RESUMO
PDE4D (Phosphodiesterase 4D) gene encodes a hydrolase of cyclic AMP. PDE4D genetic variants have been associated with asthma susceptibility. Therefore, this study aimed to investigate the association between PDE4D variants (and haplotypes) with asthma and atopy in a Brazilian population. The study comprised 1,246 unrelated participants from the SCAALA (Social Changes Asthma and Allergy in Latin America) program. Genotyping was performed using the Illumina 2.5 Human Omni bead chip. Multivariate logistic regression was used to investigate the association between PDE4D variants and asthma/atopy phenotypes in PLINK 1.09 software. Twenty-four SNVs in PDE4D were associated with atopy or asthma. The rs6898082 (A) variant increased asthma susceptibility (OR 2.76; CI 99% 1.26-6.03) and was also related to a greater PDE4D expression in the GTEx database. Also, the variant rs6870632 was further associated with asthma in meta-analysis with a replication cohort. In addition, the variants rs75699812 (C), rs8007656 (G), and rs958851 (T) were positively associated with atopy. Moreover, these variants formed an atopy risk haplotype (OR 1.82; CI 99% 1.15-2.88). Also, these variants were related to lower levels of IL-10. Functional in silico assessment showed that some PDE4D SNVs may have an impact on gene regulation and expression. Variants in the PDE4D are positively associated with asthma and allergy markers. It is possible that these variants lead to alteration in PDE4D expression and therefore impact immunity and pulmonary function.
Assuntos
Asma , Hipersensibilidade Imediata , Hipersensibilidade , Humanos , Criança , Haplótipos , Brasil/epidemiologia , Predisposição Genética para Doença , Asma/genética , Hipersensibilidade Imediata/genética , Hipersensibilidade/genética , Polimorfismo de Nucleotídeo Único , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genéticaRESUMO
BACKGROUND: Few genome-wide association studies (GWAS) on Helicobacter pylori infection susceptibility have been conducted for admixed populations from developing countries. Here, we performed a GWAS to identify genetic factors associated with H. pylori serostatus in a cohort of admixed children from a large Latin American urban center. METHODS: A cross-sectional study involving 1161 children from 4 to 11 years old living in poor areas of Salvador, in northeastern Brazil. Logistic regression analysis was performed to detect associations between single-nucleotide variants (SNVs) and H. pylori seropositivity, assuming an additive genetic model. Enrichment analyses were conducted using the MAGMA v1.10 software. RESULTS: We found 22 SNVs to be suggestively associated (p < 10-5 ) with H. pylori seropositivity. The most suggestive SNV was the rs77955022 (p = 4.83e-07) located in an intronic region of EXOC3 at 5p15.33. The second most suggestively associated SNV was rs10914996 (p = 8.97e-07), located in an intergenic region at 1p34.3. Furthermore, we were able to replicate three SNVs (p < 0.05) in the Study of Health in Pomerania (SHIP) cohort: the rs2339212 and rs4795970, both located at 17q12 near TMEM132E, as well as the rs6595814, an intronic variant of FBN2 at 5q23.3. The enrichment analysis indicated the participation of genes and metabolic pathways related to the regulation of the digestive system and gastric acid secretion in the risk of seropositivity for H. pylori. CONCLUSIONS: Additional studies are required to validate these association findings in larger population samples and to get insight into the underlying physiological mechanisms.
Assuntos
Infecções por Helicobacter , Helicobacter pylori , Humanos , Criança , Pré-Escolar , Estudo de Associação Genômica Ampla , Helicobacter pylori/genética , América Latina/epidemiologia , Infecções por Helicobacter/epidemiologia , Estudos TransversaisRESUMO
BACKGROUND: Allergen-specific immunotherapy (AIT) is the only disease-modifying treatment approach to change disease-causing allergens. Hypoallergenic derivatives show promise as potential therapeutics, amongst which BTH2 was designed to induce tolerance against Blomia tropicalis allergy. Our aim was to investigate the hypoallergenicity and immunoregulatory activity of BTH2 in vitro and its therapeutic potential in a mouse model of AIT. METHODS: Recombinant Blo t 5 and Blo t 21 allergens and their hybrid derivatives (BTH1 and BTH2) were expressed and purified. IgE binding capacity was tested by ELISA using sera from Brazilian, Colombian, and Ecuadorian subjects. Secretion of cytokines in supernatants from human cell cultures was measured following stimulation with the four recombinants and controls. The capacity of BTH2 to ameliorate allergic airway inflammation induced by B. tropicalis extract was evaluated in a murine model of AIT. RESULTS: rBlo t 5 and rBlo t 21 were identified as major allergens in Latin American patients, and BTH2 had the lowest IgE binding. In vitro stimulation of human cells induced greater levels of IL-10 and IFN-γ and reduced the secretion of Th2 cytokines. BTH2 ameliorated allergic airway inflammation in B. tropicalis-challenged A/J mice, as evidenced by the histopathological and humoral biomarkers: decreased Th2 cytokines and cellular infiltration (especially eosinophils), lower activity of eosinophil peroxidase, an increase in IgG blocking antibodies and strong reduction of mucus production by goblet cells. CONCLUSIONS: Our study shows that BTH2 represents a promising candidate for the treatment of B. tropicalis allergy with hypoallergenic, immune regulatory and therapeutic properties. Further pre-clinical studies are required in murine models of chronic asthma to further address the efficacy and safety of BTH2 as a vaccine against B. tropicalis-induced allergy.
Assuntos
Hipersensibilidade , Humanos , Camundongos , Animais , Modelos Animais de Doenças , Hipersensibilidade/terapia , Alérgenos , Inflamação , Citocinas , Dessensibilização Imunológica , Imunoglobulina ERESUMO
BACKGROUND: Allergen-specific immunotherapy (AIT) is the only clinical approach that can potentially cure some allergic diseases by inducing immunological tolerance. Dermatophagoides pteronyssinus is considered as the most important source of mite allergens worldwide, with high sensitization rates for the major allergens Der p 1, Der p 2 and Der p 23. The aim of this work is to generate a hypoallergenic hybrid molecule containing T-cell epitopes from these three major allergens. METHODS: The hybrid protein termed Der p 2231 containing T-cell epitopes was purified by affinity chromatography. The human IgE reactivity was verified by comparing those with the parental allergens. The hybrid was also characterized immunologically through an in vivo mice model. RESULTS: The hybrid rDer p 2231 stimulated in peripheral blood mononuclear cells (PBMCs) isolated from allergic patients with higher levels of IL- 2, IL-10, IL-15 and IFN-γ, as well as lower levels of IL-4, IL-5, IL-13, TNF-α and GM-CSF. The use of hybrid molecules as a therapeutic model in D. pteronyssinus allergic mice led to the reduction of IgE production and lower eosinophilic peroxidase activity in the airways. We found increased levels of IgG antibodies that blocked the IgE binding to the parental allergens in the serum of allergic patients. Furthermore, the stimulation of splenocytes from mice treated with rDer p 2231 induced higher levels of IL-10 and IFN-γ and decreased the secretion of IL-4 and IL-5, when compared with parental allergens and D. pteronyssinus extract. CONCLUSIONS: rDer p 2231 has the potential to be used in AIT in patients co-sensitized with D. pteronyssinus major allergens, once it was able to reduce IgE production, inducing allergen-specific blocking antibodies, restoring and balancing Th1/Th2 immune responses, and inducing regulatory T-cells.
Assuntos
Antígenos de Dermatophagoides , Epitopos de Linfócito T , Hipersensibilidade , Animais , Humanos , Camundongos , Alérgenos , Antígenos de Dermatophagoides/imunologia , Antígenos de Dermatophagoides/farmacologia , Antígenos de Dermatophagoides/uso terapêutico , Proteínas de Artrópodes , Dermatophagoides pteronyssinus , Epitopos de Linfócito T/química , Epitopos de Linfócito T/uso terapêutico , Hipersensibilidade/tratamento farmacológico , Imunoglobulina E , Interleucina-10 , Interleucina-4 , Interleucina-5 , Leucócitos Mononucleares , Pyroglyphidae , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imunoterapia/métodosRESUMO
Toxocariasis is an infection caused by the round worms Toxocara canis and Toxocara cati. It occurs worldwide though it is more prevalent in developing countries. For the diagnosis of toxocariasis, the most used method is the indirect enzyme-linked immunosorbent assay (indirect ELISA), based on the detection of specific antibodies using the excreted/secreted products from T. canis larvae (TES) as antigens, but it cross-reacts with several helminth infections. For this reason, there is a need to investigate species-specific immunoreactive proteins, which can be used for the development of a more sensitive and specific diagnosis. This study aims to investigate immunoreactive protein candidates to be used for the development of a more sensitive and specific diagnosis of Toxocara spp. infection in humans. We have used immunoblotting and mass spectrometry to select four Toxocara canis immunoreactive proteins that were recombinantly expressed in bacteria and evaluated as potential new diagnostic antigens (rMUC3, rTES 26, rTES32 and rCTL4). The recognition of these recombinant proteins by total serum IgG and IgG4 was assayed using the purified proteins in an isolated manner or in combination. The IgG ELISAs performed with individual recombinant antigens reached values of sensitivity and specificity that ranged from 91.7% to 97.3% and 94.0% to 97.9%, respectively. Among the analyses, the IgG4 immunoassay was proven to be more effective, revealing a sensitivity that ranged from 88.8% to 98.3% and a specificity of 97.8%-97.9%. The IgG4 ELISA was shown to be more effective and presented no cross-reactivity when using combinations of the rTES 26 and rCTL4 recombinant proteins. The combination of these two molecules achieved 100% sensitivity and specificity. The use of only two recombinant proteins can contribute to improve the current panorama of toxocariasis immunodiagnosis for, with a better optimization and reduced cost.
Assuntos
Toxocara canis , Toxocaríase , Animais , Antígenos de Helmintos , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Humanos , Immunoblotting/veterinária , Imunoglobulina G , Testes Imunológicos/veterinária , Proteômica , Proteínas Recombinantes , Toxocara , Toxocaríase/diagnósticoRESUMO
BACKGROUND: Polymorphisms in genes related to the activation and development of regulatory T cells (Tregs), such as FOXP3, may be associated with asthma and atopy development. Additionally, environmental factors such as exposure to infections can modify the effect of these associations. This study evaluated the impact of polymorphisms in the FOXP3 on the risk of asthma and atopy as also gene-environment interactions in these outcomes. METHODS: This study included 1,246 children from the SCAALA program, between 4 and 11 years of age. DNA was extracted from peripheral blood and eight SNPs (rs2280883, rs11465476, rs11465472, rs2232368, rs3761549, rs3761548, rs2232365 and rs2294021) were genotyped using the 2.5 HumanOmni Beadchip from Illumina (San Diego, California, USA) or TaqMan qRT-PCR. RESULTS: The rs2232368 (Allele T) was positively associated with asthma symptoms (OR = 1.95, CI = 1.04 to 3.66, p = 0.040) and skin prick test (SPT) reactivity to aeroallergens (OR = 2.31, CI = 1.16 to 4.59, p = 0.017). The rs3761549 (Allele T) was positively associated with SPT reactivity (OR = 1.44, CI = 1.03 to 2.02, p = 0.034). The rs2280883 (Allele C) was negatively associated with specific IgE to aeroallergens (OR = 0.83, CI = 0.70 to 0.99, p = 0.040). Furthermore, the rs2280883 played a protective role in the development of atopy only in individuals seropositive to Epstein-Barr virus (EBV) infection (OR = 0.74, CI = 0.60 to 0.92, p = 0.003 and OR = 0.74; 95% CI = 0.61-0.91, p = 0.007 for SPT and slgE respectively), but not in individuals without EBV infection. CONCLUSION: Polymorphisms in the FOXP3 gene were associated with the risk of atopy and asthma development in our population. In addition, EBV infection had an effect modifier of the observed association for rs2280883 variant.
Assuntos
Asma , Infecções por Vírus Epstein-Barr , Hipersensibilidade Imediata , Asma/genética , Brasil , Criança , Fatores de Transcrição Forkhead/genética , Interação Gene-Ambiente , Predisposição Genética para Doença , Herpesvirus Humano 4 , Humanos , Hipersensibilidade Imediata/genética , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND: Obesity is a chronic complex disease with great prevalence for children all over the world. Characterized for low-grade inflammation associated with several comorbidities such as resistance and type 2 diabetes mellitus (T2DM). OBJECTIVES: To investigate whether genetic variants in IL10, IL1RL1, IL1B, IRF4, TNF, IL6, and IL33 genes are associated with being overweight in children. METHODS: We performed the genotyping of 1004 children using Illumina 2.5 Human Omni bead chip, and association analysis on the genetic variants and the overweight through logistic regression adjusted for sex, age and components principal. RESULTS: Of the seven genes analyzed, 16 SNVs significantly associated. Eleven variants in IL1RL1, two in IL1B and one in IRF4 genes increased overweight risk and two SNVs in IL1RL1 were associated with protection against overweight. The rs2287047-A was negatively associated (OR: 0.66, CI95%: 0.19-0.45) and had a reduced IL1RL1 expression in whole blood (p 0.033) in silico eQTL. The rs12203592-T, in IRF4, was positively associated with being overweight, and led to an increased gene expression in whole blood (p < 0.001) and adipose tissue (p < 0.001). CONCLUSION: These results suggest that genetic variants in inflammatory genes may play an important role in the development of overweight in children.
Assuntos
Diabetes Mellitus Tipo 2 , Fatores Reguladores de Interferon/genética , Polimorfismo de Nucleotídeo Único , Brasil , Criança , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Interleucina-1beta/genética , Sobrepeso/genéticaRESUMO
Successful research in the wide-ranging field of allergy is usually achieved by definition not only of physicochemical and immunological properties of natural, but also recombinant allergens. Blomia tropicalis mite is a well-known source for various groups of hypersensitivity-causing proteins. The goal of the present work was to produce, purify and characterise by in silico, biochemical and immunological methods the recombinant group-12 allergen of B. tropicalis. The recombinant Blo t 12 aggregation capacity as well as the affinity to antibodies from BALB/c immunised mice and B. tropicalis-sensitised human donors were investigated through in silico analyses, dynamic light scattering, SDS-PAGE, ELISA and Western blot. The presence of Blo t 12 within B. tropicalis extracts was also determined by ELISA and Western blot. High concentrations of dimeric rBlo t 12 were detected through SDS-PAGE next to other aggregates and the results were confirmed by data from DLS and Western blot. The YITVM peptide was predicted to be the most aggregation-prone region. The IgE-reactivity of rBlo t 12 was not completely abolished by aggregate formation but it was significantly decreased compared to rBlo t 5, or B. tropicalis extracts. Natural Blo t 12 may naturally dimerises, but it was detected in non-delipidified B. tropicalis extracts in low amounts. Given that this allergen may be a specific marker for B. tropicalis allergy, the recombinant Blo t 12 herein obtained is characterised as a mid-tier allergen in Brazilian atopic patients and may be useful for the improvement in precision allergy molecular diagnostic applications.
Assuntos
Alérgenos/isolamento & purificação , Ácaros/metabolismo , Alérgenos/genética , Alérgenos/imunologia , Animais , Escherichia coli/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas RecombinantesRESUMO
BACKGROUND: Vitamin D deficiency or insufficiency, has been associated with atopy and lack of asthma control. Our objective was to investigate associations between variants in genes of vitamin D pathway with serum levels of 25-hydroxyvitamin D (25(OH)D), atopy, asthma and asthma severity in teenagers from Northeast Brazil. METHODS: This is a cross sectional study nested in a cohort population of asthma. 25(OH)D was quantified from 968 of 11-17 years old individuals by ELISA. Asthma diagnosis was obtained by using the ISAAC Phase III questionnaire. Specific IgE was determined by ImmunoCAP; genotyping was performed using the 2.5 HumanOmni Biochip from Illumina. Statistical analyses were performed in PLINK 1.07 and SPSS 22.1. RESULTS: After quality control, 104 Single Nucleotides Variants (SNVs) in vitamin D pathway genes, typed in 792 individuals, were included in the analysis. The allele A of rs10875694 on VDR was positively associated with atopy (OR = 1.35; 95% CI 1.01-1.81). The allele C of rs9279 on VDR, was negatively associated with asthma risk (OR = 0.66; 95% CI 0.45-0.97), vitamin D insufficiency (OR = 0.78; 95% CI 0.70-0.96) and higher VDR expression. Two variants in VDR were associated with asthma severity, the allele A of rs2189480 (OR = 0.34; 95% CI 0.13-0.89) and the allele G of rs4328262 (OR = 3.18; 95% CI 1.09-9.28). The combination of variants in CYP2R1 and CYP24A1 (GAC, to rs10500804, rs12794714 and rs3886163, respectively) was negatively associated with vitamin D production (ß = - 1.24; 95% CI - 2.42 to - 0.06). CONCLUSIONS: Genetic variants in the vitamin D pathway affect vitamin D serum levels and, thus, atopy and asthma.
RESUMO
Toxocariasis, a natural helminth infection of dogs and cats caused by Toxocara canis and T. cati, respectively, that are transmitted to mammals, including humans. Infection control is based currently on periodic antihelmintic treatment and there is a need for the development of vaccines to prevent this infection. MATERIALS AND METHODS: Eight potential vaccine candidate T. canis recombinant proteins were identified by in silico (rTcGPRs, rTcCad, rTcVcan, rTcCyst) and larval proteomics (rTES26, rTES32, rMUC-3 and rCTL-4) analyses. Immunogenicity and protection against infectious challenge for seven of these antigens were determined in a murine model of toxocariasis. C57BL/6 female mice were immunized with each of or combinations of recombinant antigens prior to challenge with 500 T. canis embryonated eggs. Levels of specific antibodies (IgG, IgG1, IgG2a and IgE) in sera and cytokines (IL-5, INF-ɣ and IL-10) produced by antigens-stimulated splenocytes, were measured. Presence of specific antibodies to the molecules was measured in sera of T. canis-seropositive dogs and humans. RESULTS: All seven molecules were immunogenic in immunized mice; all stimulated significantly elevated levels of specific IgG, IgG1 or IgG2a and six were associated with elevated levels of specific IgE; all induced elevated production of IFN- ɣ and IL-10 by splenocytes, but only the in silico-identified membrane-associated recombinants (rTcCad, rTcVcan, and rTcCyst) induced significantly increased IL-5 production. Vaccination with two of the latter (rTcCad and rTcVcan) reduced larval loads in the T. canis challenged mice by 54.3% and 53.9% (P < 0.0001), respectively, compared to unimmunized controls. All seven recombinants were recognized by T. canis-seropositive dog and human sera. CONCLUSION: The identification of vaccine targets by in silico analysis was an effective strategy to identify immunogenic T. canis proteins capable of reducing larval burdens following challenge with the parasite. Two recombinant proteins, rTcCad and rTcVcan, were identified as promising vaccine candidates for canine toxocariasis.