RESUMEN
BACKGROUND: IgE to galactose alpha-1,3 galactose (alpha-gal) causes alpha-gal syndrome (delayed anaphylaxis after ingestion of mammalian meat). Development of sensitization has been attributed to tick bites; however, the possible role of other parasites has not been well studied. OBJECTIVE: Our aims were to assess the presence, relative abundances, and site of localization of alpha-gal-containing proteins in common ectoparasites and endoparasites endemic in an area of high prevalence of alpha-gal syndrome, as well as to investigate the ability of ascaris antigens to elicit a reaction in a humanized rat basophil in vitro sensitization model. METHODS: Levels of total IgE, Ascaris-specific IgE, and alpha-gal IgE were measured in sera from patients with challenge-proven alpha-gal syndrome and from controls without allergy. The presence, concentration, and localization of alpha-gal in parasites were assessed by ELISA, Western blotting, and immunohistochemistry. The ability of Ascaris lumbricoides antigen to elicit IgE-dependent reactivity was demonstrated by using the RS-ATL8 basophil reporter system. RESULTS: Alpha-gal IgE level correlated with A lumbricoides-specific IgE level. Alpha-gal protein at 70 to 130 kDa was detected in A lumbricoides at concentrations higher than those found in Rhipicephalus evertsi and Amblyomma hebraeum ticks. Immunohistochemistry was used to localize alpha-gal in tick salivary acini and the helminth gut. Non-alpha-gal-containing A lumbricoides antigens activated RS-ATL8 basophils primed with serum from subjects with alpha-gal syndrome. CONCLUSION: We demonstrated the presence, relative abundances, and site of localization of alpha-gal-containing proteins in parasites. The activation of RS-ATL8 IgE reporter cells primed with serum from subjects with alpha-gal syndrome on exposure to non-alpha-gal-containing A lumbricoides proteins indicates a possible role of exposure to A lumbricoides in alpha-gal sensitization and clinical reactivity.
Asunto(s)
Ascaris lumbricoides/inmunología , Hipersensibilidad a los Alimentos/etiología , Garrapatas/inmunología , Animales , Antígenos Helmínticos/inmunología , Células Cultivadas , Disacáridos/análisis , Humanos , Inmunoglobulina E/inmunología , RatasRESUMEN
Schistosomiasis is a parasitic neglected disease with praziquantel (PZQ) utilized as the main drug for treatment, despite its low effectiveness against early stages of the worm. To aid in the search for new drugs to tackle schistosomiasis, computer-aided drug design has been proved a helpful tool to enhance the search and initial identification of schistosomicidal compounds, allowing fast and cost-efficient progress in drug discovery. The combination of high-throughput in silico data followed by in vitro phenotypic screening assays allows the assessment of a vast library of compounds with the potential to inhibit a single or even several biological targets in a more time- and cost-saving manner. Here, we describe the molecular docking for in silico screening of predicted homology models of five protein kinases (JNK, p38, ERK1, ERK2, and FES) of Schistosoma mansoni against approximately 85,000 molecules from the Managed Chemical Compounds Collection (MCCC) of the University of Nottingham (UK). We selected 169 molecules predicted to bind to SmERK1, SmERK2, SmFES, SmJNK, and/or Smp38 for in vitro screening assays using schistosomula and adult worms. In total, 89 (52.6%) molecules were considered active in at least one of the assays. This approach shows a much higher efficiency when compared to using only traditional high-throughput in vitro screening assays, where initial positive hits are retrieved from testing thousands of molecules. Additionally, when we focused on compound promiscuity over selectivity, we were able to efficiently detect active compounds that are predicted to target all kinases at the same time. This approach reinforces the concept of polypharmacology aiming for "one drug-multiple targets". Moreover, at least 17 active compounds presented satisfactory drug-like properties score when compared to PZQ, which allows for optimization before further in vivo screening assays. In conclusion, our data support the use of computer-aided drug design methodologies in conjunction with high-throughput screening approach.
Asunto(s)
Esquistosomiasis mansoni , Esquistosomiasis , Animales , Simulación del Acoplamiento Molecular , Praziquantel/farmacología , Praziquantel/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Schistosoma mansoni , Esquistosomiasis mansoni/tratamiento farmacológicoRESUMEN
Schistosomiasis is a neglected tropical disease (NTD) caused by helminthes from the Schistosoma genus. This NTD can cause systemic symptoms induced by the deposition of parasite eggs in the host liver, promoting severe complications. Functional studies to increase knowledge about parasite biology are required for the identification of new drug targets, because the treatment is solely based on praziquantel administration, a drug in which the mechanism of action is still unknown. Protein kinases are important for cellular adaptation and maintenance of many organisms homeostasis and, thus, are considered good drug targets for many pathologies. Accordingly, those proteins are also important for Schistosoma mansoni, as the parasite relies on specific environmental signals to develop into its different stages. However, the specific roles of protein kinases in S. mansoni biology are not well understood. This work aims at investigating the tyrosine-protein kinase FES (Feline Sarcoma) functions in the maintenance of S. mansoni life cycle, especially in the establishment of mammalian and invertebrate hosts' infection. In this regard, the verification of Smfes expression among S. mansoni stages showed that Smfes is more expressed in infective free-living stages: miracidia and cercariae. Schistosomula exposed to SmFES-dsRNA in vitro presented a reduction in movement and size and increased mortality. Mice infected with Smfes-knocked-down schistosomula exhibited a striking reduction in the area of liver granuloma and an increased rate of immature eggs in the intestine. Female adult worms recovered from mice presented a reduced size and changes in the ovary and vitellarium; and males exhibited damage in the gynecophoral canal. Subsequently, miracidia hatched from eggs exposed to SmFES-dsRNA presented changes in its capability to infect and to sense the snail mucus. In addition, the SmFES RNAi effect was stable from miracidia to cercariae. The establishment of infection with those cercariae reproduced the same alterations observed for the knocked-down schistosomula infection. Our findings show that SmFES tyrosine kinase (1) is important in schistosomula development and survival; (2) has a role in adult worms pairing and, consequently, female maturation; (3) might be essential for egg antigen expression, thus responsible for inducing granuloma formation and immunomodulation; and (4) is essential for miracidia infection capability. In addition, this is the first time that a gene is kept knocked down during three different S. mansoni life stages and that a tyrosine kinase is implicated in the parasite reproduction and infection establishment in the mammalian host. Accordingly, SmFES should be explored as an alternative to support schistosomiasis treatment and morbidity control.
RESUMEN
The screening of compound libraries to identify small-molecule modulators of specific biological targets is crucial in the process for the discovery of novel therapeutics and molecular probes. Considering the need for simple single-tool assay technologies with which one could monitor "all" kinases, we developed a fluorescence polarization (FP)-based assay to monitor the binding capabilities of protein kinases to ATP. We used BODIPY ATP-y-S as a probe to measure the shift in the polarization of a light beam when passed through the sample. We were able to optimize the assay using commercial Protein Kinase A (PKA) and H7 efficiently inhibited the binding of the probe when added to the reaction. Furthermore, we were able to employ the assay in a high-throughput fashion and validate the screening of a set of small molecules predicted to dock into the ATP-binding site of PKA. This will be useful to screen larger libraries of compounds that may target protein kinases by blocking ATP binding.
RESUMEN
Tuberculosis (TB) is a major public health concern worldwide; however the factors that account for resistance or susceptibility to disease are not completely understood. Although some studies suggest that the differential expression of miRNAs in peripheral blood of TB patients could be useful as biomarkers of active disease, their involvement during the inflammatory process in lungs of infected individuals is unknown. Here, we evaluated the global expression of miRNAs in the lungs of mice experimentally infected with Mycobacterium tuberculosis on 30 and 60 days post-infection. We observed that several miRNAs were differentially expressed compared to uninfected mice. Furthermore, we verified that the expression of miR-135b, miR-21, miR-155, miR-146a, and miR-146b was significantly altered in distinct leukocyte subsets isolated from lungs of infected mice, while genes potentially targeted by those miRNAs were associated with a diversity of immune related molecular pathways. Importantly, we validated the inhibition of Pellino 1 expression by miR-135b in vitro. Overall, this study contributes to the understanding of the dynamics of miRNA expression in lungs during experimental TB and adds further perspectives into the role of miRNAs on the regulation of immune processes such as leukocyte activation.
Asunto(s)
Pulmón/metabolismo , MicroARNs/genética , Transcriptoma/genética , Tuberculosis Pulmonar/genética , Animales , Células Cultivadas , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/inmunología , Pulmón/inmunología , Subgrupos Linfocitarios/inmunología , Ratones Endogámicos BALB C , MicroARNs/inmunología , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Transcriptoma/inmunología , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/metabolismo , Ubiquitina-Proteína Ligasas/biosíntesis , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Previous work has suggested that Trypanosoma cruzi diphosphohydrolase 1 (TcNTPDase-1) may be involved in the infection of mammalian cells and serve as a potential target for rational drug design. In this work, we produced recombinant TcNTPDase-1 and evaluated its nucleotidase activity, cellular localization and role in parasite adhesion to mammalian host cells. TcNTPDase-1 was able to utilize a broad range of triphosphate and diphosphate nucleosides. The enzyme's Km for ATP (0.096 mM) suggested a capability to influence the host's ATP-dependent purinergic signaling. The use of specific polyclonal antibodies allowed us to confirm the presence of TcNTPDase-1 at the surface of parasites by confocal and electron microscopy. In addition, electron microscopy revealed that TcNTPDase-1 was also found in the flagellum, flagellum insertion region, kinetoplast, nucleus and intracellular vesicles. The presence of this enzyme in the flagellum insertion region and vesicles suggests that it may have a role in nutrient acquisition, and the widespread distribution of TcNTPDase-1 within the parasite suggests that it may be involved in other biological process. Adhesion assays using anti-TcNTPDase-1 polyclonal antibodies as a blocker or purified recombinant TcNTPDase-1 as a competitor revealed that the enzyme has a role in parasite-host cell adhesion. These data open new frontiers to future studies on this specific parasite-host interaction and other unknown functions of TcNTPDase-1 related to its ubiquitous localization.