Purification and characterisation of the dimeric group 12 allergen from Blomia tropicalis heterologously expressed by Escherichia coli Top10F´.

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.

Proteomic Analysis Reveals Allergen Variability among Breeds of the Dust Mite Blomia tropicalis.

BACKGROUND: The dawn of the "omics" technologies has changed allergy research, increasing the knowledge and identification of new allergens. However, these studies have been almost restricted to Dermatophagoides spp. Although Blomia tropicalis has long been established as a clinically important source of allergens, a thorough proteomic characterization is still lacking for this dust mite. OBJECTIVE: To increase knowledge of B. tropicalis allergens through proteomic analysis. METHODS: Eleven in-bred lineages of B. tropicalis were obtained from 11 unique different pregnant females. Their somatic extracts were analyzed and compared with a commercially available extract by liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS: Considerable differences in the protein expression profiles were found among the breeds, and most of them displayed higher expression levels of major allergens than the commercially available extract. Blo t 2 was the most prominent allergenic protein in the analyzed extracts. Six identified allergens and 14 isoforms have not yet been recognized by IUIS. Conversely, 3 previously recognized B. tropicalis allergens were not found. CONCLUSIONS: The clear impact of inbreeding on allergen content shown by our study leads us to conclude that the quantification and/or identification of allergens from in-bred lines should be routinely considered for mite cultivation in order to select breeds with higher amounts of major allergens. In this sense, LC-MS/MS may be a useful method to achieve this quality control for research and commercial purposes.

Allergens of Blomia tropicalis: An Overview of Recombinant Molecules.

Allergic diseases are considered a major problem for healthcare systems in both developed and developing countries. House dust mites are well-known triggers of allergic manifestations. While the Dermatophagoides genus is widely distributed globally, Blomia tropicalis is the most prominent mite species in the tropical and subtropical regions of the world. Over the last decades, an increase in sensitization rates to B. tropicalis has been reported, leading to increased research efforts on Blomia allergens. In fact, 8 new allergens have been identified and characterized to different degrees. Here, we provide an overview of recent developments concerning the identification and production of recombinant Blomia allergens, as well as their structural and immunological characterization. Although considerable progress has been achieved, detailed molecule-based studies are still needed to better define the clinical relevance of Blomia allergens. Thus, the establishment of a well-standardized and fully characterized panel of allergens remains a challenge for the development of better diagnosis and therapy of allergic diseases induced by B. tropicalis.

Recombinant proteins of helminths with immunoregulatory properties and their possible therapeutic use.

The inverse relationship between helminth infections and the development of immune-mediated diseases is a cornerstone of the hygiene hypothesis and studies were carried out to elucidate the mechanisms by which helminth-derived molecules can suppress immunological disorders. These studies have fostered the idea that parasitic worms may be used as a promising therapeutic alternative for prevention and treatment of immune-mediated diseases. We discuss the current approaches for identification of helminth proteins with potential immunoregulatory properties, including the strategies based on high-throughput technologies. We also explore the methodological approaches and expression systems used for production of the recombinant forms of more than 20 helminth immunomodulatory proteins, besides their performances when evaluated as immunotherapeutic molecules to treat different immune-mediated conditions, including asthma and inflammatory bowel diseases. Finally, we discuss the perspectives of using these parasite-derived recombinant molecules as tools for future immunotherapy and immunoprophylaxis of human inflammatory diseases.

Toxocariasis and Toxocara vaccine: a review / Toxocariasis y vacunación para Toxocara: una revisión sistemática / Toxocaríase e vacina para controle de Toxocara: uma revisão

Abstract Based on prevalence and impact on public health, toxocariasis is an underestimated zoonosis in developing and developed countries. The transmission of Toxocara spp. involves pets, stray dogs and cats (Canis familiaris and Felis catus, respectively), which spread the parasite's eggs in their feces to the environment. One of the main risk factors for the infection and development of human toxocariasis, is to cohabit with puppies and kittens. For a long time, the preventive strategy for this parasitic infection has been the regular use of antiparasitic drugs to reduce parasite burden in the short term. A long lasting immunological protection can be achieved with vaccination, however, a vaccine is not yet available. Therefore, it is fundamental to know and to understand the state of the art of vaccine development for effective control of this zoonosis. This paper reviews the experimental studies focused on vaccine development for toxocariasis control, and special attention is given to relevant epidemiological studies on the importance of dogs in human toxocariasis.

Resumen Según la prevalencia y el impacto en la salud pública, la toxocariasis es una zoonosis subestimada en los países en desarrollo y desarrollados. La transmisión de Toxocara spp. involucra animales de compañía caninos y felinos, como también perros y gatos sin hogar (Canis familiaris y Felis catus, respectivamente), que diseminan los huevos del parásito en sus heces al medio ambiente. Uno de los principales factores de riesgo para la infección y el desarrollo de la toxocariasis humana es convivir con cachorros felinos y caninos. Durante mucho tiempo, la estrategia preventiva para esta infección parasitaria ha sido el uso regular de medicamentos antiparasitarios para reducir la carga parasitaria a corto plazo. Se puede lograr una protección inmunológica duradera con la vacunación, sin embargo, todavía no se dispone de una vacuna. Por lo tanto, es fundamental conocer y comprender el estado del arte del desarrollo de vacunas para el control efectivo de esta zoonosis. Este artículo revisa los estudios experimentales centrados en el desarrollo de vacunas para el control de la toxocariasis, y se presta especial atención a los estudios epidemiológicos relevantes sobre la importancia de los caninos domésticos en la toxocariasis humana.

Resumo Com base na prevalência e no impacto na saúde pública, a toxocaríase é uma zoonose subestimada nos países em desenvolvimento e desenvolvidos. A transmissão de Toxocara spp. envolve animais cães e gatos de estimação e vadios (Canis familiaris e Felis catus, respectivamente), que espalham os ovos do parasita nas fezes para o meio ambiente. Um dos principais fatores de risco para a infecção e desenvolvimento da toxocaríase humana é coabitar com filhotes de cachorros e gatos. Por um longo tempo, a estratégia preventiva para essa infecção parasitária tem sido o uso regular de medicamentos antiparasitários para reduzir a carga parasitária a curto prazo. Uma proteção imunológica duradoura pode ser alcançada com a vacinação, no entanto, uma vacina ainda não está disponível. Portanto, é fundamental conhecer e entender o estado da arte do desenvolvimento de vacinas para o controle efetivo dessa zoonose. Este artigo revisa os estudos experimentais focados no desenvolvimento de vacinas para o controle da toxocaríase, e atenção especial é dada a estudos epidemiológicos relevantes sobre a importância dos cães na toxocaríase humana.

DNA Vaccine Encoding the Chimeric Form of Schistosoma mansoni Sm-TSP2 and Sm29 Confers Partial Protection against Challenge Infection.

Schistosomiasis is an important parasitic disease worldwide that affects more than 207 million people in 76 countries and causes approximately 250,000 deaths per year. The best long-term strategy to control schistosomiasis is through immunization combined with drug treatment. Due to the ability of DNA vaccines to generate humoral and cellular immune responses, such vaccines are considered a promising approach against schistosomiasis. Sm29 and tetraspanin-2 (Sm-TSP2) are two proteins that are located in the S. mansoni tegument of adult worms and schistosomula and induce high levels of protection through recombinant protein immunization. In this study, we transfected BHK-21 cells with plasmids encoding Sm29, Sm-TSP2 or a chimera containing both genes. Using RT-PCR analysis and western blot, we confirmed that the DNA vaccine constructs were transcribed and translated, respectively, in BHK-21 cells. After immunization of mice, we evaluated the reduction in worm burden. We observed worm burden reductions of 17-22%, 22%, 31-32% and 24-32% in animals immunized with the pUMVC3/Sm29, pUMVC3/SmTSP-2, pUMVC3/Chimera and pUMVC3/Sm29 + pUMVC3/SmTSP-2 plasmids, respectively. We evaluated the humoral response elicited by DNA vaccines, and animals immunized with pUMVC3/Sm29 and pUMVC3/Sm29 + pUMVC3/SmTSP-2 showed higher titers of anti-Sm29 antibodies. The cytokine profile produced by the spleen cells of immunized mice was then evaluated. We observed higher production of Th1 cytokines, such as TNF-α and IFN-γ, in vaccinated mice and no significant production of IL-4 and IL-5. The DNA vaccines tested in this study showed the ability to generate a protective immune response against schistosomiasis, probably through the production of Th1 cytokines. However, future strategies aiming to optimize the protective response induced by a chimeric DNA construct need to be developed.