Search for biomarkers related to rhinitis and different asthma phenotypes by serum proteomics and immunoassays

sthma is a heterogeneous disease with several clinical phenotypes and molecular endotypes. However, the specific connection between asthma phenotypes and the underlying pathological features is difficult to explain. Thus, the overall aim of this thesis was to search for biomarkers associated with rhinitis and different phenotypes (allergic and non-allergic) and severities (intermittent-mild and moderate-severe) of asthma. To achieve this aim, we studied the role of the immune system through the analysis of certain biomarkers previously related to this disease: CD14 (innate immune system) and CD26/CD126 (adaptive immune system). In addition, in the second part of the present thesis, a prospective, non-target proteomic study aimed to identify new biomarkers associated with different phenotypes of this disease was also performed. This study consisted of the analysis of low abundance serum proteome through iTRAQ-LC-MS/MS and allowed us the identification of several potential biomarkers for allergic (e.g., IGFALS, protein AMBP, or HSPG2) and non-allergic asthma (e.g., CFI), as well as disease severity (e.g., IGFALS)

Imiquimod shows anti-viral actions in human bronchial epithelium - implications for COVID-19 treatment

Introduction: Combining anti-viral and anti-inflammatory effects in a single drug may be beneficial in treating COVID-19. We hypothesized that the TLR7 agonist imiquimod (imq) may exert these actions in human bronchial epithelial cells (HBECs), which are targets in SARS-CoV-2 mediated lung injury. Methods: Using primary HBECs from asthmatic donors (N=18), we explored actions of imq related to airway viral resistance and tolerance. HBECs were treated with imq alone or in combination with the viral mimic poly (I:C) or the SARS-CoV-2 spike protein 1 (SP1). Anti-viral and pro-inflammatory mediators were analyzed by Luminex, RT-qPCR and mRNA gene pathway analysis. Results: imq treatment alone induced IFN-ß and CCL5 (p<0.05) mRNA and reduced transcription of IL-1ß (p<0.01) at 24h. In SP1 or poly (I:C) stimulated HBECs, treatment with imq augmented IFN-ß mRNA expression by a 2-fold, respectively (p<0.05). Imq in combination with poly (I:C) decreased protein release of IL-8, CCL5, IL-1ß and IL-6 (p<0.05). Furthermore, gene pathway analysis revealed that imq enriched poly(I:C)-induced IFN signaling, IL-20 family signaling (epithelial repair), antigen presentation and cytokine signaling. Enriched cytokine signaling genes included negative regulators such as IKBKG and SIGIRR. Conclusion: imq exerts distinct anti-viral resistance effects in HBECs by increasing anti-viral signaling and improves viral infection tolerance by diminishing epithelial cytokines potentially involved in severe COVID-19. Our findings highlight a possibility of developing dual action drugs suitable for anti-SARS-CoV-2 treatment

Expansion of different subpopulations of CD26−/low T cells in allergic and non-allergic asthmatics

Asthma; Allergy; CD26Asma; Al·lèrgia; CD26Asma; Alergia; CD26CD26 displays variable levels between effector (TH17 ≫ TH1 > TH2 > Treg) and naïve/memory (memory > naïve) CD4+ T lymphocytes. Besides, IL-6/IL-6R is associated with TH17-differentiation and asthma severity. Allergic/atopic asthma (AA) is dominated by TH2 responses, while TH17 immunity might either modulate the TH2-dependent inflammation in AA or be an important mechanism boosting non-allergic asthma (NAA). Therefore, in this work we have compared the expression of CD26 and CD126 (IL-6Rα) in lymphocytes from different groups of donors: allergic (AA) and non-allergic (NAA) asthma, rhinitis, and healthy subjects. For this purpose, flow cytometry, haematological/biochemical, and in vitro proliferation assays were performed. Our results show a strong CD26-CD126 correlation and an over-representation of CD26- subsets with a highly-differentiated effector phenotype in AA (CD4+CD26-/low T cells) and NAA (CD4-CD26- γδ-T cells). In addition, we found that circulating levels of CD26 (sCD26) were reduced in both AA and NAA, while loss of CD126 expression on different leukocytes correlated with higher disease severity. Finally, selective inhibition of CD26-mRNA translation led to enhanced T cell proliferation in vitro. These findings support that CD26 down-modulation could play a role in facilitating the expansion of highly-differentiated effector T cell subsets in asthma

C57Bl/6N mice have an attenuated lung inflammatory response to dsRNA compared to C57Bl/6J and BALB/c mice

Background Lower respiratory infections caused by ssRNA viruses are a major health burden globally. Translational mouse models are a valuable tool for medical research, including research on respiratory viral infections. In in vivo mouse models, synthetic dsRNA can be used as a surrogate for ssRNA virus replication. However, studies investigating how genetic background of mice impacts the murine lung inflammatory response to dsRNA is lacking. Hence, we have compared lung immunological responses of BALB/c, C57Bl/6N and C57Bl/6J mice to synthetic dsRNA. Methods dsRNA was administered intranasally to BALB/c, C57Bl/6N and C57Bl/6J mice once/day for three consecutive days. Lactate dehydrogenase (LDH) activity, inflammatory cells, and total protein concentration were analyzed in bronchoalveolar lavage fluid (BALF). Pattern recognition receptors levels (TLR3, MDA5 and RIG-I) were measured in lung homogenates using RT-qPCR and western blot. Gene expression of IFN-β, TNF-α, IL-1β and CXCL1 was assessed in lung homogenates by RT-qPCR. ELISA was used to analyze protein concentrations of CXCL1 and IL-1β in BALF and lung homogenates. Results BALB/c and C57Bl/6J mice showed infiltration of neutrophils to the lung, and an increase in total protein concentration and LDH activity in response to dsRNA administration. Only modest increases in these parameters were observed for C57Bl/6N mice. Similarly, dsRNA administration evoked an upregulation of MDA5 and RIG-I gene and protein expression in BALB/c and C57Bl/6J, but not C57Bl/6N, mice. Further, dsRNA provoked an increase in gene expression of TNF-α in BALB/c and C57Bl/6J mice, IL-1β only in C57Bl/6N mice and CXCL1 exclusively in BALB/c mice. BALF levels of CXCL1 and IL-1β were increased in BALB/c and C57Bl/6J mice in response to dsRNA, whereas the response of C57Bl/6N was blunt. Overall, inter-strain comparisons of the lung reactivity to dsRNA revealed that BALB/c, followed by C57Bl/6J, had the most pronounced respiratory inflammatory responses, while the responses of C57Bl/6N mice were attenuated. Conclusions We report clear differences of the lung innate inflammatory response to dsRNA between BALB/c, C57Bl/6J and C57Bl/6N mice. Of particular note, the highlighted differences in the inflammatory response of C57Bl/6J and C57Bl/6N substrains underscore the value of strain selection in mouse models of respiratory viral infections

Expansion of different subpopulations of CD26 −/low T cells in allergic and non-allergic asthmatics

CD26 displays variable levels between effector (TH ≫ TH > TH > Treg) and naïve/memory (memory > naïve) CD4 T lymphocytes. Besides, IL-6/IL 6R is associated with TH -differentiation and asthma severity. Allergic/atopic asthma (AA) is dominated by TH responses, while TH immunity might either modulate the TH -dependent inflammation in AA or be an important mechanism boosting non-allergic asthma (NAA). Therefore, in this work we have compared the expression of CD26 and CD126 (IL-6Rα) in lymphocytes from different groups of donors: allergic (AA) and non-allergic (NAA) asthma, rhinitis, and healthy subjects. For this purpose, flow cytometry, haematological/biochemical, and in vitro proliferation assays were performed. Our results show a strong CD26-CD126 correlation and an over-representation of CD26 subsets with a highly-differentiated effector phenotype in AA (CD4 CD26 T cells) and NAA (CD4 CD26 γδ-T cells). In addition, we found that circulating levels of CD26 (sCD26) were reduced in both AA and NAA, while loss of CD126 expression on different leukocytes correlated with higher disease severity. Finally, selective inhibition of CD26-mRNA translation led to enhanced T cell proliferation in vitro. These findings support that CD26 down-modulation could play a role in facilitating the expansion of highly-differentiated effector T cell subsets in asthma

Association between blood eosinophil count with asthma hospital readmissions

This is an Accepted Manuscript of an article published in European Journal of Internal Medicine on 2018, available at: https://doi.org/10.1016/j.ejim.2018.02.034Introduction: The presence of eosinophils in asthma inflammation is a relevant factor in the pathophysiology of the disease, however the relationship between the blood eosinophil count (BEC) with asthma severity and prognosis is still under debate. The aim of this work is to analyze the relationship between the BEC levels and hospital readmissions in patients with asthma. Material and methods: A review was retrospectively carried out on all admissions of patients over 18 years old due to exacerbation of asthma occurring in our hospital between the years 2000 and 2010. The personal characteristics and the asthma personal history of each patient were recorded. The BEC was determined from the first blood sample taken from the patient after their arrival at the hospital. Hospital early, late and frequent readmissions were analyzed using 4 cut-off points; less than 150 eosinophils/μL vs ≥150/μL, less than 200 vs 200 /μL, less than 300 vs ≥300/μL, and less than 400 vs ≥400/μL. Results: We have included 1316 patients, 70% of whom are women, as well as a mean age of 60 years, and a mean FEV1 of 73.5% of the reference value. The mean eosinophil blood count was 201.7 cells/μL. A BEC ≥300 cells/μL showed a reduction of risk of late readmission of 42%, a BEC ≥400 cells/μL showed a reduction in late readmission risk of 41% and decrease in frequent late readmission of 63%. Conclusions: Our study appears to support that an elevated BEC is associated with a lower incidence of asthma hospital readmissions.S

Expansion of different subpopulations of CD26−/low T cells in allergic and non-allergic asthmatics

CD26 displays variable levels between effector (TH17 ≫ TH1 > TH2 > Treg) and naïve/memory (memory > naïve) CD4+ T lymphocytes. Besides, IL-6/IL−6R is associated with TH17-differentiation and asthma severity. Allergic/atopic asthma (AA) is dominated by TH2 responses, while TH17 immunity might either modulate the TH2-dependent inflammation in AA or be an important mechanism boosting non-allergic asthma (NAA). Therefore, in this work we have compared the expression of CD26 and CD126 (IL-6Rα) in lymphocytes from different groups of donors: allergic (AA) and non-allergic (NAA) asthma, rhinitis, and healthy subjects. For this purpose, flow cytometry, haematological/biochemical, and in vitro proliferation assays were performed. Our results show a strong CD26-CD126 correlation and an over-representation of CD26− subsets with a highly-differentiated effector phenotype in AA (CD4+CD26−/low T cells) and NAA (CD4−CD26− γδ-T cells). In addition, we found that circulating levels of CD26 (sCD26) were reduced in both AA and NAA, while loss of CD126 expression on different leukocytes correlated with higher disease severity. Finally, selective inhibition of CD26-mRNA translation led to enhanced T cell proliferation in vitro. These findings support that CD26 down-modulation could play a role in facilitating the expansion of highly-differentiated effector T cell subsets in asthma.This work was supported by grants from Sociedad Española de Neumología y Cirugía Torácica, (SEPAR) (121/2012) and Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (Fondo de Investigación Sanitaria, FIS; co-financed by European Union ERDF funds) (PI13/02046). JJN-F is a recipient of a Xunta de Galicia Fellowship (co-financed by European Social Fund (ESF))S

The CD14 (−159 C/T) SNP is associated with sCD14 levels and allergic asthma, but not with CD14 expression on monocytes

LPS-ligation to CD14/TLR-4 on monocytes/macrophages triggers the production of IL-12-family cytokines. IL12/18 promote TH1-differentiation, counteracting the TH2-driven asthma. Therefore, CD14 modulation could alter the TH2-differentiation and should be taken into account when studying asthma. To analyse the alteration in CD14 levels and its association with CD14 (−159 C/T) SNP (rs2569190) in Caucasian adults with stable allergic asthma, we performed a cross-sectional study (277 healthy subjects vs. 277 patients) where clinical parameters, CD14 values and the CD14 (−159 C/T) SNP were studied. Apart from typical biomarkers, we found an increment of neuron-specific enolase (NSE) in allergic asthma, probably linked to monocyte activity. Indeed, we evidenced increased monocyte numbers, but lower CD14 expression and normalised sCD14 values in patients. Moreover, we noticed an association of the T allele (P = 0.0162) and TT genotype (P = 0.0196) of the CD14 SNP with a decreased risk of allergic asthma and augmented sCD14 levels. In conclusion, monocyte CD14 expression and normalized sCD14 values were reduced in stable state asthmatics, and this could be related to the presence of an expanded CD14low monocyte subset. This study also demonstrates that the CD14 (−159 C/T) polymorphism is a risk factor for moderate-severe allergic asthma in adult CaucasiansThis study was funded by grants from Sociedad Española de Neumología y Cirugía Torácica, (SEPAR) (121/2012) and Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (Fondo de Investigación Sanitaria, FIS; co-financed by European Union ERDF funds) (PI13/02046). JJNF is a recipient of a Xunta de Galicia Fellowship (Co-financed by European Social Fund (ESF))S

Association of Rhinitis With Asthma Prevalence and Severity

Multicenter study[Abstract] Asthma and rhinitis often co-exist in the same patient. Although some authors observed a higher prevalence and/or greater severity of asthma in patients with rhinitis, this view is not homogeneous and the debate continues. The aim of our study is to describe the prevalence of rhinitis in children and adolescents and to analyse their relationship with the prevalence of asthma. A multicentre study was conducted using the methodology of the International Study of Asthma and Allergies in Childhood (ISAAC). The target population of the study was all those school children aged 6-7 and 13-14 years from 6 of the main health catchment areas of Galicia (1.9 million inhabitants). The schools required were randomly selected, and all children in the targeted age ranges were included. Multiple logistic regression was used to obtain adjusted prevalence odds ratios (OR) between asthma symptoms of the schoolchildren and rhinitis prevalence. The results were adjusted for parental smoking habits, maternal education level, cat and dog exposure, and obesity. A total of 21,420 valid questionnaires were finally obtained. Rhinitis was associated with a significant increase in the prevalence of asthma in both age groups. The highest OR were 11.375 for exercise induced asthma (EIA) for children with recent rhinoconjunctivitis and 9.807 for children with recent rhinitis in 6-7 years old group. The prevalence OR's are higher in EIA and severe asthmatics. Rhinitis in children and adolescents is associated with a higher prevalence and severity of asthma.This work was funded by the Maria Jose Jove Foundatio

Proteomic analysis of food allergens

Food allergies are a growing health problem that generate high costs for health systems. Food allergies have a prevalence of 6-8% in children and 2-3% in adults, which is increasing in the last 10 years due to industrialization. The wide variety of ingredients containing food allergens, in combination with the high number of product formulations and processing methods to produce food makes allergen detection a challenge. It is important to highlight that even trace amounts of allergens are able to elicit allergic reactions and the commercialization of food products with a potential health risk is forbidden (European Union Regulation (EU) 178/2002). Hence, precautionary allergen labeling must be provided in the different food products. In addition, this creates a real need for the development of highly sensitive and reliable techniques that allow the detection and quantification of multiple allergens present in trace amounts. This, together with the possibility to perform new allergen discovery studies (shotgun proteomics), makes Proteomic approaches a widely used methodology in this field. In this chapter we summarize the current knowledge regarding food allergies and the proteomic studies performed for the analysis of the different allergens. We briefly describe immunological processes underlying the different types of food allergies, the causative allergens involved in these processes, as well as the different proteomic approaches developed to identify (e.g., LC-MS/MS) and quantify (e.g., targeted proteomics such as selected/multiple reaction monitoring, SRM/MSM) these allergens in different conditions (e.g., complex mixtures, ultra-processed food, etc). Moreover, we have performed a comprehensive review of the different allergens and proteomic studies carried out in the field of plant food allergies, including gluten related disorders (GRDs), pollen-fruit allergy syndrome (PFAS), legumes allergy, and tree-nuts allergy, as well as animal food allergies, including cow´s milk, red meat, egg, fish, and shellfish allergies