Vaz-Rodrigues R

Is Zebrafish a Good Model for the Alpha-Gal Syndrome?

The alpha-Gal syndrome (AGS) is an underdiagnosed tick-borne allergy characterized by both immediate and delayed IgE-mediated anaphylactic reactions to the galactose-alpha-1,3-galactose (alpha-Gal) epitope. Common manifestations include gastrointestinal, cutaneous, and respiratory symptoms appearing 2-6 h after the consumption of mammalian meat or derived products. Zebrafish (Danio rerio) are emerging as essential animal models in biomedical studies, due to their anatomical, genetic, and physiological similarities to humans, with significant applications in toxicology, behavioral research, oncology, and inflammation studies. The mechanisms associated with AGS are sustained by studies in the humanized α1,3GalT-KO C57BL/6 mouse (Mus musculus) and zebrafish animal models for the production of anti-alpha-Gal antibodies in response to tick saliva, the development of allergic reactions in animals sensitized with tick protein extracts following mammalian meat consumption, and the identification of immune mechanisms. The immune mechanisms characterized in both models are associated with a skewed type 2 immune response, triggering Toll-Like receptor (TLR) signaling pathways, IL-4 production, and humoral activity. These results support the use of both models rather than a single one for a more comprehensive characterization of AGS-associated immune mechanisms. In this study, we focused on the use of zebrafish as a model for biomedicine research in immunity, infectious, and allergic diseases, with a particular emphasis on the AGS and the identification of candidate therapeutic interventions. Based on insights from multiple studies, we concluded that zebrafish is a suitable model for studying the AGS, considering the addressed limitations and in combination with the α1,3GalT-KO mouse model.

Tick salivary proteins metalloprotease and allergen-like p23 are associated with response to glycan α-Gal and mycobacterium infection.

The alpha-Gal syndrome (AGS) evolved as a catastrophic selection associated with anti-α-Gal IgM/IgG protective response against pathogen infection and tick-borne food allergy caused by IgE-type antibodies against this glycan present in glycoproteins and glycolipids from mammalian meat and derived products. The immune response to α-Gal is modulated by tick salivary proteins with and without α-Gal modifications in combination with tick saliva non-protein fraction. Herein, we characterized the role of tick salivary proteins, metalloprotease and allergen-like p23 in AGS and protection against tuberculosis in the AGS zebrafish animal model. Metalloprotease and p23 are involved in allergic reactions after mammalian meat consumption through upregulation of pro-inflammatory protein-coding genes prkdc, tlr2, tnfα and il1b. Challenge with Mycobacterium marinum activated Th1-mediated immune protective response with reduced pathogen infection, ameliorating Th2-associated allergic reactions associated with AGS. These results highlight molecular mechanisms modulated by tick proteins in response to α-Gal and provide insights to reduce AGS impact on human health.

Publisher Correction: Tick salivary proteins metalloprotease and allergen-like p23 are associated with response to glycan α-Gal and mycobacterium infection.

Allergic reactions associated with tsetse fly bites.

Allergic reactions in response to arthropod bites are a growing concern worldwide. Alpha-Gal syndrome (AGS) is a red meat allergy associated with IgE antibodies against galactose-alpha-1,3-galactose (alpha-Gal), typically linked to tick bites but other arthropods may also play a role. The objective of this study was to investigate the potential association between tsetse fly ( spp.) bites and AGS by analyzing clinical cases, citizen science data, and the possible production of alpha-Gal by tsetse flies. Data from five case series involving tsetse fly bites included demographic information such as age, gender and geographical location, as well as clinical details like symptoms and diagnosis. The production of alpha-Gal by tsetse flies focused on epitope content analysis by ELISA and the expression of several galactosyltransferases using RT-qPCR in the whole body of Ugandan tsetse flies and midgut tissues. The cases included patients bitten by tsetse flies in Uganda-Rwanda and, with different symptoms. Individuals resided in Spain, the USA or Uganda, with no record of or reaction to bites from ticks or other arthropods. Cases 1 and 2 were diagnosed with AGS, whereas Ugandan Case 3 presented symptoms consistent with AGS. Ugandan participative or citizen science survey identified one individual (Case 4) who experienced allergic reactions to red meat following multiple tsetse fly bites, while eight individuals (summarized as grouped Case 5) exhibited only local skin reactions to several bites. The expression of galactosyltransferases suggested the possible production of alpha-Gal modification in these arthropods. Allergic anaphylactic reactions to tsetse fly bites may occur in some individuals, potentially linking tsetse fly bites to AGS. Diagnosis and monitoring should consider factors beyond anti-alpha-Gal IgE antibody levels, such as arthropod salivary compounds and human immune biomarkers.

Public perspectives on tick bite exposure, healthcare visits and associated allergies in iberia.

Tick bites and tick-borne diseases (TBDs) are a worldwide concern, with growing evidence of an ongoing spread and emergence of new cases. This study applied a participatory citizen science across Spain and Portugal to gather public perspectives on the impact of tick bites, the presence of TBDs and a potential connection with the α-Gal syndrome (AGS). Data collected from the questionnaire (380 participants) was geographically represented using QGIS Geographic Information System and fitted into multiple generalized linear models (GLMs). Statistical analysis identified factors influencing the occurrence of local skin reactions post-tick bite, AGS-compatible symptoms and visits to health centers following tick exposure. Results showed that the probability of developing localized skin reactions to tick bites rises with increasing age ( = 0.006,  < 0.05), the occurrence of multiple bites ( = 0.006,  = 0.063) and among individuals bitten in the center-north part of the peninsula ( = 0.006,  = 0.058). Seeking medical care following tick bite was reported by 21.6% of respondents, being more common in first-time bite cases ( = 0.002, p < 0.01), men ( = 0.002,  < 0.01) or individuals presenting localized skin symptoms ( = 0.002,  < 0.05). Although 38 inquiries (10.0%) showed signs of AGS, only 3 had received a formal diagnosis, with the odds of developing this disease marginally rising with advancing age ( = 0.215,  = 0.066). This study provides valuable insights that should be considered to improve TBDs surveillance and diagnostic strategies, as well as developing preventive measures to reduce tick bite exposure and TBD cases.Developing local skin reactions to tick bites increases with age, multiple bites and in individuals bitten in central-northern Iberia.Seeking medical care following a tick bite is more common in first-time bite cases, men or individuals with local skin symptoms.Although 38 inquiries (10.0%) showed signs of AGS, only 3 were formally diagnosed, with risk increasing with age.

Transcriptomics insights into the functional role of tick Ixodes ricinus proteins metalloprotease and antigen p23.

Ixodes ricinus ticks (Acari: Ixodidae) are hematophagous ectoparasites and a major European vector of zoonotic diseases affecting global health. Tick salivary and midgut proteins antigen p23 (A0A0K8RKR7) and metalloprotease (A0A0K8RCY8) were previously implicated in the pathophysiology of the tick-borne allergy alpha-Gal syndrome (AGS). This study aimed to functionally characterize these two biomolecules, focusing on their role in I. ricinus tick feeding and reproduction through gene knockdown by RNA interference and midgut transcriptomic analysis. Validation of RNA-seq data was conducted using RT-qPCR analysis on tick midgut and salivary gland tissues. Silencing the expression of p23 and metalloprotease did not result in any significant differences in tick engorgement and egg batch weights compared to the control group. Gene set enrichment analysis following antigen p23 gene knockdown identified significantly upregulated pathways associated with protein production and suppressed routes mostly correlated with ion transport, lipid metabolism, catalytic activity, protein modification, and G-protein activity. Partial knockdown of the metalloprotease led to the upregulation of several biological and functional pathways associated with RNA splicing and significantly suppressed routes connected with detoxification, protein modification, catalytic activity and molecule binding. Antigen p23 appears to play a functional role in tick midgut cell homeostasis, primarily by participating in regulatory and signaling processes essential for cell viability. Metalloprotease is potentially involved in regulating midgut response to oxidative stress, thereby reducing tissue damage and promoting regular cell proliferation, growth and behavior. These results provide insights into tick physiology and bases for further research on tick-host interactions and AGS pathogenesis.

Tick salivary proteome and lipidome with low glycan content correlate with allergic type reactions in the zebrafish model.

Ticks, as hematophagous ectoparasites, can manipulate host immune and metabolic processes, causing tick-borne allergies such as α-Gal syndrome (AGS). Glycolipids with bound galactose-alpha-1-3-galactose (α-Gal) are potential allergenic molecules associated with AGS. Nevertheless, proteins and lipids lacking α-Gal modifications may contribute to tick salivary allergies and be linked to AGS. In this study, we characterized the effect of deglycosylated tick salivary proteins without lipids on treated zebrafish fed with dog food formulated with mammalian (beef, lamb, pork) meat by quantitative proteomics analysis of intestinal samples. The characterization and functional annotations of tick salivary lipids with low representation of glycolipids was conducted using a lipidomics approach. Results showed a significant effect of treatment with saliva and saliva deglycosylated protein fraction on zebrafish abnormal or no feeding (p < 0.005). Treatment with this fraction affected multiple metabolic pathways, defense responses to pathogens and protein metabolism, which correlated with abnormal or no feeding. Lipidomics analysis identified 23 lipid classes with low representation of glycolipids (0.70% of identified lipids). The lipid class with highest representation was phosphatidylcholine (PC; 26.66%) and for glycolipids it corresponded to diacylglycerol (DG; 0.48%). Qualitative analysis of PC antibodies revealed that individuals bitten by ticks were more likely to produce PC-IgG antibodies (p < 0.001). DG levels were significantly higher in tick salivary glands (p < 0.05) compared with tick saliva and salivary fractions. The α-Gal content was higher in tick saliva than in deglycosylated saliva and lipid fractions. These results support a possible role for tick salivary proteins and lipids without α-Gal modifications in AGS.

Zebrafish gut microbiota composition in response to tick saliva biomolecules correlates with allergic reactions to mammalian meat consumption.

The α-Gal syndrome (AGS) is an IgE-mediated tick borne-allergy that results in delayed anaphylaxis to the consumption of mammalian meat and products containing α-Gal. Considering that α-Gal-containing microbiota modulates natural antibody production to this glycan, this study aimed to evaluate the influence on tick salivary compounds on the gut microbiota composition in the zebrafish (Danio rerio) animal model. Sequencing of 16 S rDNA was performed in a total of 75 zebrafish intestine samples, representing different treatment groups: PBS control, Ixodes ricinus tick saliva, tick saliva non-protein fraction (NPF), tick saliva protein fraction (PF), and tick saliva protein fractions 1-5 with NPF (F1-5). The results revealed that treatment with tick saliva and different tick salivary fractions, combined with α-Gal-positive dog food feeding, resulted in specific variations in zebrafish gut microbiota composition at various taxonomic levels and affected commensal microbial alpha and beta diversities. Metagenomics results were corroborated by qPCR, supporting the overrepresentation of phylum Firmicutes in the tick saliva group, phylum Fusobacteriota in group F1, and phylum Cyanobacteria in F2 and F5 compared to the PBS-control. qPCRs results at genus level sustained significant enrichment of Plesiomonas spp. in groups F3 and F5, Rhizobium spp. in NPF and F4, and Cloacibacterium spp. dominance in the PBS control group. This study provides new results on the role of gut microbiota in allergic reactions to tick saliva components using a zebrafish model of AGS. Overall, gut microbiota composition in response to tick saliva biomolecules may be associated with allergic reactions to mammalian meat consumption in AGS.

Netosis and trained immunity in tick-borne diseases: a possible pathogenetic role.

Various types of pathogens transmitted by ticks elicit distinct immune responses just like the emerging α-Gal syndrome that is associated with allergic reactions to tick bites. The mechanisms of Neutrophil Extracellular Traps release (called NETosis) and trained immunity in response to tick-borne microbes have not been extensively investigated. In our paper, we explored the intricate interplay of NETosis and trained immunity within the realm of infectious diseases triggered by tick bites and their possible pathogenetic role in autoimmunity. We conducted an extensive literature search to identify studies for this review, considering articles and reviews published in English within the last years. Additionally, we scrutinized the references of all included papers and relevant review articles to ensure comprehensive coverage. We shed light on a plausible correlation between these innate immune responses and their potential implication in certain pathological conditions, with a specific focus on some autoimmune diseases. These findings offer new perspectives for a more profound comprehension of the immunopathogenesis of certain autoimmune-like signs where clinicians should include Tick-Borne Diseases (TBDs) in their differential diagnoses, in those geographical areas of tick infestation.

Citizen Science on Lyme Borreliosis in Spain Reveals Disease-Associated Risk Factors and Control Interventions.

Lyme borreliosis (LB) caused by lato complex spirochetes is one of the tick-borne diseases with high prevalence and social/economic burden in the United States, Spain, and other European countries. The objective is to address limited information available about the incidence, prevalence, and symptoms of LB, current prevention, and treatment interventions that are not adequately focused and thus not very effective against this disease. To address these limitations, in this study, we used a citizen science approach to evaluate the LB-associated risks and implementation of control interventions in Spain. A total of 405 participants in the survey were included in the analysis. Responses to the questionnaire were received during January-July 2022. The questionnaire contained qualitative and quantitative questions. Homogeneity among binary variables was analyzed using a Fisher's exact test. Despite limitations of the study associated with response to the questionnaire and information on tick species, the results evidenced the effect of factors such as age, gender, tick bites, disease clinical signs, comorbidities such as alpha-gal syndrome, health care services, and treatment effectiveness affecting LB. The main conclusions of the study highlight the need for better surveillance of tick infestations, pathogen infection, and diagnosis of LB and related comorbidities. To advance in disease prevention, diagnosis, and treatment, new interventions need to be developed and implemented in both public and private health care services. Providing access to these results to the society, health care system, and scientists is important to further advance in disease surveillance, diagnosis, control, and prevention.

Allergic reactions to tick saliva components in zebrafish model.

Alpha-Gal syndrome (AGS) is a tick-borne food allergy caused by IgE antibodies against the glycan galactose-alpha-1,3-galactose (α-Gal) present in glycoproteins and glycolipids from mammalian meat. To advance in the diagnosis and treatment of AGS, further research is needed to unravel the molecular and immune mechanisms underlying this syndrome. The objective of this study is the characterization of tick salivary components and proteins with and without α-Gal modifications involved in modulating human immune response against this carbohydrate. Protein and α-Gal content were determined in tick saliva components, and proteins were identified by proteomics analysis of tick saliva fractions. Pathophysiological changes were recorded in the zebrafish (Danio rerio) model after exposure to distinct Ixodes ricinus tick salivary components. Serum samples were collected from zebrafish at day 8 of exposure to determine anti-α-Gal, anti-glycan, and anti-tick saliva protein IgM antibody titers by enzyme-linked immunosorbent assay (ELISA). Zebrafish treated with tick saliva and saliva protein fractions combined with non-protein fractions demonstrated significantly higher incidence of hemorrhagic type allergic reactions, abnormal behavioral patterns, or mortality when compared to the phosphate-buffered saline (PBS)-treated control group. The main tick salivary proteins identified in these fractions with possible functional implication in AGS were the secreted protein B7P208-salivary antigen p23 and metalloproteases. Anti-α-Gal and anti-tick salivary gland IgM antibody titers were significantly higher in distinct saliva protein fractions and deglycosylated saliva group when compared with PBS-treated controls. Anti-glycan antibodies showed group-related profiles. Results support the hypothesis that tick salivary biomolecules with and without α-Gal modifications are involved in modulating immune response against this carbohydrate.

Serum biomarkers for nutritional status as predictors in COVID-19 patients before and after vaccination.

The aim of this study was to characterize serum protein biomarkers for nutritional status that may be used as predictors for disease symptomatology in COVID-19 patients before and after vaccination. In pre-vaccine cohorts, proteomics analysis revealed significant differences between groups, with serum proteins alpha-1-acid glycoproteins (AGPs) 1 and 2, C-reactive protein (CRP) and retinol binding protein (RBP) increasing with COVID-19 severity, in contrast with serum albumin, transthyretin (TTR) and serotransferrin (TF) reduction as the symptomatology increased. Immunoassay reproduced and validated proteomics results of serum proteins albumin and RBP. In post-vaccine cohorts, the results showed the same pattern as in pre-vaccine cohorts for serum proteins AGPs, CRP, albumin and TTR. However, TF levels were similar between groups and RBP presented a slight reduction as COVID-19 symptomatology increased. In these cohorts, immunoassay validated proteomics results of serum proteins albumin, TTR and TF. Additionally, immune response to α-Gal in pre-vaccine cohorts varied in predominant immunoglobulin type profile, while post-vaccine groups presented mainly anti-α-Gal protective IgG antibodies. The study identified serum nutritional biomarkers that could potentially predict an accurate prognostic of COVID-19 disease to provide an appropriate nutritional care and guidance in non-vaccinated and vaccinated individuals against SARS-CoV-2. These results highlight the importance of designing personalized nutrition protocols to improve diet along with the application of prebiotics or probiotics for the control of COVID-19 and other infectious diseases.

Multi-omics analysis of zebrafish response to tick saliva reveals biological processes associated with alpha-Gal syndrome.

The alpha-Gal syndrome (AGS) is a tick-borne allergy. A multi-omics approach was used to determine the effect of tick saliva and mammalian meat consumption on zebrafish gut transcriptome and proteome. Bioinformatics analysis using R software was focused on significant biological and metabolic pathway changes associated with AGS. Ortholog mapping identified highly concordant human ortholog genes for the detection of disease-enriched pathways. Tick saliva treatment increased zebrafish mortality, incidence of hemorrhagic type allergic reactions and changes in behavior and feeding patterns. Transcriptomics analysis showed downregulation of biological and metabolic pathways correlated with anti-alpha-Gal IgE and allergic reactions to tick saliva affecting blood circulation, cardiac and vascular smooth muscle contraction, behavior and sensory perception. Disease enrichment analysis revealed downregulated orthologous genes associated with human disorders affecting nervous, musculoskeletal, and cardiovascular systems. Proteomics analysis revealed suppression of pathways associated with immune system production of reactive oxygen species and cardiac muscle contraction. Underrepresented proteins were mainly linked to nervous and metabolic human disorders. Multi-omics data revealed inhibition of pathways associated with adrenergic signaling in cardiomyocytes, and heart and muscle contraction. Results identify tick saliva-related biological pathways supporting multisystemic organ involvement and linking α-Gal sensitization with other illnesses for the identification of potential disease biomarkers.

Allergic reactions associated with medically relevant arthropods.

Vector-borne diseases are a growing burden worldwide. In particular, the risks of allergic reactions to bites are associated with growing arthropod populations in contact with the public. The diversity of allergic reactions associated with host and arthropod factors difficult disease diagnosis, prognosis and prevention. Therefore, arthropod-associated allergies are underdiagnosed and require better surveillance of arthropod populations and disease diagnosis and management. To face these challenges, in this study, we describe five cases to illustrate arthropod-associated allergies with different symptomatology, including alpha-gal syndrome (AGS) associated with anti-alpha-gal IgE antibody titres. Information on symptoms in response to arthropod bites was collected from patients and medical doctors. The five cases included patients bitten by a robber fly and different tick species. Cases were in Spain or U.S.A. Two cases were diagnosed with AGS and one case was diagnosed with anaphylaxis in response to tick bite with high anti-alpha-gal IgE levels. The symptoms in response to arthropod bites vary between different cases. Allergic reactions and symptoms in response to arthropod bites vary in association with host and arthropod factors. Herein we propose recommendations to control allergic symptoms, associated disease risk factors and the way forward to advance in the prevention and control of arthropod-associated allergies.

Current and Future Strategies for the Diagnosis and Treatment of the Alpha-Gal Syndrome (AGS).

The α-Gal syndrome (AGS) is a pathognomonic immunoglobulin E (IgE)-mediated delayed anaphylaxis in foods containing the oligosaccharide galactose-α-1,3-galactose (α-Gal) such as mammalian meat or dairy products. Clinical presentation of AGS can also comprise immediate hypersensitivity due to anticancer therapy, gelatin-containing vaccines or mammalian serum-based antivenom. The IgE initial sensitization is caused by hard-bodied tick bites and symptomatic individuals typically develop delayed pruritus, urticaria, angioedema, anaphylaxis, malaise or gut-related symptoms. Due to inapparent presentation, delayed reactions and a wide variety of patients´ clinical history, the AGS diagnosis and treatment remain challenging. This review covers not only current diagnostic methods used for AGS such as the skin prick test (SPT), the oral food challenge (OFC), anti-α-Gal IgE levels measurement and the basophil activation test (BAT), but also potentially relevant next-generation diagnostic tools like the mast cell activation test (MAT), the histamine-release (HR) assay, omics technologies and model-based reasoning (MBR). Moreover, it focuses on the therapeutical medical and non-medical methods available and current research methods that are being applied in order to elucidate the molecular, physiological and immune mechanisms underlying this allergic disorder. Lastly, future treatment and preventive tools are also discussed, being of utmost importance for the identification of tick salivary molecules, with or without α-Gal modifications, that trigger IgE sensitivity as they could be the key for further vaccine development. Bearing in mind climate change, the tick-host paradigm will shift towards an increasing number of AGS cases in new regions worldwide, which will pose new challenges for clinicians in the future.