[Immunophenotyping by spectral cytometry reveals a profile of lymphopenia associated with deregulation with an increase in effector memory lymphocytes in a patient with a mutation in the ITPR3 gene]. / Inmunofenotipo por citometría espectral, revela un perfil de linfopenia asociado a desregulación con aumento de linfocitos de memoria efectora en paciente con mutación en el gen ITPR3.

BACKGROUND: Variants in intracellular calcium transport genes have been associated with syndromic immunodeficiencies with a SCID phenotype. CASE REPORT: Seven-year-old girl of non-consanguineous parents, in Cartagena-Colombia. At two months of age, he presented hematochezia and was diagnosed with alimentary proctolitis without improvement with restriction to milk, wheat and eggs, and malnutrition developed. At eight months, a colon biopsy shows chronic lymphoid hyperplasia, presenting with anemia, eosinophilia, but total and specific IgE to normal foods. After four years, the Immunology Service found her asymptomatic, nutritionally recovered and without allergic sensitization, but eosinophilia and elevated calprotectin persisted, suggesting an early-onset inflammatory bowel disease. Immunoglobulins were normal, lymphocyte populations with CD3, CD4 and CD8 lymphopenia. At six years old, she presented atopic dermatitis, still had elevated calprotectin and was lymphopenic. Immunophenotyping by spectral cytometry using Cytek®cFluor®Immunoprofiling-Kit14 showed lymphopenia and CD4/CD8 inversion. Naïve CD4+ and CD8+ T lymphocytes were decreased, while T-CD8+CD45RA-CCR7- and T-CD8+CD45RA+CCR7- effector memory populations were expanded. Effector and central memory CD4+ T-lymphocytes were also increased1 (Image 1). The exome revealed a heterozygous variant in the ITPR3 gene (carrier father), c.7571G>A, p.(Arg2524His); predictors classify it as having a potential eliminating effect. CONCLUSIONS: The clinical features and immunophenotype of this candidate variant differ from others related to intracellular calcium transport. They are functional studies necessary to validate their causality. A patient with a potentially deleted variant presents an immunophenotype with CD3 lymphopenia and persistent lymphocyte activation.

ANTECEDENTES: Las variantes en genes del transporte de calcio intracelular han sido asociadas a inmunodeficiencias sindrómicas con un fenotipo IDCG. REPORTE DE CASO: Niña de siete años, de padres no consanguíneos, en Cartagena-Colombia. A los dos meses de vida, presenta hematoquecia y se diagnostica con proctolitis alimentaria sin mejoría con restricción a leche, trigo y huevo, desarrollando desnutrición. A los ocho meses, una biopsia de colon muestra hiperplasia linfoide crónica, cursa con anemia, eosinofilia, pero IgE total y específica a alimentos normales. A los cuatro años, el Servicio de Inmunología la encuentra asintomática, recuperada nutricionalmente y sin sensibilización alérgica, pero persiste eosinofilia y calprotectina elevada, sugiriendo una enfermedad inflamatoria intestinal de inicio temprano. Las inmunoglobulinas fueron normales, poblaciones linfocitarias con linfopenia CD3, CD4 y CD8. A los seis años, presenta dermatitis atópica, sigue con calprotectina elevada y linfopénica. El inmunofenotipo por citometría espectral mediante Cytek®cFluor®Immunoprofiling-Kit14, mostró linfopenia e inversión CD4/CD8. Los linfocitos T-vírgenes CD4+ y CD8+ estaban disminuidos, en cambio las poblaciones de memoria efectora T-CD8+CD45RA-CCR7- y T-CD8+CD45RA+CCR7­ estaban expandidas. Los linfocitos T-CD4+ de memoria efectora y central, también estaban aumentados1 (Imagen 1). El exoma reveló una variante heterocigótica en el gen ITPR3 (padre portador), c.7571G>A, p.(Arg2524His); los predictores la clasifican como de potencial efecto deletéreo. CONCLUSIONES: La clínica y el inmunofenotipo de esta variante candidata difiere de otras relacionadas con el transporte del calcio intracelular. Son necesarios estudios funcionales para validar su causalidad. Una paciente con una variante potencialmente deletérea, presenta un inmunofenotipo con linfopenia CD3 y activación persistente de los linfocitos.

[Cytokine measurement using a multiplex technique with spectral flow cytometry: application in the study of the immunobiology of ascariasis]. / Medición de citoquinas mediante una técnica multiplex por citometría espectral: aplicación en el estudio de la inmunobiología de la ascariasis.

OBJECTIVE: To quantify the production of Th1/Th2/Th17 cytokines induced by Ascaris lumbricoides antigens in peripheral blood mononuclear cells (PBMCs) using a multiplex technique. METHODS: PBMCs were cultured from individuals with mild A. lumbricoides infection (n = 20) and uninfected individuals (n = 21) and stimulated with A. lumbricoides extract (ExtAscaris), a mix of anti-CD2/CD3/CD28 (CDmix) as a positive control, and only medium (negative control). Cytokines in the supernatants were measured using the BD™ Cytometric Bead Array Human Th1/Th2/Th17 kit, to identify IFN-γ, TNF, IL-10, IL-6, IL-4, IL-2, and IL-17A. Readings were taken on a spectral cytometer (Northern Lights, Cytek, USA), and analysis was performed using R software with packages "tidyverse," "beadplexr," "flowCore," and "arsenal." Cytokine concentrations were calculated using a 5-parameter logistic curve. The t-test was used to compare cases and controls, and statistical significance was set at p < 0.05. The study was approved by the Ethics Committee of the University of Cartagena and the participants provided informed consent. This study was financially supported by the Colombian Sistema General de Regalías under the BPIN2020000100405 - BPIN2020000100364. RESULTS: Efficient fluorescence intensity extraction for each cytokine was achieved using detection channel R8 and the "mclust" clustering model (Figure 1). No significant differences were found in the levels of the seven cytokines between cases and controls (Figure 2). Although the IFN-γ response to ExtAscaris was higher in cases than in controls (252.5 ng/mL vs. 173.1 ng/mL), the difference was not significant. IL-17A (detection limit: 18.9 pg/mL) was more detectable in cases than controls (5 cases, 23% vs. 2 controls, 9.5%). IL-4 was only detected in the supernatants from CDmix-stimulated cultures but not with the Ascaris extract (Figure 2). CONCLUSIONS: The multiplex technique using spectral flow cytometry combined with open-source software analysis proved applicable for quantifying cytokines induced by A. lumbricoides antigens in PBMCs. However, a more sensitive method is needed to evaluate IL-4 response in the context of ascariasis. The results did not reveal significant differences in cytokine production between cases and controls for the evaluated stimuli.

OBJETIVOS: Cuantificar la producción de citoquinas Th1/Th2/Th17, inducida por antígenos de Ascaris lumbricoides en PBMCs, utilizando una técnica de multiplex. MÉTODOS: Se realizaron cultivos de PBMCs de personas con infección leve por A. lumbricoides (n = 20), y no infectadas (n = 21), y se estimularon con extracto de A. lumbricoides (ExtAscaris), un mix de anti-CD2/CD3/CD28 (CDmix), como control positivo, y solo medio (control negativo). Las citoquinas en los sobrenadantes, se midieron usando el estuche BD™ Cytometric Bead Array Human Th1/Th2/Th17, para identificar IFN-γ, TNF, IL-10, IL-6, IL-4, IL-2 e IL-17A. La lectura se realizó en un citómetro espectral (Northern Lights, Cytek, USA), y el análisis en software R, usando los paquetes tidyverse, beadplexr, flowCore y arsenal. Se calculó la concentración de citoquinas mediante ajuste de curva logística de cinco parámetros. Se empleó la prueba t para comparar casos y controles y una p < 0,05, se consideró como significativa. Se contó con autorización del Comité de Ética de la Universidad de Cartagena para hacer la investigación y con el consentimiento informado por parte de los participantes. Este trabajo fue financiado por el Sistema General de Regalías de Colombia, bajo el BPIN2020000100405 - BPIN2020000100364. RESULTADOS: Al utilizar el canal de detección R8 para identificar las citoquinas y el modelo de agrupamiento mclust, se extrajo eficientemente la intensidad de fluorescencia para cada citoquina (Figura 1). No se encontraron diferencias significativas en los niveles de las siete citoquinas entre casos y controles (Figura 2). Aunque la respuesta de IFN-, γ hacia ExtAscaris fue más alta en los casos de controles (252,5 ng/mL vs 173,1 ng/mL), la diferencia no fue significativa. La IL-17A (límite de detección: 18,9 pg/mL) fue más detectable en casos que en controles (cinco casos, 23% vs dos controles, 9,5%). La IL-4 solo se detectó en los sobrenadantes de cultivos estimulados con CDmix, pero no con el extracto de Ascaris (Figura 2). CONCLUSIONES: La técnica multiplex por citometría espectral, combinada con el análisis en software de licencia libre, se mostró aplicable para cuantificar citoquinas inducidas por antígenos de A. lumbricoides en PBMCs. Sin embargo, se requiere de un método más sensible para evaluar la respuesta de IL-4 en el contexto de la ascariasis. Los resultados no revelaron diferencias significativas en la producción de citoquinas entre casos y controles para los estímulos evaluados.

[Characterization of peripheral blood cellular populations in relation to Ascaris Lumbricoides infection in rural areas of Bolívar]. / Caracterización de poblaciones celulares en sangre periférica, con relación a la infección por Ascaris lumbricoides en zonas rurales de Bolívar.

OBJECTIVE: To compare the relative frequencies of immune cell populations in the peripheral blood according to A. lumbricoides infection status. METHODS: Peripheral blood samples were collected from participants infected (n = 35) and uninfected with A. lumbricoides (n=27) residing in different rural municipalities of Bolívar. Infection was diagnosed using two coprological examinations and the Kato-Katz technique. Immunophenotyping was performed using two panels of markers and staining in fresh blood. The flow cytometry reading was performed on a spectral cytometer (Northern Lights, Cytek, USA). The populations identified in the first panel (Figure 1) were T lymphocytes (CD45+ CD3+), CD4+ or CD8+, B lymphocytes (CD45+ SSClow CD3- CD19+), neutrophils (CD45+ SSChi CD3- CD16+), and eosinophils (CD45+ SSChi CD3- CD16low). Monocytes were identified in another panel (Figure 2): classical (CD14++ CD16 -), intermediate (CD14++ CD16+), and non-classical (CD14+ CD16++). Dendritic cells, including CD123 + + CD303 + (plasmacytoid), HLA-DR + + CD1c + (myeloid CD1c +), and CD14-CD141 + + (myeloid CD141 +), were also identified. The study received approval from the Ethics Committee of the University of Cartagena, and participants provided informed consent. Funding was provided by the Colombian Sistema General de Regalías under BPIN2020000100405 - BPIN2020000100364. RESULTS: No significant differences were observed in age [mean cases: 35.69 (SD: 17.7) vs. controls: 37.04 (SD: 15.6) years] or sex (cases: 62.9% vs. controls: 74.1%) (Table 1). All infections were mild, with a median of 96 eggs (IQR, 48-216). A marginally significant difference was observed only in the percentage of neutrophils (45.37% in cases vs. 54.79% in controls, p=0.041) (Figure 3). Although the frequency of eosinophils was higher in the cases (8.1% vs. 6%), this difference was not significant (p=0.138) (Figure 3). No significant differences were observed in the populations of monocytes or dendritic cells between cases and controls (Figure 4). CONCLUSION: Mild A. lumbricoides infection appears to affect the number of neutrophils in peripheral blood. The low infection intensity in the studied samples may explain the lack of a significant impact on other cellular populations.

OBJETIVO: Comparar las frecuencias relativas de poblaciones de células inmunes en sangre periférica de acuerdo con el estado de infección por A. lumbricoides. MÉTODOS: Se recolectaron muestras de sangre periférica de participantes infectados (n=35) y no infectados con A. lumbricoides (n=27), residentes en distintos municipios rurales de Bolívar. La infección se diagnosticó por dos métodos coprológicos y la técnica de Kato-Katz. El inmunofenotipo se determinó con dos baterías de marcadores y tinciones en sangre fresca. La lectura fue realizada en un citómetro espectral (Northern Lights, Cytek, USA). Las poblaciones identificadas en la primera batería (Figura 1) fueron linfocitos T (CD45+ CD3+) CD4+ o CD8+, linfocitos B (CD45+ SSClow CD3- CD19+), neutrófilos (CD45+ SSChi CD3- CD16+), y eosinófilos (CD45+ SSChi CD3- CD16low). Los monocitos se identificaron en otra batería (Figura 2): clásicos (CD14++ CD16­), intermedios (CD14++ CD16+), y no clásicos (CD14+ CD16++). También se identificaron células dendríticas, tales como: CD123++ CD303+ (plasmocitoides), HLA-DR++ CD1c+ (mieloides CD1c+), y CD14- CD141++ (mieloides CD141+). El estudio recibió la aprobación del Comité de Ética de la Universidad de Cartagena, y los participantes otorgaron su consentimiento informado. La financiación fue proporcionada por el Sistema General de Regalías de Colombia, bajo el BPIN2020000100405 - BPIN2020000100364. RESULTADOS: No se observaron diferencias significativas en edad [media = casos: 35,69 (DE: 17,7) vs controles: 37,04 (DE: 15,6 años] o sexo (casos: 62,9% vs. controles: 74,1%). Todas las infecciones fueron leves con una mediana de huevos de 96 (RIC: 48 - 216). Solo se encontró diferencia significativa marginal en el porcentaje de neutrófilos (45,37% en los casos vs 54,79% en controles, p=0,041). Si bien la frecuencia de eosinófilos fue más alta en los casos (8,1% vs. 6%), esta diferencia no alcanzó la significancia (p=0,138). No se observaron diferencias significativas en las poblaciones de monocitos o células dendríticas entre casos y controles (Figura 4). CONCLUSIÓN: La infección leve por A. lumbricoides parece afectar el número de neutrófilos en sangre periférica. Es posible que por la baja intensidad de la infección en la muestra estudiada, no se detecte un impacto importante de la misma sobre el resto de las poblaciones celulares. Palabras claves: Helmintos; Ascaris lumbricoides; Citometría de flujo; Inmunofenotipado; Neutrófilos.

[Characterization of asthma phenotypes in children of the tropics]. / Caracterización de fenotipos de asma en niños del trópico.

OBJECTIVE: Determine the main asthma phenotypes in a population of asthmatic children in Cartagena, Colombia. METHODS: 107 children (7 to 17 years old) with a previous diagnosis of asthma were recruited. Biomarkers of T2 inflammation were evaluated by measuring FeNO, eosinophil count in peripheral blood by hemocytometry, and determination of specific IgE to mite allergens by ELISA. The study was approved by the ethics committee of the University of Cartagena (SGR, Grant BPIN2020000100405). RESULTS: The average age of patients was 10,9 years. 19,6% of the children did not show elevation of any of the T2 inflammation biomarkers evaluated (FeNO<20ppb, eos<300/ul, negative specific IgE), so they were considered patients with non-allergic asthma (non-T2). 71,9% of all patients were sensitized to at least one allergen, this phenotype was considered allergic asthma. 30,8% of the patients presented the three elevated biomarkers (FeNO>20ppb + eos >300/ul + positive specific IgE), this phenotype was classified as high T2 allergic asthma. A moderate correlation (Spearman rho=0,44, p<0,0001) was found between FeNO values and eosinophil counts. CONCLUSION: In this study, the following phenotypes were found: allergic asthma, high T2 asthma, and non-allergic asthma. Most patients presented a type 2 inflammatory phenotype with allergic sensitization. In addition to the measurement of specific IgE, the use of FeNO and eosinophil count in peripheral blood help to accurately determine those patients with high T2 asthma phenotypes.

OBJETIVO: Determinar los fenotipos principales de asma en una población de niños asmáticos en Cartagena, Colombia. MÉTODOS: Se reclutaron 107 niños (entre 7 y 17 años), con diagnóstico previo de asma. Se evaluaron biomarcadores de inflamación T2 mediante la medición de FeNO, conteo de eosinófilos en sangre periférica mediante hemocitometría, y la determinación de IgE específica a alergenos de ácaros mediante ELISA. El estudio fue aprobado por el Comité de Ëtica de la Universidad de Cartagena (SGR, Grant BPIN2020000100405). RESULTADOS: La edad media de los pacientes fue de 10,9 años. El 19,6% de los niños no mostró elevación de ninguno de los biomarcadores de inflamación T2 evaluados (FeNO<20 ppb, eos<300/ul, IgE específica negativa), por lo que se consideraron como pacientes con asma no alérgica (no-T2). El 71,9% de todos los pacientes estaban sensibilizados al menos a un alergeno considerándose este fenotipo como asma alérgica. El 30,8% de los pacientes presentaron los tres biomarcadores elevados (FeNO>20 ppb + eos >300/ul + IgE específica positiva), clasificando este fenotipo como asma alérgica T2 alta. Se encontró una correlación moderada (Spearman rho=0,44, p<0,0001) entre los valores de FeNO y los conteos de eosinófilos. CONCLUSIÓN: En este estudio se encontraron los siguientes fenotipos de asma alérgica: asma T2 alta y asma no alérgica. La mayoría de los pacientes presentó un fenotipo inflamatorio tipo 2 con sensibilización alérgica. Además de la medición de la IgE específica, el uso del FeNO y los conteos de eosinófilos en sangre periférica ayudan a determinar con mayor exactitud a aquellos pacientes con fenotipos de asma T2 alto.

[Circulating populations of CD4+ CD25+ CD127- regulatory t lymphocytes in peripheral blood of allergic asthmatic children]. / Determinación de poblaciones circulantes en sangre periférica de linfocitos T, reguladores CD4+ CD25+ CD127-, en niños asmáticos alérgicos.

OBJECTIVE: To carry out a preliminary analysis on the Treg lymphocyte counts present in the peripheral blood of allergic asthmatic children from the city of Cartagena, Colombia, compared to healthy controls. METHODS: We compared cytometry counts of ten asthmatic patients (age 7-16 years) and seven healthy controls (6-12 years), recruited in the city of Cartagena. Peripheral blood samples were stained using Cytek's 14-color cFluor Immunoprofiling kit (Cytek® cFluor® Immunoprofiling Kit 14 Color RUO kit), and analyzed on a Northern Lights™ spectral cytometer (Cytek® Biosciences, Fremont, CA, USA), to read 50.000 events per sample. The data obtained were analyzed in SpectroFlo® and FlowJo. The study was approved by the ethics committee of the University of Cartagena (SGR, Grant BPIN2020000100405). RESULTS: The frequency of CD3+, CD4+, CD25+, CD127- Tregs was 11% of all CD4+ T cells, with a range of minimum 8,1% and maximum 17,7%. There was no significant difference in the proportion of Tregs between allergic asthmatic patients and healthy controls (P = 0,2). CONCLUSIONS: With this preliminary sample size, no significant differences were found in the Treg lymphocyte population between allergic asthmatic patients and healthy controls. The 14-color multiplexed panel is a useful tool not only to count CD3+ and CD4+ populations, but also to obtain the percentage of regulatory T cells using cell surface markers.

OBJETIVO: Realizar un análisis preliminar sobre los conteos de linfocitos Tregs presentes en sangre periférica de niños asmáticos alérgicos de la ciudad de Cartagena, comparado con controles sanos. MÉTODOS: Se compararon los conteos de citometría de diez pacientes asmáticos (entre 7 y16 años) y siete controles sanos (entre 6 y12 años), reclutados en la ciudad de Cartagena. La muestra de sangre periférica fue teñida empleando el kit de inmunofenotipo multiplexado de 14 colores de Cytek (Cytek® cFluor® Immunoprofiling Kit 14 Color), y analizada en un citómetro espectral Northern Lights™ (Cytek® Biosciences, Fremont, CA, USA), a lectura de 50.000 eventos por muestra. Los datos obtenidos fueron analizados en SpectroFlo® y FlowJo. El estudio fue aprobado por el Comité de Ética de la Universidad de Cartagena. RESULTADOS: El panel de tinción funcionó apropiadamente y dentro de los parámetros apropiados. Se obtuvo un promedio de células Tregs CD3+, CD4+, CD25+ y CD127- del 11% de todos los CD4+ en las muestras estudiadas, con un rango de mínimo de 8,1% y un máximo de 17,7%. No hubo diferencias significativas en la proporción de linfocitos Tregs entre los pacientes asmáticos alérgicos y los controles sanos (P = 0.2). CONCLUSIONES: Con este tamaño de muestra preliminar, no se encontraron diferencias significativas en la población de linfocitos Tregs entre los pacientes asmáticos alérgicos y los controles sanos. El panel multiplexado de 14 colores es una herramienta útil no solo para derivar las poblaciones CD3+ y CD4+, sino también para obtener el porcentaje de células T reguladoras empleando marcadores de superficie celular.

Cystatin from the helminth Ascaris lumbricoides upregulates mevalonate and cholesterol biosynthesis pathways and immunomodulatory genes in human monocyte-derived dendritic cells.

Background: Ascaris lumbricoides cystatin (Al-CPI) prevents the development of allergic airway inflammation and dextran-induced colitis in mice models. It has been suggested that helminth-derived cystatins inhibit cathepsins in dendritic cells (DC), but their immunomodulatory mechanisms are unclear. We aimed to analyze the transcriptional profile of human monocyte-derived DC (moDC) upon stimulation with Al-CPI to elucidate target genes and pathways of parasite immunomodulation. Methods: moDC were generated from peripheral blood monocytes from six healthy human donors of Denmark, stimulated with 1 µM of Al-CPI, and cultured for 5 hours at 37°C. RNA was sequenced using TrueSeq RNA libraries and the NextSeq 550 v2.5 (75 cycles) sequencing kit (Illumina, Inc). After QC, reads were aligned to the human GRCh38 genome using Spliced Transcripts Alignment to a Reference (STAR) software. Differential expression was calculated by DESEq2 and expressed in fold changes (FC). Cell surface markers and cytokine production by moDC were evaluated by flow cytometry. Results: Compared to unstimulated cells, Al-CPI stimulated moDC showed differential expression of 444 transcripts (|FC| ≥1.3). The top significant differences were in Kruppel-like factor 10 (KLF10, FC 3.3, PBH = 3 x 10-136), palladin (FC 2, PBH = 3 x 10-41), and the low-density lipoprotein receptor (LDLR, FC 2.6, PBH = 5 x 10-41). Upregulated genes were enriched in regulation of cholesterol biosynthesis by sterol regulatory element-binding proteins (SREBP) signaling pathways and immune pathways. Several genes in the cholesterol biosynthetic pathway showed significantly increased expression upon Al-CPI stimulation, even in the presence of lipopolysaccharide (LPS). Regarding the pathway of negative regulation of immune response, we found a significant decrease in the cell surface expression of CD86, HLA-DR, and PD-L1 upon stimulation with 1 µM Al-CPI. Conclusion: Al-CPI modifies the transcriptome of moDC, increasing several transcripts encoding enzymes involved in cholesterol biosynthesis and SREBP signaling. Moreover, Al-CPI target several transcripts in the TNF-alpha signaling pathway influencing cytokine release by moDC. In addition, mRNA levels of genes encoding KLF10 and other members of the TGF beta and the IL-10 families were also modified by Al-CPI stimulation. The regulation of the mevalonate pathway and cholesterol biosynthesis suggests new mechanisms involved in DC responses to helminth immunomodulatory molecules.

Frequent IgE recognition of Blomia tropicalis allergen molecules in asthmatic children and young adults in equatorial Africa.

Background: Asthma is not well investigated in equatorial Africa and little is known about the disease-associated allergen molecules recognized by IgE from patients in this area. The aim was to study the molecular IgE sensitization profile of asthmatic children and young adults in a semi-rural area (Lambaréné) of an equatorial African country (Gabon), to identify the most important allergen molecules associated with allergic asthma in equatorial Africa. Methods: Fifty-nine asthmatic patients, mainly children and few young adults, were studied by skin prick testing to Dermatophagoides pteronyssinus (Der p), D. farinae (Der f), cat, dog, cockroach, grass, Alternaria and peanut. Sera were obtained from a subset of 35 patients, 32 with positive and 3 with negative skin reaction to Der p and tested for IgE reactivity to 176 allergen molecules from different allergen sources by ImmunoCAP ISAC microarray technology and to seven recombinant Blomia tropicalis (Blo t) allergens by IgE dot blot assay. Results: Thirty-three of the 59 patients (56%) were sensitized to Der p and 23 of them (39%) were also sensitized to other allergen sources, whereas 9 patients (15%) were only sensitized to allergen sources other than Der p. IgE serology analyses (n=35) showed high IgE-binding frequencies to the Blo t allergens Blo t 5 (43%), Blo t 21 (43%) and Blo t 2 (40%), whereas the Der p allergens rDer p 2, rDer p 21 and rDer p 5 (34%, 29% and 26%) were less frequently recognized. Only few patients showed IgE reactivity to allergens from other allergen sources, except to allergens containing carbohydrate determinants (CCDs) or to wasp venom allergens (i.e., antigen 5). Conclusion: Our results thus demonstrate that IgE sensitization to mite allergens is very prevalent in asthmatics in Equatorial Africa with B. tropicalis allergen molecules representing the most important ones associated with allergic asthma.

EAACI Molecular Allergology User's Guide 2.0.

Since the discovery of immunoglobulin E (IgE) as a mediator of allergic diseases in 1967, our knowledge about the immunological mechanisms of IgE-mediated allergies has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of the allergic disease for individual patients. The first edition of the "EAACI Molecular Allergology User's Guide" (MAUG) in 2016 rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine. Nevertheless, the field of molecular allergology is moving fast, and after 6 years, a new EAACI Taskforce was established to provide an updated document. The Molecular Allergology User's Guide 2.0 summarizes state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care professionals through the overwhelming list of different allergen molecules available for testing. Further, it provides diagnostic algorithms on the clinical relevance of allergenic molecules and gives an overview of their biology, the basic mechanisms of test formats, and the application of tests to measure allergen exposure.

Understanding the Cellular Sources of the Fractional Exhaled Nitric Oxide (FeNO) and Its Role as a Biomarker of Type 2 Inflammation in Asthma.

Fractional exhaled nitric oxide (FeNO) has gained great clinical importance as a biomarker of type 2 inflammation in chronic airway diseases such as asthma. FeNO originates primarily in the bronchial epithelium and is produced in large quantities by the enzyme inducible nitric oxide synthase (iNOS). It should be noted that nitric oxide (NO) produced at femtomolar to picomolar levels is fundamental for respiratory physiology. This basal production is induced in the bronchial epithelium by interferon gamma (IFNγ) via Janus kinases (JAK)/STAT-1 signaling. However, when there is an increase in the expression of type 2 inflammatory cytokines such as IL-4 and IL-13, the STAT-6 pathway is activated, leading to overexpression of iNOS and consequently to an overproduction of airway NO. Increased NO levels contributes to bronchial hyperreactivity and mucus hypersecretion, increases vascular permeability, reduces ciliary heartbeat, and promotes free radical production, airway inflammation, and tissue damage. In asthmatic patients, FeNO levels usually rise above 25 parts per billion (ppb) and its follow-up helps to define asthma phenotype and to monitor the effectiveness of corticosteroid treatment and adherence to treatment. FeNO is also very useful to identify those severe asthma patients that might benefit of personalized therapies with monoclonal antibodies. In this review, we revised the cellular and molecular mechanisms of NO production in the airway and its relevance as a biomarker of type 2 inflammation in asthma.

Longitudinal analyses of development of the immune system during the first five years of life in relation to lifestyle.

BACKGROUND: Changes in immune cell composition during the immunological window within the first years after birth are not fully understood, especially the effect that different lifestyles might have on immune cell functionality. METHODS: Peripheral blood mononuclear cells from mothers and their children at birth and at two anvd five years were analyzed by mass cytometry. Immune cell composition and functionality was analyzed according to family lifestyle (anthroposophic and non-anthroposophic). RESULTS: We found no significant differences in the proportions of major immune lineages between anthroposophic and non-anthroposophic children at each time point, but there were clear changes over time in the proportions of mononuclear leukocytes, especially in B-cells and T lymphocytes. Phenotypic distances between cord blood and maternal blood were high at birth but decreased sharply the first two years, indicating strong phenotypic convergence with maternal cells. We found that children exhibited similar stimulation responses at birth, but subsequently segregated into two discrete functional trajectories. Trajectory 1 was associated with a decrease in tumor necrosis factor alpha (TNFa) production by CD4+ T- and NK-cells, while Trajectory 2 depicted an increase in the production of IL-2 and interferon gamma (INFg) by T-cells. In both trajectories, there was an increase in IL-17A production by T-cells resulting in prominent differences at five years of age. CONCLUSIONS: This exploratory study suggests that leukocyte frequencies and cell phenotypes change with age in the same way across all children, while functional development follows one of two discrete trajectories that largely segregate by family lifestyle, supporting the hypothesis that early environmental exposures imprint immune cell function which may contribute to IgE sensitization. Our results also support that the first two years are critical for the environmental exposures to imprint the immune cells. Further studies with larger sample sizes are required to validate our findings.

Multiethnic genome-wide and HLA association study of total serum IgE level.

BACKGROUND: Total serum IgE (tIgE) is an important intermediate phenotype of allergic disease. Whole genome genetic association studies across ancestries may identify important determinants of IgE. OBJECTIVE: We aimed to increase understanding of genetic variants affecting tIgE production across the ancestry and allergic disease spectrum by leveraging data from the National Heart, Lung and Blood Institute Trans-Omics for Precision Medicine program; the Consortium on Asthma among African-ancestry Populations in the Americas (CAAPA); and the Atopic Dermatitis Research Network (N = 21,901). METHODS: We performed genome-wide association within strata of study, disease, and ancestry groups, and we combined results via a meta-regression approach that models heterogeneity attributable to ancestry. We also tested for association between HLA alleles called from whole genome sequence data and tIgE, assessing replication of associations in HLA alleles called from genotype array data. RESULTS: We identified 6 loci at genome-wide significance (P < 5 × 10-9), including 4 loci previously reported as genome-wide significant for tIgE, as well as new regions in chr11q13.5 and chr15q22.2, which were also identified in prior genome-wide association studies of atopic dermatitis and asthma. In the HLA allele association study, HLA-A∗02:01 was associated with decreased tIgE level (Pdiscovery = 2 × 10-4; Preplication = 5 × 10-4; Pdiscovery+replication = 4 × 10-7), and HLA-DQB1∗03:02 was strongly associated with decreased tIgE level in Hispanic/Latino ancestry populations (PHispanic/Latino discovery+replication = 8 × 10-8). CONCLUSION: We performed the largest genome-wide association study and HLA association study of tIgE focused on ancestrally diverse populations and found several known tIgE and allergic disease loci that are relevant in non-European ancestry populations.

Chronic Obstructive Pulmonary Disease Patients Have Increased Levels of Plasma Inflammatory Mediators Reported Upregulated in Severe COVID-19.

Background: Chronic obstructive pulmonary disease (COPD) is associated with increased risk of severe COVID-19, but the mechanisms are unclear. Besides, patients with severe COVID-19 have been reported to have increased levels of several immune mediators. Methods: Ninety-two proteins were quantified in 315 plasma samples from 118 asthmatics, 99 COPD patients and 98 healthy controls (age 40-90 years), who were recruited in Colombia before the COVID-19 pandemic. Protein levels were compared between each disease group and healthy controls. Significant proteins were compared to the gene signatures of SARS-CoV-2 infection reported in the "COVID-19 Drug and Gene Set Library" and with experimentally tested protein biomarkers of severe COVID-19. Results: Forty-one plasma proteins showed differences between patients and controls. Asthmatic patients have increased levels in IL-6 while COPD patients have a broader systemic inflammatory dysregulation driven by HGF, OPG, and several chemokines (CXCL9, CXCL10, CXCL11, CX3CL1, CXCL1, MCP-3, MCP-4, CCL3, CCL4 and CCL11). These proteins are involved in chemokine signaling pathways related with response to viral infections and some, were found up-regulated upon SARS-CoV-2 experimental infection of Calu-3 cells as reported in the COVID-19 Related Gene Sets database. An increase of HPG, CXCL9, CXCL10, IL-6, MCP-3, TNF and EN-RAGE has also been experimentally detected in patients with severe COVID-19. Conclusions: COPD patients have altered levels of plasma proteins that have been reported increased in patients with severe COVID-19. Our study suggests that COPD patients have a systemic dysregulation in chemokine networks (including HGF and CXCL9) that could make them more susceptible to severe COVID-19. Also, that IL-6 levels are increased in some asthmatic patients (especially in females) and this may influence their response to COVID-19. The findings in this study depict a novel panel of inflammatory plasma proteins in COPD patients that may potentially associate with increased susceptibility to severe COVID-19 and might be useful as a biomarker signature after future experimental validation.

Genotyping of Ascaris spp. infecting humans and pigs in Italy, Slovakia and Colombia.

BACKGROUND: The systematics and taxonomy of Ascaris lumbricoides and Ascaris suum, two of the world's most widespread nematodes, still represent a highly debated scientific issue. Two different transmission scenarios have been described according to endemicity: separated host-specific transmission cycles in endemic regions, and a single pool of infection shared by humans and pigs in non-endemic regions. The swine roundworm A. suum is now recognized as an important cause of human ascariasis also in endemic areas such as China, where cross-infections and hybridization have also been reported, as well as in non-endemic regions like Italy. This study aimed to investigate the molecular epidemiology of human and pig ascariasis in three countries representing different epidemiological scenarios: Italy as a non-endemic country, Colombia as an endemic country, and Slovakia as a non-endemic country, but with a poor socio-economic context linked to some focal populations of Roma settlements. MATERIALS AND METHODS: A total of 237 nematodes were analysed: 46 from Colombia (13 from humans, 33 from pigs), 114 from Slovakia (20 from humans, 94 from pigs) and 77 from Italy (17 from humans and 60 from pigs). Genotyping by PCR-RFLP of nuclear (ITS) and sequencing of mitochondrial (cox1) target regions were performed. ITS genotypes were used to estimate the Hardy-Weinberg (HW) equilibrium according to hosts and country of origin. The partial cox1 sequences were used to analyse genetic polymorphisms according to hosts and country of origin, as well as to infer the network of haplotypes, their evolutionary relationships and geographical distribution. RESULTS: 110 quality cox1 sequences were obtained. Haplotype network revealed three main groups corresponding to clade A, B and C. Clade C included most of the human cases from Italy, while those from Slovakia and Colombia were grouped in clade B. Ascaris from Italian and Colombian pigs showed HW equilibrium at the ITS marker, while disequilibrium was found in A. lumbricoides from Slovak pigs, which suggest a high unexpected amount of roundworms of human origin circulating also in pigs. CONCLUSIONS: This study updates and extends the current understanding of Ascaris species and genotypes circulating in different epidemiological scenarios, with particular attention to the inclusion of human-derived Ascaris in the phylogenetic cluster C. Despite the evidence of HW equilibrium in the ITS in pig-derived Italian samples, the amount of genetic variation seems to support the existence of two closely related species.

High-resolution targeted bisulfite sequencing reveals blood cell type-specific DNA methylation patterns in IL13 and ORMDL3.

BACKGROUND: Methylation of DNA at CpG sites is an epigenetic modification and a potential modifier of disease risk, possibly mediating environmental effects. Currently, DNA methylation is commonly assessed using specific microarrays that sample methylation at a few % of all methylated sites. METHODS: To understand if significant information on methylation can be added by a more comprehensive analysis of methylation, we set up a quantitative method, bisulfite oligonucleotide-selective sequencing (Bs-OS-seq), and compared the data with microarray-derived methylation data. We assessed methylation at two asthma-associated genes, IL13 and ORMDL3, in blood samples collected from children with and without asthma and fractionated white blood cell types from healthy adult controls. RESULTS: Our results show that Bs-OS-seq can uncover vast amounts of methylation variation not detected by commonly used array methods. We found that high-density methylation information from even one gene can delineate the main white blood cell lineages. CONCLUSIONS: We conclude that high-resolution methylation studies can yield clinically important information at selected specific loci missed by array-based methods, with potential implications for future studies of methylation-disease associations.

Perinatal and Early-Life Nutrition, Epigenetics, and Allergy.

Epidemiological studies have shown a dramatic increase in the incidence and the prevalence of allergic diseases over the last several decades. Environmental triggers including risk factors (e.g., pollution), the loss of rural living conditions (e.g., farming conditions), and nutritional status (e.g., maternal, breastfeeding) are considered major contributors to this increase. The influences of these environmental factors are thought to be mediated by epigenetic mechanisms which are heritable, reversible, and biologically relevant biochemical modifications of the chromatin carrying the genetic information without changing the nucleotide sequence of the genome. An important feature characterizing epigenetically-mediated processes is the existence of a time frame where the induced effects are the strongest and therefore most crucial. This period between conception, pregnancy, and the first years of life (e.g., first 1000 days) is considered the optimal time for environmental factors, such as nutrition, to exert their beneficial epigenetic effects. In the current review, we discussed the impact of the exposure to bacteria, viruses, parasites, fungal components, microbiome metabolites, and specific nutritional components (e.g., polyunsaturated fatty acids (PUFA), vitamins, plant- and animal-derived microRNAs, breast milk) on the epigenetic patterns related to allergic manifestations. We gave insight into the epigenetic signature of bioactive milk components and the effects of specific nutrition on neonatal T cell development. Several lines of evidence suggest that atypical metabolic reprogramming induced by extrinsic factors such as allergens, viruses, pollutants, diet, or microbiome might drive cellular metabolic dysfunctions and defective immune responses in allergic disease. Therefore, we described the current knowledge on the relationship between immunometabolism and allergy mediated by epigenetic mechanisms. The knowledge as presented will give insight into epigenetic changes and the potential of maternal and post-natal nutrition on the development of allergic disease.

Helminth-derived cystatins: the immunomodulatory properties of an Ascaris lumbricoides cystatin.

Helminth infections such as ascariasis elicit a type 2 immune response resembling that involved in allergic inflammation, but differing to allergy, they are also accompanied with strong immunomodulation. This has stimulated an increasing number of investigations, not only to better understand the mechanisms of allergy and helminth immunity but to find parasite-derived anti-inflammatory products that could improve the current treatments of chronic non-communicable inflammatory diseases such as asthma. A great number of helminth-derived immunomodulators have been discovered and some of them extensively analysed, showing their potential use as anti-inflammatory drugs in clinical settings. Since Ascaris lumbricoides is one of the most successful parasites, several groups have focused on the immunomodulatory properties of this helminth. As a result, several excretory/secretory components and purified molecules have been analysed, revealing interesting anti-inflammatory activities potentially useful as therapeutic tools. One of these molecules is A. lumbricoides cystatin, whose genomic, cellular, molecular, and immunomodulatory properties are described in this review.

DNA Methylation Levels in Mononuclear Leukocytes from the Mother and Her Child Are Associated with IgE Sensitization to Allergens in Early Life.

DNA methylation changes may predispose becoming IgE-sensitized to allergens. We analyzed whether DNA methylation in peripheral blood mononuclear cells (PBMC) is associated with IgE sensitization at 5 years of age (5Y). DNA methylation was measured in 288 PBMC samples from 74 mother/child pairs from the birth cohort ALADDIN (Assessment of Lifestyle and Allergic Disease During INfancy) using the HumanMethylation450BeadChip (Illumina). PBMCs were obtained from the mothers during pregnancy and from their children in cord blood, at 2 years and 5Y. DNA methylation levels at each time point were compared between children with and without IgE sensitization to allergens at 5Y. For replication, CpG sites associated with IgE sensitization in ALADDIN were evaluated in whole blood DNA of 256 children, 4 years old, from the BAMSE (Swedish abbreviation for Children, Allergy, Milieu, Stockholm, Epidemiology) cohort. We found 34 differentially methylated regions (DMRs) associated with IgE sensitization to airborne allergens and 38 DMRs associated with sensitization to food allergens in children at 5Y (Sidak p ≤ 0.05). Genes associated with airborne sensitization were enriched in the pathway of endocytosis, while genes associated with food sensitization were enriched in focal adhesion, the bacterial invasion of epithelial cells, and leukocyte migration. Furthermore, 25 DMRs in maternal PBMCs were associated with IgE sensitization to airborne allergens in their children at 5Y, which were functionally annotated to the mTOR (mammalian Target of Rapamycin) signaling pathway. This study supports that DNA methylation is associated with IgE sensitization early in life and revealed new candidate genes for atopy. Moreover, our study provides evidence that maternal DNA methylation levels are associated with IgE sensitization in the child supporting early in utero effects on atopy predisposition.

Risk Factors Associated With Health Care Utilization in Preschool Recurrent Wheezers in a Tropical Environment.

Introduction: The severity of wheezing episodes is related with the need for health services, but the factors associated with health care utilization in preschool recurrent wheezers in underdeveloped regions are unclear. Objective: To evaluate the factors associated with health care utilization in preschool recurrent wheezers in Cartagena, Colombia. Methods: One hundred twenty-seven recurrent wheezers (age 2-6 years old) who were admitted to the emergency room (ER) due to wheezing in a Pediatric reference hospital in Cartagena were included. Children were evaluated by means of questionnaires and classified according to the number of ER visits, need for hospitalization and history of intensive care unit (ICU) admission due to wheezing within the last year. Total serum IgE and specific IgE to house dust mite allergens (HDM) were measured by ImmunoCAP® and allergen sensitization was evaluated by skin prick tests (SPT). Results: The maternal report of nocturnal cough without fever in their children increased the risk to have ≥5 ER visits in the last year due to wheezing. The use of montelukast was negatively associated with hospitalization, while a history of pneumonia and lack of tap water, increased the risk of hospitalization due to wheezing. A history of bronchiolitis, family history of asthma, cohabiting with two or more siblings, passive exposure to smoke and lack of sewage facilities increased the risk of ICU admission due to wheezing. The presence of atopy evaluated by SPT reactivity, total IgE levels or specific IgE to HDM were not associated with health care utilization. We also found that seroprevalence of positive IgE (≥0.35 kU/L) was 27% to B. tropicalis and 20.3% to D. pteronyssinus but the prevalence of positive IgE sensitization to these allergens was below 2% and 8% when evaluated by SPT, respectively. Conclusions: Poverty indicators are associated with ICU admission in a group of preschool recurrent wheezers and should be considered as aggravating factors for wheezing. These factors must be systematically assessed in the medical approach in underdeveloped regions in the tropics. Nocturnal cough without fever is a symptom associated with frequent ER visits while atopy was not associated with health care utilization in preschool recurrent wheezers.

Epigenetic alterations in skin homing CD4+CLA+ T cells of atopic dermatitis patients.

T cells expressing the cutaneous lymphocyte antigen (CLA) mediate pathogenic inflammation in atopic dermatitis (AD). The molecular alterations contributing to their dysregulation remain unclear. With the aim to elucidate putative altered pathways in AD we profiled DNA methylation levels and miRNA expression in sorted T cell populations (CD4+, CD4+CD45RA+ naïve, CD4+CLA+, and CD8+) from adult AD patients and healthy controls (HC). Skin homing CD4+CLA+ T cells from AD patients showed significant differences in DNA methylation in 40 genes compared to HC (p < 0.05). Reduced DNA methylation levels in the upstream region of the interleukin-13 gene (IL13) in CD4+CLA+ T cells from AD patients correlated with increased IL13 mRNA expression in these cells. Sixteen miRNAs showed differential expression in CD4+CLA+ T cells from AD patients targeting genes in 202 biological processes (p < 0.05). An integrated network analysis of miRNAs and CpG sites identified two communities of strongly interconnected regulatory elements with strong antagonistic behaviours that recapitulated the differences between AD patients and HC. Functional analysis of the genes linked to these communities revealed their association with key cytokine signaling pathways, MAP kinase signaling and protein ubiquitination. Our findings support that epigenetic mechanisms play a role in the pathogenesis of AD by affecting inflammatory signaling molecules in skin homing CD4+CLA+ T cells and uncover putative molecules participating in AD pathways.