Induction of CD4+CD25+FOXP3+ Regulatory T Cells during Human Hookworm Infection Modulates Antigen-Mediated Lymphocyte Proliferation

Hookworm infection is considered one of the most important poverty-promoting neglected tropical diseases, infecting 576 to 740 million people worldwide, especially in the tropics and subtropics. These blood-feeding nematodes have a remarkable ability to downmodulate the host immune response, protecting themselves from elimination and minimizing severe host pathology. While several mechanisms may be involved in the immunomodulation by parasitic infection, experimental evidences have pointed toward the possible involvement of regulatory T cells (Tregs) in downregulating effector T-cell responses upon chronic infection. However, the role of Tregs cells in human hookworm infection is still poorly understood and has not been addressed yet. In the current study we observed an augmentation of circulating CD4+CD25+FOXP3+ regulatory T cells in hookworm-infected individuals compared with healthy non-infected donors. We have also demonstrated that infected individuals present higher levels of circulating Treg cells expressing CTLA-4, GITR, IL-10, TGF-β and IL-17. Moreover, we showed that hookworm crude antigen stimulation reduces the number of CD4+CD25+FOXP3+ T regulatory cells co-expressing IL-17 in infected individuals. Finally, PBMCs from infected individuals pulsed with excreted/secreted products or hookworm crude antigens presented an impaired cellular proliferation, which was partially augmented by the depletion of Treg cells. Our results suggest that Treg cells may play an important role in hookworm-induced immunosuppression, contributing to the longevity of hookworm survival in infected people

Variabilidade molecular e aAnálise filogeográfica de populações Brasileiras de Ancylostoma caninum.

Exportado OPUSMade available in DSpace on 2019-08-12T18:14:28Z (GMT). No. of bitstreams: 1 tese_corrigida_rodrigo_miranda.pdf: 2824265 bytes, checksum: 245a516c1df329ecdd369709ce41afb4 (MD5) Previous issue date: 20O Ancylostoma caninum (Ercolani, 1859) possui uma ampla distribuição geográfica, representando um risco para a saúde animal e humana. Além disso, representa um importante modelo de estudo para as demais espécies antropofílicas de ancilostomídeos. Presentemente, está sendo realizado um projeto para desenvolver uma vacina anti-ancilostomídeos o qual está sendo executado pelo Sabin Vaccine Institute em conjunto com a George Washington University / EUA e outras instituições. Considerando os grandes esforços e investimentos financeiros que estão sendo empregados na tentativa de se formular uma vacina contra ancilostomídeos, se faz necessário a implementação de projetos para estudar a variabilidade molecular e a diversidade e estrutura genética populacional destes patógenos. Este trabalho teve como objetivo investigar a estrutura genética e a variabilidade molecular de populações brasileiras de A. caninum, utilizando-se marcadores moleculares mitocondriais e nucleares, incluindo um gene codificante de uma proteína importante para estratégias imunoprofiláticas, a Ancylostoma secreted protein 2, Ac-ASP-2. Foram coletadas amostras de A. caninum em centros de controle de zoonoses de cinco localidades brasileiras: Belo Horizonte/MG (BH = 36 indivíduos), Campo Grande/MS (CG = 46 indivíduos), Curitiba/PR (CT = 35 indivíduos), Ribeirão Preto/SP (11 indivíduos) e São Luís/MA (49 indivíduos). Os vermes foram identificados morfologicamente e submetidos individualmente à extração de DNA. Posteriormente, avaliou-se a diversidade e estrutura genética de populações brasileiras de A. caninum utilizando o marcador mitocondrial citocromo c oxidase subunidade I (COI), marcadores nucleares conhecidos como espaços transcritos internos (ITS-1 e ITS-2) e microssatélites de DNA. Os níveis de diversidade molecular foram intermediários e constantes nas populações brasileiras de A. caninum. Os resultados revelaram ainda moderada diferenciação entre as populações avaliadas. Os dados obtidos com os marcadores ribossomais ITS-1 e ITS-2 se mostraram inadequados para o estudo de genética populacional em A. caninum devido ao baixo número de sítios polimórficos encontrados. Alguns fatores foram discutidos para explicar a estrutura genética observada nas populações brasileiras de A. caninum: (i) distância geográfica evitando o evento de panmixia entre as localidades avaliadas; (ii) fluxos gênicos parciais entre subpopulações amostradas, incluindo a influência de movimentos recentes de hospedeiros; (iii) a presença de subpopulações distintas geneticamente melhores adaptadas à diferentes hospedeiros e/ou a presença de espécies crípticas dentro de uma mesma localização geográfica; e (iv) outros eventos genéticos (por exemplo, deriva genética) ocorrendo independentemente em cada subpopulação. Além dos estudos de Biologia Populacional com marcadores neutros, um fragmento genômico do gene Ac-ASP-2 foi também seqüenciado utilizando-se os vermes das cinco localidades brasileiras amostradas. As análises reveleram um considerável polimorfismo nucleotídico. A maior parte da variabilidade foi encontrada nos íntrons devido a menor pressão seletiva destes segmentos. As regiões codificantes também revelaram polimorfismos nucleotídicos e aminoacídicos distribuídos de forma não homogênea entre os éxons avaliados. Estes resultados contribuem para uma melhor compreensão da ecologia, padrões de transmissão, desenvolvimento de resistência à drogas e desenvolvimento de vacinas contra ancilostomídeos

Dominance of P-glycoprotein 12 in phenotypic resistance conversion against ivermectin in Caenorhabditis elegans.

While diseases caused by nematodes remains a considerable drawback for the livestock, agriculture and public health, anthelmintics drug resistance has been observed over the past years and is a major concern for parasite control. Ivermectin, initially considered as a highly potent drug, currently presents a reduced anti-helminthic efficacy, which is influenced by expression of several ATP-binding cassette transporters (ABC), among them the P-glycoproteins (Pgps). Here we present some evidences of Pgps dominance during Ivermectin resistance/susceptibility using Pgps double silencing in C. elegans and the phylogenetic relationship of Pgps among nematodes, which strengthen the use of this model for study of drug resistance in nematodes. Firstly, we evaluated the quantitative gene expression of 12 out the 15 known Pgps from resistant and WT strains of C. elegans, we demonstrated the upregulation of Pgps 12 and 13 and downregulation of all remaining Pgps in ivermectin resistant strain. By using an RNAi loss-of-function approach we observed that Pgp 12 gene silencing reverts the resistance phenotype to ivermectin, while Pgp 4 gene silencing does not alter the resistance phenotype but induces a resistance in wild type strain. Interestingly, the dual silencing of Pgp 12 and Pgp 4 expression demonstrates the dominance of phenotype promoted by Pgp 12 silencing. Finally, in silico analysis reveals a close relationship between Pgps from C. elegans and several nematodes parasites. Taken together, our results indicate that Pgp 12 is crucial for the resistance to ivermectin and thus a good candidate for further studies aiming to develop specific inhibitors to this transporter, allowing the continuous use of ivermectin to control the burden on animal and human health inflicted by nematode parasites globally

Identification of candidate antigens from adult stages of Toxocara canis for the serodiagnosis of human toxocariasis

In the present work, we identified adult Toxocara canis antigens through sodium dodecyl sulfate-polyacrylamide gel electrophoresis for potential use in human toxocariasis immunodiagnosis. The sensitivity and specificity of several semi-purified antigens, as well as their cross-reactivity with other parasitic infections, were assessed by IgM and IgG-enzime linked immunosorbent assay. Whilst we found that the crude extract of the parasite presented limited sensitivity, specificity and high cross-reactivity against other parasites, we identified 42, 58, 68 and 97-kDa semi-purified antigens as the most promising candidates for immunodiagnosis. Moreover, the 58 and 68-kDa antigens presented the lowest IgM cross-reactivity. When tested as a combination, a mixture of the 58 and 68-kDa antigens presented 100% sensitivity and specificity, as well as minor cross-reactivity. Although the combination of the 42, 58, 68 and 97-kDa antigens presented 100% sensitivity at a dilution of 1:40, the low specificity and high cross-reactivity observed suggested a limited use for diagnostic purposes. Our data suggested that the 58 and 68-kDa antigens might be most suitable for the immunodiagnosis of human toxocariasis

Primers and sequence information.

<p>Primers and sequence information.</p

<i>In silico</i> analysis revealing a close relationship between Pgps from <i>C</i>. <i>elegans</i> and nematodes parasites represented.

<p>As follow: <i>Ascaris suum</i> (A), <i>Brugia malayi</i> (B), <i>Cooperia oncophora</i> (C), <i>Haemonchus contortus</i> (H), <i>Loa loa</i> (L), <i>Onchocerca volvulus</i> (O), <i>Parascaris equorum</i> (P), <i>Strongyloides ratti</i> (S), <i>Trichuris trichiura</i> (T), <i>Wuchereria bancrofti</i> (W), <i>C</i>. <i>brenneri</i> (1), <i>C</i>. <i>briggsae</i> (2), <i>C</i>. <i>remanei</i> (4), Pgp 4 of <i>C</i>. <i>elegans</i> (Δ), Pgp 12 of <i>C</i>. <i>elegans</i> (•), all remaining Pgps from <i>C</i>. <i>elegans</i> (2).</p

<i>C</i>. <i>elegans</i> Pgps genes profile using 2^-ΔΔCt method.

<p>Pgps gene expression for WT strains are represented by dashed line. P values were obtained with One sample t test, comparing sample value to WT expression reference value.</p

Evaluation of RNAi-mediated impairment of Pgp 4 and 12 expression in the WT and IVR30 strain.

<p>(A) Percentage of L₃ paralysis percentage after 48 hours ivermectin exposure for groups Pgps 4 and 12 silenced [RNAi⁺], Pgps 4 and 12 non silenced [RNAi^-] and negative control [CT], cultured only in M9 medium and not exposed to ivermectin treatment. (B) Assessement of L₃ viability cell using propidium iodide marker after 48 hours ivermectin exposure in Pgps 4 and 12 silenced [RNAi⁺] and Pgps 4 and 12 non silenced [RNAi^-] groups. P value refers to one-way ANOVA test with Turkey’s multiple comparisons test.</p

Evaluation of RNAi-mediated impairment of Pgp 12 expression in the WT and IVR30 strain.

<p>(A) Percentage of L₃ paralysis after 48 hours of ivermectin exposure for groups Pgp 12 silenced [RNAi⁺], Pgp 12 non silenced [RNAi^-] and negative control [CT], cultured only in M9 medium and not exposed to ivermectin treatment. (B) Assessment L₃ viability cell using propidium iodide marker after 48 hours ivermectin exposure in Pgp 12 silenced [RNAi⁺] and Pgp 12 non silenced [RNAi^-] groups. P value refers to one-way ANOVA test with Turkey’s multiple comparisons test.</p

Application of natural rubber latex as scaffold for osteoblast to guided bone regeneration

Natural rubber latex (NRL) from Hevea brasiliensis is a colloidal system composed of cis-1,4-polyisoprene. Its applications have grown due its angiogenic and wound healing activity. NRL has been used in guided bone regeneration as barrier, enhancing bone formation. However, there has been no study reported so far which shows its in vitro biocompatibility with osteoblasts. Thus, the aim of this work was to apply thermally induced phase separation under several temperatures to induce porosity in NRL; and test its mechanical properties, cytotoxicity, cell adhesion, and mineralization with MC3T3-E1. Only biomembranes submitted at -20 and -10 degrees C presented porosity. Fourier transform infrared spectroscopy showed no change in cis-1,4-isoprene spectra. Biomembranes were elastic (Young's modulu