Canine leishmaniasis (CanL), caused by Leishmania infantum , is a chronic zoonotic disease characterized by a strong humoral immune response that is typically associated with an inability to control infection, leading to progression to severe symptomatic disease. The exacerbated response leads to overproduction of antibodies, which often occurs in the presence of high antigen load, leading to the formation of circulating immune complexes (CIC). These CIC are central to the immunopathogenesis of CanL, as their deposition in tissues contributes to vasculitis, uveitis, dermatitis, glomerulonephritis, and renal failure. Quantification of CIC by a specific ELISA has shown that dogs with advanced clinical signs exhibit significantly higher CIC concentrations in their serum than asymptomatic or early-stage cases. Recent studies have demonstrated a positive correlation between CIC levels and disease severity, suggesting their potential as biomarkers for diagnosis, staging, and monitoring treatment responses in the management of CanL. Beyond their levels, the protein composition of CIC may also reflect the stage of the disease. Therefore, the aim of this study was to characterize the protein composition of CIC and evaluate its association with disease severity. The results have demonstrated that CIC composition varies according to disease stage in dogs with CanL, reflecting the immune status of the host.

A functional allergen quantification kit requires two key components: a pair of monoclonal antibodies (mAbs) that do not interfere with each other at the protein binding site, and a reference standard with sufficient purity and stability. Despite the clinical relevance of Jun a 1 in patients with Cupressaceae pollinosis, no commercial kits are currently available for its quantification in allergenic extracts. The aim of this study was to develop and validate a sandwich ELISA kit for the quantification of Jun a 1 in Juniperus ashei extracts, using appropriately selected mAbs and a stable standard. To this end, Jun a 1 was purified to >95% purity, as confirmed by SDS-PAGE, Western blot, and mass spectrometry analysis. Its stability was demonstrated after 48 months of storage. The linear B-cell epitopes recognized by the mAbs were identified and mapped onto the Jun a 1 structure, confirming that they are non-overlapping. Following validation, the ELISA method proved to be specific, linear, accurate, and precise. In conclusion, we successfully developed and validated an ELISA assay for the quantification of Jun a 1 in J. ashei extracts, supported by the generation of suitable monoclonal antibodies and a stable reference standard.

BACKGROUND AND OBJECTIVES:Defining a patient´s allergic profile by identifying the allergens that cause symptoms is essential for the design of specific, tailored immunotherapy, ensuring greater effectiveness in cases of polyallergy. Objective: This observational, multicenter study aimed to demonstrate the heterogeneity of the Spanish population with respiratory allergy by analyzing specific immunoglobulin E (sIgE) patterns for aeroallergens and exploring the relationship between sensitization and clinical symptoms. PATIENTS AND METHODS:A total of 474 patients with respiratory allergy from different regions of Spain were recruited based on their case histories and skin prick test data for aeroallergens. Serum samples were analyzed using ImmunoCAPTM assays to determine sIgE levels for 33 allergenic sources and 43 molecular allergens. RESULTS:Ninety percent of patients were polysensitized. Pollen was the most common cause of sensitization, followed by mites. Interestingly, polysensitization was significantly associated with the severity of rhinitis symptoms. The molecular diagnosis revealed Phl p 1 as the most prevalent pollen sensitizer. Sensitization to mites was mainly driven by Der p 2. Fel d 1 and Alt a 1 were the most frequently recognized allergens among patients sensitized to animal epithelia and molds, respectively. Moreover, sensitization to mite and animal epithelia allergens was significantly associated with more severe asthma symptoms. CONCLUSIONS:Our findings show a high prevalence of polysensitization and highlight the importance of molecular diagnosis for improving diagnostic accuracy and designing immunotherapy tailored to patients in the region.

Circulating immune complexes (CIC) cause different organ lesions in canine leishmaniosis. This study aimed to measure CIC in dogs infected with Leishmania (L.) infantum in a non-endemic country and to analyze associations with disease parameters. Measurement of Leishmania-specific CIC was performed by a polyethylene glycol ELISA (cut-off: 0.274 optical density (OD)) every three months during a one-year study period in 52 L. infantum-infected dogs. Each appointment included a physical examination, complete blood count, serum biochemistry including C-reactive protein, urinalysis, L. infantum PCR and antibody ELISA. Statistical analyses included Mann-Whitney U tests, a multivariable robust linear regression, Spearman correlation, univariable logistic regression, and a receiver operating characteristic (ROC) curve. CIC levels differed significantly between dogs with and without lymphadenopathy (p < 0.01; β=-0.38; 95% CI: -0.52, -0.24), uveitis (p < 0.01; β=-0.52; 95% CI: -0.89, -0.15), seborrhea/hypotrichosis (p = 0.04; β=-0.11; 95% CI: -0.21, -0.01), and skin nodules (p < 0.01; β = 0.22; 95% CI: 0.13, 0.31). Significant moderate to strong correlations with CIC levels were found for L. infantum antibodies (p < 0.01; rs = 0.65; 95% CI: 0.57, 0.72), globulin (p < 0.01; rs = 0.60; 95% CI: 0.51, 0.68), albumin-to-globulin ratio (p < 0.01; rs=-0.56; 95% CI: -0.65, -0.47) and total protein (p < 0.01; rs = 0.46; 95% CI: 0.35, 0.56). CIC levels were significantly higher in dogs with positive lymph node PCR (p = 0.04) as well as in dogs with disease relapses (p < 0.01). The ROC curve analysis revealed one highly specific cut-off value at 1.668 OD for differentiating between dogs with and without disease relapse (98% specificity; 60% sensitivity). Measurement of Leishmania-specific CIC is useful for monitoring dogs with L. infantum infections and for indicating disease relapse.

Background: Most patients with respiratory allergy are polyallergic. Combining different allergen extracts in the same allergen-specific immunotherapy is a common practice. However, it should be justified. Objective: To analyze the stability, safety, and immune response of allergen extract mixtures from nonhomologous groups. Methods: We analyzed 2 depigmented-polymerized mixture extracts (DPmixEs): cat dander–grass pollen and Alternaria alternata–grass pollen. The stability of the mixtures was investigated by studying proteolysis and degradation effects. The allergenicity and humoral and cellular immune responses of DPmixEs were also evaluated using various technical approaches, including the Bradford assay, enzyme-linked immunosorbent assay, rabbit immunization, peripheral blood mononuclear cell culture, and flow cytometry. The results were compared with those of individual depigmented-polymerized extracts (DPEs) and native mixture extracts (NmixEs). Results: The proteolytic activity of DPmixEs was lower than that of NmixEs. The protein content of DPmixEs remained stable for 18 months, whereas that of NmixEs decreased significantly during the first month. The allergenicity of DPmixEs was similar to that of DPEs and lower than that of NmixEs. Regarding the immune response, DPmixEs induced functional specific IgG antibodies in rabbits and blocked sIgEallergen binding. Moreover, DPmixEs induced IL-10 secretion in peripheral blood mononuclear cells from polyallergic patients, improving the Treg/TH2 cell balance. Conclusion: These findings support the use of DPmixE as a promising formulation for allergen immunotherapy, combining stability, reduced enzymatic activity, and enhanced immunological stimulation, while preserving in vitro safety and efficacy comparable to separated depigmented-polymerized extracts.

BACKGROUND:Cupressaceae pollen increasingly causes respiratory allergies worldwide. Carbohydrates are abundant in extracts of these pollens, and the associated allergens are highly glycosylated. However, the contribution of saccharides to the allergenicity of these species remains unknown. METHODS:Juniperus ashei pollen extract was deglycosylated and characterised using SDS-PAGE and immunoblotting. Additionally, N- and O-glycans were purified from the extract, identified, used as inhibitors in IgE-immunoblotting and further analysed via basophil activation tests. The interactions between IgE and J. ashei glycans were analysed using a glycan array. Purified Jun a 1 was treated with β-N-acetylglucosaminidase S and analysed using immunoblotting. The native pollen extract was used to immunise rabbits, and the IgG response was analysed using ELISA and glycan array. RESULTS:Deglycosylation of J. ashei proteins abolished the interaction between IgE and allergens. This effect primarily depends on N-glycans. Purified N-glycans triggered basophil activation in some patients. A biantennary N-glycan with terminal GlcNAc, β-1,2 xylose and core α-1,3 fucose (GnGnXF3) was the most abundant glycan identified. The glycan array confirmed its interaction with IgE. The contribution of terminal N-acetylglucosamines (GlcNAc) to IgE-Jun a 1 interaction was validated. Moreover, effective immunisation of rabbits with the native extract confirmed the immunogenicity of their N-glycans. CONCLUSIONS:The IgE-J. ashei allergen interaction is broadly controlled through N-glycans different from MUXF3. GnGnXF3 exerts an immunogenic effect in humans and rabbits; terminal GlcNAc residues influence its recognition by IgE. These discoveries reinforce the role of N-glycans in the allergic response to J. ashei.

INTRODUCTION:Allergen immunotherapy is the only modifying treatment of the natural course of respiratory allergic diseases; however, the lack of evidence leads to little inconclusive results. Real life studies are on the rise and are becoming a valuable tool to confirm and complement findings from clinical trials. The objective of this study was to evaluate the effectiveness and safety of a depigmented-polymerized undiluted subcutaneous extract of grass and olive pollen, under routine clinical practice conditions. METHODS:This was an observational, retrospective, longitudinal, single-center study on the use of a 2-pollen (grass mix and Olea europaea) undiluted subcutaneous extract over at least 3 consecutive years. Data were collected from 76 patients (n = 44 female; median age: 12.5 years old) diagnosed with allergic rhinoconjunctivitis with/without allergic asthma due to sensitization to both grasses and O. europaea. Primary and secondary effectiveness endpoints were symptom severity, concomitant medication, and immunological profile before and after completing the immunotherapy. A 2-year follow-up of patients' symptoms and medication history after completing the subcutaneous immunotherapy (SCIT) was performed. RESULTS:There was a significant improvement of symptoms and medication consumption after 3 years of SCIT treatment, and a significant decrease in specific IgE levels for grasses and O. europaea was observed after finishing the treatment. CONCLUSION:Three years treatment of allergic patients using an undiluted mixture of two allergen extracts was shown to be safe and effective for rhinitis and asthma, with efficacy maintained for at least 2 years after finishing SCIT. These results reinforce the importance of real life clinical data in addition to those from clinical trials, helping to individualize allergic treatments.

BACKGROUND:Studying the sensitisation profiles of patients with allergies allows for a deeper understanding of the disease which may facilitate the selection of the best-personalised allergen immunotherapy. This observational, cross-sectional, multicentre study aimed to demonstrate the heterogeneity of the German population with allergies by analysing specific immunoglobulin E (sIgE) patterns towards aeroallergens and exploring the relationship between sensitisation and clinical symptoms. METHODS:In total, 500 patients with allergies from different regions of Germany were recruited based on their case histories, clinical allergic symptoms and skin prick test data for aeroallergens. Serum samples were analysed using ImmunoCAP assays to determine sIgE levels for 33 allergenic sources and 43 molecular allergens. RESULTS:Most patients (81%) were polysensitised. Betula verrucosa pollen was the most common cause of sensitisation (59%), followed by Phleum pratense (58%) and Dermatophagoides pteronyssinus (44%). The highest prevalence rates of molecular allergens were observed for Bet v 1 (84%) from birch pollen, Phl p 1 from grass pollen (82%), Der p 2 (69%) from mites and Fel d 1 (69%) from cat. Polysensitisation was significantly associated with the presence of asthma and the severity of rhinitis symptoms. CONCLUSIONS:Our findings show a high rate of polysensitisation and emphasise the importance of molecular diagnosis for more precise and comprehensive insights into sensitisation patterns and their association with clinical symptoms. These data may help improve personalised diagnosis and immunotherapy adapted to the needs of individual patients in the region.

Aerobiological studies constitute a relevant tool to predict the most influential parameters over the pollen seasons with significant clinical relevance in the allergic populations. The aim of this study was to describe the aerobiological behaviour of the most relevant allergenic sources in the semi-arid area of southeast of Spain (Almería) and to investigate the correlation with meteorological factors and clinical symptoms of allergic patients. Daily pollen count and meteorological parameters of Almería, Spain, were compiled for ten years. The clinical symptoms of 248 allergic patients were also recorded. Descriptive statistics and correlations between variables were assessed. Multivariate analyses were performed to predict the influence of meteorological factors on pollen concentration and the risk of suffer respiratory symptoms. Eight pollen families were identified as the most relevant allergenic sources. Temperature correlated with main pollen season evolution of all taxa whereas rainfall and relative humidity only correlated in Oleaceae, Pinaceae, Amaranthaceae, Asteraceae and Urticaceae. Rainfall and relative humidity were the most influential predictors of pollen concentration, except in Amaranthaceaea and Poaceae families, while temperature only influenced on Cupressaceae and Urticaceae pollen concentrations. A significant positive influence was observed between maximum temperature and rainfall with the appearance of allergic symptoms in patients sensitized to grasses, Parietaria sp. and Olea sp. This study, highlight the main aerobiological features in the region and establish a suitable tool for clinical follow-up and management of allergic patients. Further studies are needed to establish an accurate measurement aimed to control and prevent pollinosis in sensitized patients.

Eight different allergens have been identified until now. However, IgE epitopes remains unknown and the patter of sensitization of allergic patients has not been fully investigated. This knowledge could provide valuable information to develop more efficient AIT.

BACKGROUND AND OBJECTIVE:Peach allergy is a prevalent cause of food allergy. Despite the repertoire of allergens available for molecular diagnosis, there are still patients with undetectable IgE levels to peach allergens but presenting symptoms after its ingestion. The objective of this study was to investigate the allergenic profile in a patient population with symptoms produced by peach. METHODS:An exploratory retrospective study was performed with patients presenting symptoms after the ingestion of peach. Forty-two patients were included in the study. The allergenic profile of individual patients was investigated by immunoblot. A serum pool was prepared with the sera that recognized a 70 kDa band. This pool was used to detect this protein in peach peel and pulp and to identify the 70 kDa protein in 2D immunoblot. Spots recognized in the 2D immunoblot were sequenced by LC-MS/MS. Inhibition studies were performed between peach peel and almond. RESULTS:Twenty-two patients (52.4%) recognized the 70 kDa protein in immunoblot. This protein was recognized in peel and pulp. Two different spots were observed in 2D-PAGE, both were identified as (R)-mandelonitrile lyases (RML) with high amino acid similarity with Pru du 10. Peach RML were partially inhibited with an almond extract. No association was found between any reported symptom and sensitization to RML. RML-sensitized patients were older and reported pollen associated respiratory symptoms more frequently than negative patients. CONCLUSION:A new peach allergen, a RML, homologous of Pru du 10, recognized by 52% of the population has been identified.

IntroductionCanine leishmaniosis (CanL) is a systemic disease that affects dogs. When multiplication of the parasite cannot be controlled, dogs consistently show high levels of antigen and IgG antibodies, which lead to the formation of circulating immune complexes (CIC). Timely intervention to reduce the parasite load and CIC levels is crucial for preventing irreversible organ damage. However, a diagnostic test to quantify CIC levels is currently lacking.MethodsIn this real-world study, we aimed to examine the performance of a new ELISA to measure CIC levels in dogs naturally infected with Leishmania infantum. Thirty-four dogs were treated according to their clinical condition and followed for 360 days. Before (day 0) and after treatment (days 30, 90, 180, 270, and 360), all dogs underwent a physical examination, and blood samples were obtained for CBC, biochemical profile, serum protein electrophoresis and IFAT. Serum PEG-precipitated CIC were determined by ELISA.ResultsOur results indicate higher CIC levels in dogs in advanced disease stages showing higher antibody titres (p < 0.0001, r = 0.735), anemia (p < 0.0001), dysproteinemia (p < 0.0001), and proteinuria (p = 0.004). Importantly, dogs responding well to treatment exhibited declining CIC levels (p < 0.0001), while in poor responders and those experiencing relapses, CIC were consistently elevated. CIC emerged as a robust discriminator of relapse, with an area under the curve (AUC) of 0.808. The optimal cut-off to accurately identify relapse was an optical density of 1.539.DiscussionOur findings suggest that declining CIC levels should be expected in dogs showing a favorable treatment response. Conversely, in dogs displaying a poor response and recurrent clinical relapses, CIC levels will be high, emphasizing the need for vigilant monitoring. These findings suggest that CIC could serve as a valuable biomarker for disease progression, treatment efficacy, and relapse detection in CanL. Our study contributes to enhancing diagnostic approaches for CanL and underscores the potential of CIC as a complementary tool in veterinary practice. As we move forward, larger studies will be essential to confirm these findings and establish definitive cut-offs for clinical application.