive NPT to grass pollen was predicted by an index combining the specific activity of IgE towards Phl p 5 and Cyn d 1 (AUC: 0.82; p < 0.01; best cut-off ≥7.25%, sensitivity 70.5%, specificity: 90.9%). VAS results also predicted NPT positivity, although with less precision (AUC: 0.77, p < 0.01; best cut-off ≥7, sensitivity: 60.7%, specificity: 81.8%). An index combining the specific activity of IgE to rPhl p 5 and nCyn d 1 predicted with moderate sensitivity and high specificity the outcome of a grass pollen NPT in complex, poly-sensitized pediatric patients with seasonal allergic rhinitis. Further studies are needed to improve the index sensitivity and to assess its usefulness for NPT allergen selection or as an alternative to this demanding test procedure.

Efficacy and safety of sublingual immunotherapy for allergic rhinitis: A network meta-analysis.

To systematically evaluate the clinical efficacy and safety of sublingual immunotherapy for allergic rhinitis (AR) and provide evidence for clinical treatment. A literature search was performed on the China National Knowledge Infrastructure (CNKI), Wanfang database, PubMed, Web of Science, Cochrane Library, and Embase database. Data from randomized controlled trials (RCTs) of sublingual immunotherapy for AR were screened and extracted from the establishment of those databases to November 2022. Subsequently, a network meta-analysis was performed using a statistical software R 4.2. Totally 22 RCTs that met the inclusion and exclusion criteria and screened from 1,164 literature were included. A total of 4,941 AR patients were involved in the 22 trials, as well as five interventions including placebo, pharmacotherapy, subcutaneous immunotherapy_dust mite, sublingual immunotherapy_dust mite, and sublingual immunotherapy_ grass mix plus pollen extract. The results of network meta-analysis showed that, based on symptom scores after different interventions for AR, the most effective treatments for AR were in order as follows: sublingual immunotherapy_dust mite, subcutaneous immunotherapy_dust mite, sublingual immunotherapy_ grass mix plus pollen extract, placebo, and pharmacotherapy. Importantly, sublingual immunotherapy had fewer adverse reactions and higher safety. Sublingual immunotherapy_dust mite for AR has the best efficacy, whereas traditional medicine has the worst. More high-quality studies with a large sample and multiple centers are needed to verify this conclusion in the future, so as to further provide more reliable evidence-based medical evidence for the clinical treatment options of AR patients.

Molecular allergy diagnosis is sensitive and avoids misdiagnosis in patients sensitized to seasonal allergens.

The specificity of extract-based pollen allergy diagnosis is decreased due to cross-reactivity via cross-reactive carbohydrate determinants (CCDs) or panallergens such as profilins or polcalcins. This study aimed to explore the prevalence of sensitization to seasonal extracts, CCDs, profilin and polcalcin and investigate the sensitivity and specificity of seasonal molecular allergy diagnosis (MAD) using commercially available test methods. 2948 patients were screened for specific immunoglobulin E to ash, birch, mugwort, ragweed and timothy grass pollen extracts and grouped according to the number of positive tests (1-5). 100 patients from each group and a control group were randomly selected to calculate the prevalence of CCD and panallergen sensitization. With 742 patients, sensitivity and specificity of MAD (Alt a 1, Fra/Ole e 1, Bet v 1, Phl p 1, Art v 1, and Amb a 1) was determined. 1627 patients (55.2%) were positive to at least one, and 1002 patients (34.0%) were positive to multiple of the five pollen allergens investigated; 18.5% of the pollen-sensitized patients had sensitization to CCDs or panallergens. Specifically, sensitization to CCDs, profilins, and polcalcins was observed in 8.7%, 10.9%, and 2.9% of these patients, respectively. The sensitivity of MAD was high, with sensitivities between 96.2% and 100% using ImmunoCAP and 91.5% and 100% using ALEX2 . Specificity was 100% for both assays. Due to cross-reactivity, about one-fifth of pollen-sensitized patients is at risk of misdiagnosis. However, MAD is sensitive, specific and helps to avoid misdiagnosis and select primary allergen sources for immunotherapy.

Single-cell RNA-Seq identifies precise tolerogenic cellular and molecular pathways induced by depigmented-polymerized grass pollen allergen extract

Single-cell RNA-Seq (scRNA-seq) provides a novel approach to identify novel tolerogenic molecular pathways induced by allergen immunotherapy. We sought to investigate the underpinning tolerogenic molecular and cellular mechanisms of depigmented-polymerized grass pollen extract for allergen immunotherapy.

Extract-Shaped Immune Repertoires as Source for Nanobody-Based Human IgE in Grass Pollen Allergy.

The presence of allergen-specific IgE in serum is a biomarker for allergic disease. Specific IgE antibodies for research and diagnostics, however, remain scarce. In contrast to prototypic antibodies, camelid species have evolved single domains as moiety for antigen recognition. These so-called nanobodies represent a versatile platform for the development of diagnostic and therapeutic approaches. In this study, we aimed for generating nanobodies and derived IgE formats from an extract-shaped immune repertoire. Timothy grass pollen represents a complex, but well-defined mixture of individual allergens. Therefore, a repertoire library from a timothy grass pollen extract immunised llama was established. The selection by phage display yielded 3 nanobodies with immunoreactivity to the extract. IgE-like nanobody-based human IgE (nb-hIgE) antibodies were produced in mammalian cells and assessed in different immunoassays and commercial platforms. Immunoblotting and diagnostic ImmunoCap analysis of single timothy grass pollen allergens identified the major allergens Phl p 6 and Phl p 4 as targets. Assessment of immunoreactivity further documented significant molecular cross-reactivity with pollen extract of different grass species and variant presence of allergens within extracts of Pooideae grasses. In summary, our study shows that extract-based immunisation enables the generation of allergen-specific nanobodies and derived nb-hIgE formats linking nanobody technologies with allergological applications.

House Dust Mite and Grass Pollen Allergen Extracts for Seasonal Allergic Rhinitis Treatment: A Systematic Review.

Background: The treatment of allergic rhinitis is important due to the burden that the disease causes globally. The objective of this review is to explore the efficiency of house dust mite and grass pollen extracts in allergic rhinitis treatment.Methods: We performed research in electronic databases and searched relevant articles on PubMed, CINAHL, OVID, ScienceDirect, Cochrane CENTRAL, and MEDLINE. We used keywords such as ‘allergic rhinitis', ‘sublingual immunotherapy’, ‘randomized controlled trials', ‘grass pollen’, 'allergen immunotherapy’, and ‘house dust mite’. We included nine randomized controlled trials (RCTs). Quality assessment of included studies was performed independently by two authors.Results: We included nine eligible RCTs in this review. Five RCTs were about grass pollen extracts and four RCTs were about house dust mite extracts. Most of the studies reported positive results and suggested further evaluation of sublingual immunotherapy (SLIT) treatment. Grass pollen extracts mostly used were Dactylis glomerata, Poa pratensis, Lolium perenne, Anthoxanthum odoratum, Phleum pratense, and Parietaria. House dust mite extracts used were from Dermatophagoides pteronyssinus and Dermatophagoides farina. According to the quality assessment, no bias was observed in the included studies.Conclusions: Although sublingual allergen immunotherapy shows a benefit compared to placebo in the treatment of allergic rhinitis and rhino-conjunctivitis in adults, the results are interpreted with caution due to the high heterogenicity among studies in treatment protocols and dosing. More standardization among studies is needed.

Ex Vivo Immunomodulatory Effects of Lactobacillus-, Lacticaseibacillus-, and Bifidobacterium-Containing Synbiotics on Human Peripheral Blood Mononuclear Cells and Monocyte-Derived Dendritic Cells in the Context of Grass Pollen Allergy

Sensing of the intestinal microbiota by the host immune system is important to induce protective immune responses. Hence, modification of the gut microbiota might be able to prevent or treat allergies, mediated by proinflammatory Th2 immune responses. The aim was to investigate the ex vivo immunomodulatory effects of the synbiotics Pollagen® and Kallergen®, containing the probiotic bacterial strains Lactobacillus, Lacticaseibacillus and Bifidobacterium, in the context of grass pollen allergy. Peripheral blood mononuclear cells (PBMCs) from grass pollen–allergic patients and healthy controls were stimulated with grass pollen extract (GPE) and synbiotics and Gata3 expression and cytokine secretion analyzed. Monocyte-derived dendritic cells (MoDCs) cells were matured in the presence of GPE and synbiotics, co-cultured with autologous naïve T cells and maturation markers and cytokine secretion analyzed. GPE stimulation of PBMCs from grass pollen–allergic patients resulted in a significant higher production of the Th2 cytokines IL-4, IL-5, IL-9 and IL-13 compared to healthy controls. Gata3+CD4+ T cell induction was independent of the allergic status. The synbiotics promoted IL-10 and IFN-γ secretion and downregulated the GPE-induced Th2-like phenotype. Co-culturing naïve T cells with MoDCs, matured in the presence of GPE and synbiotics, shifted the GPE-induced Th2 cytokine release towards Th1-Th17-promoting conditions in allergic subjects. The investigated synbiotics are effective in downregulating the GPE-induced Th2 immune response in PBMCs from grass pollen–allergic patients as well as in autologous MoDC-T cell stimulation assays. In addition to increased IL-10 release, the data indicates a shift from a Th2- to a more Th1- and Th17-like phenotype.

Serum IgE profiles in Chinese pollinosis patients with grass pollen sensitisation

PurposePollen from trees, grasses, and weeds is a common allergen source. The characteristics of pollen allergy in China are obviously different from Europe. Most studies have focused on tree and weed pollen, but there is a paucity of data on grass pollen sensitisation in China. Therefore, we used component-resolved diagnostics to investigate the serum-specific immunoglobulin E (sIgE) to grass pollen in Chinese patients with pollinosis. MethodsWe retrospectively analysed 547 patients with pollen allegy from an outpatient Allergy Department in Beijing, China. All the patients answered questionnaires about their clinical allergy histories. Total immunoglobulin E (IgE) and sIgE levels to grass pollen (Bermuda, Timothy grass) were quantified by ImmunoCAP using 0.35 kUA/L as a threshold for positivity. ResultsOf the 547 pollinosis patients, 389 (71.1%) showed a positive sIgE reaction to either grass pollen, or both. The prevalence of food allergy was significantly lower in patients with grass pollen sensitisation. Among the 389 patients with grass pollen sensitisation, the prevalence of sIgE to allergen extracts of bermuda, mugwort, ragweed, plane, hop, ash, birch, and timothy grass was 97%, 96%, 94%, 88%, 88%, 84%, 78%, and 78%, respectively. However, only 134/389 (34%) were positive for Cyn d 1, 29/389 (7%) for Phl p 1, and 8/389 (2%) for Phl p 5b. For pollinosis patients, 62/547 (11%) were sIgE-positive for cross-reactive carbohydrate determinants (CCDs), and their grass pollen-sIgE was also positive. ConclusionsThe prevalence of in vitro IgE sensitisation to grass pollen extract is high in Chinese patients with pollinosis. But mostly spurious and characterized by IgE sensitisation to profilins and CCD, induced by other pollen. Component-resolved diagnostics is an extremely useful tool precise diagnostics of pollen allergy in China.

Standardisation of allergen products: 4. Validation of a candidate European Pharmacopoeia standard method for quantification of major grass pollen allergen Phl p 5.

The aim of the BSP090 project is the establishment of European Pharmacopoeia Chemical Reference Substances (CRSs) in combination with corresponding standard ELISA methods for quantification of major allergens in allergen products. Here, we present data of a Phlp5-specific sandwich ELISA that proved suitable for the quantification of Phlp5, one of the major Timothy grass (Phleum pratense) pollen allergens. A Phlp5-specific ELISA system was assessed with respect to accuracy, precision, inter-assay (within laboratory) and inter-laboratory variations, in a ring trial including 14laboratories in Europe and the USA. Model samples containing recombinant Phlp5a CRS as well as native grass pollen extracts were analysed. Each participant was instructed to perform at least one preliminary assay to familiarise with the protocol, followed by three independent assays. The candidate standard ELISA proved suitable to quantify recombinant and native Phlp5 with satisfactory precision (93% of results within ±30% acceptance range). Inter-assay variation (max. GCV 24%) and especially inter-laboratory variation (max. GCV 13%) showed conclusive results. When assessing accuracy by means of recovery of recombinant spikes from a grass pollen extract matrix, similarly satisfactory spike recovery results were observed for the two spikes with higher concentrations (all within ±30% acceptance range), whereas recovery of the lowest concentration spike was slightly poorer with mean results of six laboratories exceeding acceptance range. Based on the collaborative study results, the assessed Phlp5-specific immunoassay is appropriate to be proposed as European Pharmacopoeia standard method.

Molecular allergy diagnosis using pollen marker allergens and pollen panallergens: Five patterns seen in multiple test reactions topollen extracts.

Polysensitizations to tree, grass, and weed pollen are found in ~ 20% of pollen-allergic individuals. They are often based on broad IgE cross-reactivities to pollen panallergens belonging to highly conserved protein families: 1. profilins, 2. polcalcins (calcium-binding proteins in pollen), 3. cyclophilins. They represent highly conserved cross-reactive minor allergens present in all pollen species, but also in plant foods and other organisms. Despite being rarely clinically relevant they can hamper allergy diagnostic tests with extracts. In this situation, molecular allergy diagnosis is able to distinguish broad cross-reactivity due to allergen-specific IgE to pollen panallergens (i.e. profilins Bet v 2 or Phl p 12; polcalcins Bet v 4 or Phl p 7; and, in the future, cyclophilins Bet v 7 or Ole e 15) from primary IgE sensitizations to so-called marker allergens represented by important pollen major allergens: Bet v 1 for the birch and beech family (Fagales), Ole e 1 for olive and ash (Oleaceae), Phl p 1 for temperate climate grasses (Poaceae), Art v 1 for mugwort (Artemisia), Amb a 1 for Ambrosia species (Ambrosia). Five typical cases (A – E) with positive skin prick test results to tree, grass, and weed pollen extracts demonstrate typical patterns of IgE sensitization with a variable impact of pollen panallergens: A – profilins, B – polcalcins, C – profilins and polcalcins, D – presumably cyclophilins, E – primary polysensitization to tree, grass, and weed pollen without interference from profilins or polcalcins. Differences between pollen extract-based skin prick test diagnosis and molecular allergen-specific IgE testing are explained using the presented concept. This approach allows to reduce the number of allergen extracts – presuming they are also clinically relevant – for allergen immunotherapy (i.e., only tree and/or grass pollen extracts), particularly in pollen-polysensitized patients.

Evaluation of safety and tolerability of a rush up-dosing allergen-specific immunotherapy with grass pollen, birch, hazel, and alder allergoid in children with allergic rhinoconjunctivitis, with or without asthma.

Background:Usually, the number of injections required to achieve the maintenance dose in subcutaneous immunotherapy (SCIT) is relatively small for some of the currently used allergens, but this may still be uncomfortable for patients, thus compromising adherence and compliance.Objective:The purpose of this study was to evaluate the safety and tolerability of a dose acceleration of a conventional induction schedule using an allergoid extract of grass pollen, birch, hazel, and alder, needed to achieve the ideal maintenance dose.Methods:In this open-label study, 34 patients with allergic rhinoconjunctivitis, with or without asthma, were treated with SCIT using an allergoid for grass pollen or birch or mix trees with an increase in accelerated induction dose comprising only 3 injections, one per week, compared to a conventional induction pattern in five injections (once a week). Safety determination was assessed by evaluating local and systemic adverse events. Tolerability was evaluated by patients and physicians who performed the treatment.Results:No treatment-related adverse events were observed in any of the patients undergoing rush SCIT. No local reactions, no systemic reactions of any degree (WAO Grade) have been observed. Tolerability has always been rated as very good by both patients and physician.Conclusions:The induction phase, needed to achieve the monthly maintenance dose for a pollen extract, can be greatly accelerated, ensuring a tolerability comparable to that of the conventional schedule. (www.actabiomedica.it)

Repetitive nasal allergen challenge in allergic rhinitis: Priming and Th2-type inflammation but no evidence of remodelling.

Local tissue eosinophilia and Th2 cytokines are characteristic features of seasonal allergic rhinitis. Airway remodelling is a feature of asthma whereas evidence for remodelling in allergic rhinitis (AR) is conflicting. By use of a novel human repetitive nasal allergen challenge (RAC) model, we evaluated the relationship between allergic inflammation and features of remodelling in AR. Twelve patients with moderate-severe AR underwent 5 alternate day challenges with diluent which after 4weeks were followed by 5 alternate day challenges with grass pollen extract. Nasal symptoms, Th1/Th2 cytokines in nasal secretion and serum were evaluated. Nasal biopsies were taken 24hours after the 1st and 5th challenges with diluent and with allergen. Sixteen healthy controls underwent a single challenge with diluent and with allergen. Using immunohistochemistry, epithelial and submucosal inflammatory cells and remodelling markers were evaluated by computed image analysis. There was an increase in early and late-phase symptoms after every allergen challenge compared to diluent (both P<.05) with evidence of both clinical and immunological priming. Nasal tissue eosinophils and IL-5 in nasal secretion increased significantly after RAC compared to corresponding diluent challenges (P<.01, P=.01, respectively). There was a correlation between submucosal mast cells and the early-phase clinical response (r=0.79, P=.007) and an association between epithelial eosinophils and IL-5 concentrations in nasal secretion (r=0.69, P=.06) in allergic rhinitis. No differences were observed after RAC with regard to epithelial integrity, reticular basement membrane thickness, glandular area, expression of markers of activation of airway remodelling including α-SMA, HSP-47, extracellular matrix (MMP7, 9 and TIMP-1), angiogenesis and lymphangiogenesis for AR compared with healthy controls. Novel repetitive nasal allergen challenge in participants with severe persistent seasonal allergic rhinitis resulted in tissue eosinophilia and increases in IL-5 but no structural changes. Our data support no link between robust Th2-inflammation and development of airway remodelling in AR.

Development of a protein microarray-based diagnostic chip mimicking the skin prick test for allergy diagnosis

Protein microarrays have been successfully used for detection of allergen-specific IgE in patient sera. Here, we demonstrate proof-of-concept of a solid-phase technique coupling the high-throughput potential of protein microarrays with the biologically relevant readout provided by IgE reporter cells, creating a novel allergic sensitization detection system. Three proteins (κ-casein, timothy grass pollen extract, polyclonal anti-human IgE) were printed onto three different polymer-coated surfaces (aldehyde-, epoxy- and NHS ester-coated). ToF–SIMs analysis was performed to assess printed protein stability and retention during washing steps. NFAT-DsRed rat basophil leukemia cell attachment and retention during washing steps was assessed after treatment with various extracellular matrix proteins. NFAT-DsRed IgE reporter cells were sensitized with serum of an allergic donor, incubated on the printed slides, and cell activation determined using a microarray laser scanner. NFAT DsRed IgE reporter cell binding was significantly increased on all polymer surfaces after incubation with fibronectin and vitronectin, but not collagen or laminin. All surfaces supported printed protein stability during washing procedure, with epoxy- and NHS ester-coated surfaces showing best protein retention. Cell activation was significantly higher in NHS ester-coated slides after timothy grass pollen extract stimulation appearing a suitable substrate for further development of an automated allergy diagnosis system.

Protein microarrays: The future of allergy diagnosis? Optimization of coating and printing of allergen arrays used with fluorescent humanised basophil reporter cell line NFAT-DsRed RBL

Up-to-date, in vitro allergy diagnosis techniques rely on the detection of allergen-specific Immunoglobulin E (sIgE). Protein microarrays enable the simultaneous screening of hundreds of sIgEs using only a small amount of serum. However, they do not always report clinically relevant sIgE. Combining protein arrays with humanised rat basophilic leukaemia (RBL) reporter cells that provide a biological readout, we aim to develop a high-throughput amenable, clinically relevant allergy diagnosis platform. Here, we demonstrate the effectiveness of different extracellular matrix (ECM) proteins to facilitate RBL cell attachment on various polymer-coated slide surfaces. We also determine printed protein stability using time-of flight secondary ion mass spectrometry (ToF-SIMS). Lastly, we demonstrate the ability of this system to detect an allergic response. Aldehyde-, epoxy- and amine-coated glass slides were coated with 10 μg/mL vitronectin, fibronectin or laminin for 48 hours at 37 ͦ C and with 10 μg/mL collagen for 48 hours at 4 ͦ C. After incubation, NFAT-DsRed RBL cells were cultured on each surface for 72 hours. The number of adherent cells was assessed every 24 hours using 1 μg/mL Hoechst 33342 solution. Next, the following proteins diluted in PBS to a final concentration of 1mg/mL, goat anti-human IgE, timothy grass pollen extract, human serum albumin, k-casein from bovine milk or the above mentioned proteins diluted with 10% fibronectin, were printed in triplicate onto the different polymer-coated slides using a XYZ3200 dispensing workstation (Biodot). Printed proteins were characterized by ToF-SIMS before and after washing the surfaces with deionized water. Finally, cells were sensitized overnight with human IgE. Cell activation was assessed using fluorescence microscopy after 24h incubation onto the protein printed surfaces. Treatment of the aldehyde- and epoxy-coated slides with different ECM proteins had no significant effect on cell adhesion compared to control (cells only). The amine coated surfaces showed higher printed protein stability compared to aldehyde and epoxy ones. Lastly, cell activation was higher on the epoxy slides after printing 10% fibronectin containing protein solution compared to control (protein only). The epoxy-coated slides would consist the optimum platform to assess cell activation and potentially detect an allergic response.

Reacción tipo penfigoide por inmunoterapia alérgeno-específica: una reacción adversa inusual

For the most part, adverse reactions caused by subcutaneous allergen-specific immunotherapy (AIT) are directly induced by the administration of an allergen; there is usually redness and induration at the area of the injection. Other skin lesions are extremely unusual. A 40-year-old woman with allergic rhinoconjunctivitis and asthma due to sensitization to grass pollen had received monthly doses of 0.5mL of a subcutaneous polymerized extract of grass pollen with glutaraldehyde and adsorbed in aluminum hydroxide. Five hours after the seventh dose, the patient presented stinging and itching at the area of the injection of the immunotherapy, together with a blistering reaction that appeared later. Although adverse reactions by allergen-specific immunotherapy are rare and difficult to verify, it is important to perform the necessary analyses to identify the causes or their relation to the symptoms to avoid reactions in the future.

Sensitization to subtropical grass pollens in patients with seasonal allergic rhinitis from Bahia Blanca, Argentina

BackgroundGrass pollinosis is an important contributor to allergic diseases, with varying patterns and frequency of allergens according to the geographical location studied. Our study aims to provide a better understanding of subtropical grass pollinosis in Argentinian patients with seasonal allergic rhinitis. MethodsWe conducted a retrospective cross-sectional study involving 894 patients with seasonal allergic rhinitis from Bahía Blanca, Argentina. Skin prick tests were performed to selected pollen species belonging to three subfamilies of the Poaceae genera. Frequency of sensitization to specific grass pollen extracts, cross-reactivity of allergens assessed by skin prick test, and possible associations between allergen extracts and asthma or allergic conjunctivitis were analyzed. ResultsSensitization to the Pooideae subfamily was the most frequent, encompassing 86.8% (CI: 84.4%–88.9%) of the studied population. Positive reactions to allergen extracts from the Chloridoideae and the Panicoideae subfamilies showed smaller papule size than allergen extracts from the Pooideae subfamily (χ2(5) = 83.75, p < 0.001). Patients with a positive skin prick test (SPT) to a specific extract were more likely to present some degree of cross-reactivity to the remaining pollens when compared to patients with negative SPT to the same specific extract. Even though the proportion of patients presenting with asthma (46.9%) was higher than those with conjunctivitis (22.6%), there was only a statistically significant association between sensitization to Festuca arundinacea (φ = 0.089, p = .009), Phalaris arundinacea (φ = 0.074, p = .032) and Paspalum notatum (φ = 0.070, p = .038) and the presence of conjunctivitis. ConclusionsOur results suggest a high frequency of sensitization to grass pollen extracts from the Poaceae family among patients with seasonal allergic rhinitis. Overall, sensitization to the Pooidae subfamily was the most common, where Phalaris arundinacea presented the highest frequency.

Therapeutic efficacy of orally administered pollen for nonallergic diseases: An umbrella review.

Pollen has been used for centuries as a tonic and a multipurpose remedy in traditional medicine. The present umbrella review aims to qualitatively assess the therapeutic efficacy of orally administered pollen in the management of nonallergic diseases. MEDLINE via PubMed, Embase, Scopus, Web of Science, CINAHL, Cochrane Library, and Google Scholar were systematically searched for relevant systematic reviews and meta-analyses. Articles were independently screened and selected, then quality of evidence of included studies was evaluated with a dedicated NIH tool. Retrieved evidence was critically appraised and discussed. Two hundred four articles were found and, after selection process, five systematic reviews were included in the present work, including one with a meta-analysis. Evidence from these reviews supports the use of grass pollen extracts for symptomatic benign prostatic hyperplasia (BPH) and chronic prostatitis (CP). Additional preliminary evidence on the topic indicates the potential use of grass pollen extracts for vasomotor symptoms in women. Overall, results of the present review suggest that flower pollen extracts may be useful as a complementary remedy for the management of BPH, CP, and vasomotor symptoms. Evidence regarding bee pollen is too limited to draw any conclusion on its clinical efficacy. Further studies are needed.