Browsing by Author "Anes, Elsa"
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- Anti-inflammatory effects of phosphatidylcholinePublication . Treede, Irina; Braun, Annika; Sparla, Richard; Kuehnel, Mark; Giese, Thomas; Turner, Jerrold R.; Anes, Elsa; Kulaksiz, Hasan; Fuellekrug, Joachim; Stremmel, Wolfgang; Griffiths, Gareth; Ehehalt, RobertWe recently showed that mucus from patients with ulcerative colitis, a chronic inflammatory disorder of the colon, is characterized by a low level of phosphatidylcholine (PC) while clinical studies reveal that therapeutic addition of PC using slow release
- Antimycobacterial evaluation and preliminary phytochemical investigation of selected medicinal plants traditionally used in MozambiquePublication . Luo, Xuan; Pires, David; Aínsa, José; Gracia, Begoña; Mulhovo, Silva; Duarte, Aida; Anes, Elsa; Ferreira, Maria José UmbelinoEthnopharmacological relevance Several medicinal plants are traditionally used in Mozambique to treat tuberculosis and related symptoms. Aims of the study It was aimed to assess the in vitro antimycobacterial activity of crude extracts from fifteen medicinal plants and to reveal main classes of compounds which may account for the activity of extracts. Methods and materials The plant materials were sequentially extracted by n-hexane, dichloromethane, ethyl acetate, and 70% ethanol. Decoction of each plant material was also prepared according to traditional use. Broth microdilution method was employed to screen extracts against two mycobacterial species: Mycobacterium smegmatis ATCC 607 and Mycobacterium tuberculosis H37Rv. The extracts with minimum inhibitory concentration(s) (MIC) below 125 μg/mL were considered active and further tested against different mycobacterial species and strains, namely Mycobacterium tuberculosis H37Ra, Mycobacterium bovis BCG ATCC 35734, Mycobacterium smegmatis mc2 155, Mycobacterium avium DSM 44156 and DSM 44157. Cytotoxic effect was evaluated against human macrophages from the monocytic THP-1 cells. Main classes of compounds in these active extracts were proposed from their 1H NMR spectroscopic characterizations. Results n-Hexane extracts of Maerua edulis and Securidaca longepedunculata, ethyl acetate extract of Tabernaemontana elegans and dichloromethane extract of Zanthoxylum capense were found to possess considerable activity against Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra with MIC 15.6–62.5 μg/mL. Tabernaemontana elegans ethyl acetate extract displayed strong activity against Mycobacterium tuberculosis H37Rv (MIC 15.6 μg/mL). Except for Tabernaemontana elegans ethyl acetate extract which presented potent cytotoxic effects in THP-1 cells (IC50 < 4 μg/mL), the other three plant extracts showed moderate to none toxicity. Based on 1H NMR spectroscopic analysis, major components in both Maerua edulis and Securidaca longepedunculata n-hexane extracts were linear chain unsaturated fatty acids. Zanthoxylum capense dichloromethane extract contained more complex constituents (mostly phenolic compounds). In the most potent extract, Tabernaemontana elegans ethyl acetate extract, the prominent compounds were identified as indole alkaloids. Conclusions The pronounced antimycobacterial activity of the medicinal plants Maerua edulis, Securidaca longepedunculata, Zanthoxylum capense, and Tabernaemontana elegans suggested that they might provide compounds which could be potential anti-TB drug leads.
- Application of confocal microscopy for quantification of intracellular mycobacteria in macrophages.Publication . Bettencourt, Paulo; Pires, David; Carmo, Nuno; Anes, ElsaThis chapter describes the techniques used to prepare a uniform and consistent mycobacterial culture and for the infection of macrophages in vitro. Here, protocols are described for the achievement of a certain number of single cell bacilli per macrophage. Confocal microscopy in combination with the software ImageJ are highlighted, and these techniques will be correlated with quantification by FACS and confirmed by colony forming units (CFU) the classical method to validate the intracellular survival of Mycobacterium tuberculosis. Conventional CFU for quantification of intracellular slow growing mycobacteria is labour-intensive, with incubation requirements that can take up to several weeks. New alternatives and fast methods are required for a rapid assessment of the immune response as well to test new antibacterial drugs in high- throughput screens.
- cAMP synthesis and degradation by phagosomes regulate actin assembly and fusion eventsPublication . Kalamidas, Stefanos A.; Kuehnel, Mark P.; Peyron, Pascale; Rybin, Vladimir; Rauch, Susanne; Kotoulas, Othon B.; Houslay, Miles; Hemmings, Brian A.; Gutierrez, Maximiliano G.; Anes, Elsa; Griffiths, GarethWe showed recently that actin assembly by phagosomal membranes facilitates fusion with late endocytic organelles in macrophages. Moreover, lipids that induced phagosomal actin also stimulated this fusion process. In macrophages infected with pathogenic my
- Development of Chitosan Particles Loaded with siRNA for Cystatin C to Control Intracellular Drug-Resistant Mycobacterium tuberculosisPublication . Pires, David; Mandal, Manoj; Matos, Ana I.; Peres, Carina; Catalão, Maria João; Azevedo-Pereira, José M.; Satchi-Fainaro, Ronit; Florindo, Helena F; Anes, ElsaThe golden age of antibiotics for tuberculosis (TB) is marked by its success in the 1950s of the last century. However, TB is not under control, and the rise in antibiotic resistance worldwide is a major threat to global health care. Understanding the complex interactions between TB bacilli and their host can inform the rational design of better TB therapeutics, including vaccines, new antibiotics, and host-directed therapies. We recently demonstrated that the modulation of cystatin C in human macrophages via RNA silencing improved the anti-mycobacterial immune responses to Mycobacterium tuberculosis infection. Available in vitro transfection methods are not suitable for the clinical translation of host-cell RNA silencing. To overcome this limitation, we developed different RNA delivery systems (DSs) that target human macrophages. Human peripheral blood-derived macrophages and THP1 cells are difficult to transfect using available methods. In this work, a new potential nanomedicine based on chitosan (CS-DS) was efficiently developed to carry a siRNA-targeting cystatin C to the infected macrophage models. Consequently, an effective impact on the intracellular survival/replication of TB bacilli, including drug-resistant clinical strains, was observed. Altogether, these results suggest the potential use of CS-DS in adjunctive therapy for TB in combination or not with antibiotics.
- Efeito de anti-inflamatórios não esteróides no Mycobacterium tuberculosisPublication . Simões, Marta Filpa; Anes, Elsa
- Effects of omega-3 and-6 fatty acids on Mycobacterium tuberculosis in macrophages and in micePublication . Jordao, Luisa; Lengeling, Andreas; Bordat, Yann; Boudou, Frederic; Gicquel, Brigitte; Neyrolles, Olivier; Becker, Pablo D.; Guzman, Carlos A.; Griffiths, Gareth; Anes, ElsaWe recently showed that treatment of macrophages prior to Mycobacterium tuberculosis infection with the pro-inflammatory omega-6 lipid, arachidonic acid (AA) enhanced bacterial killing whereas the anti - inflammatory, omega-3 lipid eicosapentaenoic acid (. - FCT ; FEDER [POCI/BIA-BCM/55327/2004, SFRWBD/14284/2003]. - We thank Cecilia Rodrigues, Maximilliano Gutierrez, Luis Mayorga and Sabrina Marion for their constructive support and discussion. This work was financed by FCT with co-participation of FEDER (POCI/BIA-BCM/55327/2004 and SFRWBD/14284/2003).
- ESAT-6 a Major Virulence Factor of Mycobacterium tuberculosisPublication . Anes, Elsa; Pires, David; Mandal, Manoj; Azevedo-Pereira, José MiguelMycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis (TB), is one of the most successfully adapted human pathogens. Human-to-human transmission occurs at high rates through aerosols containing bacteria, but the pathogen evolved prior to the establishment of crowded populations. Mtb has developed a particular strategy to ensure persistence in the host until an opportunity for transmission arises. It has refined its lifestyle to obviate the need for virulence factors such as capsules, flagella, pili, or toxins to circumvent mucosal barriers. Instead, the pathogen uses host macrophages, where it establishes intracellular niches for its migration into the lung parenchyma and other tissues and for the induction of long-lived latency in granulomas. Finally, at the end of the infection cycle, Mtb induces necrotic cell death in macrophages to escape to the extracellular milieu and instructs a strong inflammatory response that is required for the progression from latency to disease and transmission. Common to all these events is ESAT-6, one of the major virulence factors secreted by the pathogen. This narrative review highlights the recent advances in understanding the role of ESAT-6 in hijacking macrophage function to establish successful infection and transmission and its use as a target for the development of diagnostic tools and vaccines.
- Esters of pyrazinoic acid are active against pyrazinamide-resistant strains of Mycobacterium tuberculosis and other naturally resistant mycobacteria in vitro and ex vivo within macrophagesPublication . Pires, David; Valente, Emília; Simões, Marta Filipa; Carmo, Nuno; Testa, Bernard; Constantino, Luis; Anes, ElsaPyrazinamide (PZA) is active against major Mycobacterium tuberculosis species (M. tuberculosis, M. africanum, and M. microti) but not against M. bovis and M. avium. The latter two are mycobacterial species involved in human and cattle tuberculosis and in HIV coinfections, respectively. PZA is a first-line agent for the treatment of human tuberculosis and requires activation by a mycobacterial pyrazinamidase to form the active metabolite pyrazinoic acid (POA). As a result of this mechanism, resistance to PZA, as is often found in tuberculosis patients, is caused by point mutations in pyrazinamidase. In previous work, we have shown that POA esters and amides synthesized in our laboratory were stable in plasma (M. F. Simões, E. Valente, M. J. Gómez, E. Anes, and L. Constantino, Eur J Pharm Sci 37:257-263, 2009, http://dx.doi.org/10.1016/j.ejps.2009.02.012). Although the amides did not present significant activity, the esters were active against sensitive mycobacteria at concentrations 5- to 10-fold lower than those of PZA. Here, we report that these POA derivatives possess antibacterial efficacy in vitro and ex vivo against several species and strains of Mycobacterium with natural or acquired resistance to PZA, including M. bovis and M. avium. Our results indicate that the resistance probably was overcome by cleavage of the prodrugs into POA and a long-chain alcohol. Although it is not possible to rule out that the esters have intrinsic activity per se, we bring evidence here that long-chain fatty alcohols possess a significant antimycobacterial effect against PZA-resistant species and strains and are not mere inactive promoieties. These findings may lead to candidate dual drugs having enhanced activity against both PZA-susceptible and PZA-resistant isolates and being suitable for clinical development.
- Exosomal Hsp70 induces a pro-inflammatory response to foreign particles including mycobacteriaPublication . Anand, Paras K.; Anand, Ellis; Bleck, Christopher K. E.; Anes, Elsa; Griffiths, GarethBackground: Exosomes are endosome-derived vesicles that are released when multi-vesicular bodies (MVBs) fuse with the plasma membrane. Exosomes released from mycobacteria-infected cells have recently been shown to be pro-inflammatory. A prominent host molecule that is found within these exosomes is Hsp70, a member of the heat-shock family of proteins. Methodology/Principal Findings: We first characterized the exosomes purified from control and mycobacteria-infected cells. We found that relative to uninfected cells, macrophages infected with M. smegmatis and M. avium release more exosomes and the exosomes they released had more Hsp70 on their surface. Both exosomes and exogenous Hsp70 treatment of macrophages led to NF-kB activation and TNFa release in uninfected macrophages; Hsp70 levels were elevated in mycobacteria-infected cells. Macrophage treatment with Hsp70 also led to increase in the phagocytosis and maturation of latex-bead phagosomes. Finally, Hsp70 pre-incubation of M. smegmatis- and M. avium-infected cells led to increased phago-lysosome fusion, as well as more killing of mycobacteria within macrophages. Conclusions/Significance: Our results fit into an emerging concept whereby exosomes-containing Hsp70 are effective inducers of inflammation, also in response to mycobacterial infection.
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