[In silico analysis of Cit s 2: A highly conserved profilin]. / Análisis in silico de cit s 2: una profilina altamente conservada.

OBJECTIVE: Analyze phylogenetic relationships and molecular mimicry of Cit s 2 and other plant profilins. METHODS: Online bioinformatics tools including Basic Local Alignment Search Tool (BLASTP), PRALINE and MEGA were used for multiple alignments and phylogenetic analysis. A 3D-homology model of Cit s 2 was predicted. Models were calculated with MODELLER. The best model was selected with the model scoring option of MAESTRO. Conserved regions between Cit s 2 and other profilins were located on the 3D model and antigenic regions were predicted by ElliPro server (3-5). RESULTS: Cit s 2 amino acid sequence (Uniprot code:P84177) was compared with other 30 profilins from different allergenic sources. The identity between Cit s 2 and other profilins ranged between 82 and 99%. The highest identity was observed with Cucumis melo (99%) followed by Prunus persica (98%) and Malus domestica (92%). High conserved antigenic regions were observed on the 3D predicted model. Seven lineal and six discontinuous epitopes were found in Cit s 2. CONCLUSION: High conserved antigenic regions were observed on the 3D predicted model of Cit s 2, which might involve potential cross-reactivity between Cit s 2 and other profilins. Future studies are needed to further analyze these results.

OBJETIVO: Analizar las relaciones filogenéticas y el mimetismo molecular de Cit s 2 y otras profilinas vegetales. MÉTODOS: Se utilizaron herramientas bioinformáticas en línea, incluida la de búsqueda de alineación local básica (BLASTP), PRALINE y MEGA, para alineamientos múltiples y análisis filogenético. Se predijo un modelo de homología 3D de Cit s 2. Los modelos se calcularon con MODELLER. El mejor modelo fue seleccionado con la opción de puntuación de modelo de Maestro. Las regiones conservadas entre Cit s 2 y otras profilinas se ubicaron en el modelo 3D y las regiones antigénicas fueron predichas por el servidor ElliPro (3-5). RESULTADOS: La secuencia de aminoácidos de Cit s 2 (código Uniprot: P84177), se comparó con otras 30 profilinas de diferentes fuentes alergénicas. La mayor identidad se observó con Cucumis melo (99%) seguida de Prunus persica (98%) y Malus domestica (92%). Se observaron regiones antigénicas altamente conservadas en el modelo predicho en 3D. Se encontraron siete epítopes lineales, y seis epítopes discontinuos en Cit s 2. CONCLUSIÓN: Se observaron regiones antigénicas altamente conservadas en el modelo 3D predicho de Cit s 2, lo que podría implicar una posible reactividad cruzada entre Cit s 2 y otras profilinas. Se necesitan estudios futuros para analizar más a fondo estos resultados.

Comparison of Antibody Responses against Two Molecules from Ascaris lumbricoides: The Allergen Asc l 5 and the Immunomodulatory Protein Al-CPI.

Immunity to Ascaris lumbricoides influences the pathogenesis of allergic diseases. Antibody responses to its proteins have been found to be associated with asthma presentation; however, helminth products that induce immunosuppression have been reported, which also raise specific antibodies. We aimed to evaluate antibody responses (IgE, IgG4 and IgG) to two A. lumbricoides molecules, Asc l 5 and Al-CPI (an anti-inflammatory Cysteine Protease Inhibitor), in an endemic population, exploring their relationships with the infection and asthma. The two molecules were produced as recombinant proteins in E. coli expression systems. Specific antibodies were detected by ELISA. Lower human IgE, but higher IgG4 and IgG antibody levels were observed for Al-CPI than for rAsc l 5. The IgE/IgG4 isotype ratio was significantly higher for Asc l 5 than for Al-CPI. In humans Al-CPI did not induce basophil activation as has been previously described for Asc l 5. In mice, Al-CPI induced fewer IgE responses, but more IgG2a antibody titers than rAsc l 5. Our results suggest that these molecules elicit different patterns of immune response to A. lumbricoides.

Identification and Physicochemical Characterization of a New Allergen from Ascaris lumbricoides.

To analyze the impact of Ascaris lumbricoides infection on the pathogenesis and diagnosis of allergic diseases, new allergens should be identified. We report the identification of a new Ascaris lumbricoides allergen, Asc l 5. The aim of this study was to evaluate the physicochemical and immunological features of the Asc l 5 allergen. We constructed an A. lumbricoides cDNA library and Asc l 5 was identified by immunoscreening. After purification, rAsc l 5 was physicochemically characterized. Evaluation of its allergenic activity included determination of Immunoglobulin E (IgE) binding frequency (in two populations: 254 children and 298 all-age subjects), CD203c based-basophil activation tests (BAT) and a passive cutaneous anaphylaxis (PCA) mouse model. We found by amino acid sequence analysis that Asc l 5 belongs to the SXP/RAL-2 protein family of nematodes. rAsc l 5 is a monomeric protein with an alpha-helical folding. IgE sensitization to rAsc l 5 was around 52% in general population; positive BAT rate was 60%. rAsc l 5 induced specific IgE production in mice and a positive PCA reaction. These results show that Asc l 5 has structural and immunological characteristics to be considered as a new allergen from A. lumbricoides.

Ascaris lumbricoides Cystatin Prevents Development of Allergic Airway Inflammation in a Mouse Model.

Severe helminth infections are negatively associated to allergic diseases like asthma; therefore, the immunomodulatory properties of parasite-derived components have been analyzed, raising the possibility of their use as anti-inflammatory molecules. We evaluated the immunomodulatory properties of Ascaris lumbricoides recombinant cysteine protease inhibitor (rAl-CPI) in a mouse model of allergic airway inflammation induced by the house dust mite (HDM) Blomia tropicalis and its effects on human monocyte-derived dendritic cells (HmoDCs). The B. tropicalis sensitized/challenged mice developed extensive cellular airway inflammatory response, which was significantly reduced upon treatment with rAl-CPI prior to B. tropicalis sensitization, affecting particularly the perivascular/peribronchial infiltrate cells, eosinophils/neutrophils, and goblet cells. A significant decrease of Th2 cytokines, total, and specific IgE antibodies was observed in rAl-CPI treated mice. The antibody response was biased to IgG, mainly IgG2a. Administration of rAl-CPI-alone and rAl-CPI before mite sensitization were associated with a significant increase of regulatory T cells (Tregs) in spleen and elevated IL-10 levels in BAL and splenocytes culture supernatants, which was partially affected by anti-IL10 receptor use. In vitro, rAl-CPI showed a modulatory effect on HmoDCs, lowering the expression of HLA-DR, CD83, and CD86, while inducing IL-10 and IL-6 production. This suggests an inhibition of HmoDC maturation and a possible link with the inhibition of the allergic response observed in the murine model.

Hygienic conditions influence sensitization to Blomia tropicalis allergenic components: Results from the FRAAT birth cohort.

BACKGROUND: In tropical zones, perennial exposure to house dust mite (HDM) allergens and helminth infections is present. Studying the impact of these conditions on the inception and evolution of allergic diseases is necessary to have an accurate view of their natural history. We aimed to evaluate the dynamics of genuine sensitization to Blomia tropicalis and Ascaris in children from the FRAAT birth cohort and the effects of helminth infection, environmental HDM allergen levels, and sociodemographic factors. METHODS: Children were followed up to 6 years old. Specific IgE to recombinant allergens from B. tropicalis (Blo t 5 and Blo t 12) and Ascaris spp (Asc l 3, Asc l 13 and Asc s 1) was measured by ELISA at different time points. Allergen levels were measured in dust when children were 6 months old. RESULTS: IgE sensitization increased over time up to 3 years old. Correlation among the specific IgE levels to B. tropicalis and Ascaris components is poor at year 1, but coefficients are high and significant (Spearman's rho coefficients >0.70) at year 6. Unhygienic conditions increased the odds of sensitization to B. tropicalis allergenic components. Blo t 5 levels were lower in the poorest. IgE response to Blo t 5 and Blo t 12 was less intense in children with high exposure to Blo t 5 (levels >80th percentile). CONCLUSION: In this tropical community, the pattern of childhood IgE sensitization is different from that in developing countries and is influenced by the hygienic conditions.

Early life IgE responses in children living in the tropics: a prospective analysis.

BACKGROUND: There are few birth cohort studies analyzing IgE sensitization in the tropics. OBJECTIVES: We aimed to describe the evolution of total IgE and specific IgE responses to house-dust mite (HDM) allergens and Ascaris in a birth cohort (Risk Factors for Asthma and Allergy in the Tropics, FRAAT), analyzing their relationships with wheezing. METHODS: Total and specific IgE were measured by ImmunoCap in mothers and children at four different time points (S1-S4) between 0 and 42 months. Parasite infection was evaluated by stool examination. RESULTS: Maternal total IgE (aOR: 2.43, 95% CI: 1.09-5.43; p = 0.03) and socio-demographic factors were associated with high cord blood (CB) total IgE. High CB total IgE was positively associated with higher Blomia tropicalis and Ascaris-specific IgE values during lifetime, but protected from recurrent wheezing (aOR: 0.26, 95% CI: 0.08-0.88, p = 0.03). Prevalence rates of IgE sensitization were high; at around 3 yr old, they were 33.3, 18.6, and 26.5% for B. tropicalis, Dermatophagoides pteronyssinus, and Ascaris, respectively. Indicators of unhygienic conditions were risk factors for HDM and Ascaris sensitization in children. A weak statistical association between B. tropicalis-specific IgE and ever wheezing was found (aOR: 1.47 95% CI: 1.00-2.28, p = 0.05). CONCLUSIONS: In a socioeconomically deprived community from the tropics, sensitization to HDM allergens was very frequent at early life, especially to B. tropicalis. In contrast to expected according to the hygiene hypothesis, unhygienic/poverty conditions were risk factors for allergen sensitization. High CB total IgE levels were a risk factor for allergen sensitization but protected from recurrent wheezing.

Prevalence of asthma and other allergic conditions in Colombia 2009-2010: a cross-sectional study.

BACKGROUND: While it is suggested that the prevalence of asthma in developed countries may have stabilized, this is not clear in currently developing countries. Current available information for both adults and children simultaneously on the burden and impact of allergic conditions in Colombia and in many Latin American countries is limited. The objectives of this study were to estimate the prevalence for asthma, allergic rhinitis (AR), atopic eczema (AE), and atopy in six colombian cities; to quantify costs to the patient and her/his family; and to determine levels of Immunoglobulin E (IgE) in asthmatic and healthy subjects. METHODS: We conducted a cross-sectional, population-based study in six cities during the academic year 2009-2010. We used a school-based design for subjects between 5-17 years old. We carried out a community-based strategy for subjects between 1-4 years old and adults between 18-59 years old. Serum samples for total and antigen-specific (IgE) levels were collected using a population-based, nested, case-control design. RESULTS: We obtained information on 5978 subjects. The largest sample of subjects was collected in Bogotá (2392). The current prevalence of asthma symptoms was 12% (95% CI, 10.5-13.7), with 43% (95% CI, 36.3-49.2) reporting having required an emergency department visit or hospitalization in the past 12 months. Physician diagnosed asthma was 7% (95% CI, 6.1-8.0). The current prevalence of AR symptoms was 32% (95% CI, 29.5-33.9), and of AE symptoms was 14% (95% CI, 12.5-15.3). We collected blood samples from 855 subjects; 60.2% of asthmatics and 40.6% of controls could be classified as atopic. CONCLUSIONS: In Colombia, symptom prevalence for asthma, AR and AE, as well as levels of atopy, are substantial. Specifically for asthma, symptom severity and absence from work or study due to symptoms are important. These primary care sensitive conditions remain an unmet public health burden in developing countries such as Colombia.