Dogs are highly valued companions and work animals that are susceptible to many life-threatening conditions such as canine leishmaniosis (CanL). Plasma-derived extracellular vesicles (EVs), exploited ...extensively in biomarker discovery, constitute a mostly untapped resource in veterinary sciences. Thus, the definition of proteins associated with plasma EVs recovered from healthy and diseased dogs with a relevant pathogen would be important for biomarker development. For this, we recovered, using size-exclusion chromatography (SEC), EVs from 19 healthy and 20 CanL dogs' plasma and performed proteomic analysis by LC-MS/MS to define their core proteomic composition and search for CanL-associated alterations. EVs-specific markers were identified in all preparations and also non-EVs proteins. Some EVs markers such as CD82 were specific to the healthy animals, while others, such as the Integrin beta 3 were identified in most samples. The EVs-enriched preparations allowed the identification of 529 canine proteins that were identified in both groups, while 465 and 154 were only identified in healthy or CanL samples, respectively. A GO enrichment analysis revealed few CanL-specific terms.
spp. protein identifications were also found, although with only one unique peptide. Ultimately, CanL-associated proteins of interest were identified and a core proteome was revealed that will be available for intra- and inter-species comparisons.
Natural products are a very rich source for obtaining new compounds with therapeutic potential. In the search for new antiparasitic and antimicrobial agents, molecular hybrids were designed based on ...the structures of antimicrobial marine quinazolinones and eugenol, a natural phenolic compound. Following reports of the therapeutic potential of quinazolinones and eugenol derivatives, it was expected that the union of these pharmacophores could generate biologically relevant substances. The designed compounds were obtained by classical synthetic procedures and were characterized by routine spectrometric techniques. Nine intermediates and final products were then evaluated in vitro against Trypanosoma brucei and Leishmania infantum. Antifungal and antibacterial activity were also evaluated. Six compounds (9b, 9c, 9d, 10b, 10c, and 14) showed mild activity against T. brucei with IC50 in the range of 11.17–31.68 μM. Additionally, intermediate 9c showed anti-Leishmania activity (IC50 7.54 μM) and was six times less cytotoxic against THP-1 cells. In conclusion, novel derivatives with a simple quinazolinone scaffold showing selectivity against parasites without antibacterial and antifungal activities were disclosed, paving the way for new antitrypanosomal agents.
Sleeping sickness and leishmaniasis are neglected tropical diseases that threaten millions of people. The currently available therapies present several limitations, including high toxicity, lack of ...efficacy, and emerging drug resistance, prompting a search for novel therapeutic agents. In this work, we designed, synthesized, and in vitro evaluated the activity of new pyrimido5,4-dpyrimidines against Trypanosoma brucei and Leishmania infantum (promastigote and amastigote forms). The cytotoxicity of the compounds against the THP1 cell line was also assessed. Most tested compounds presented low micromolar activity against T. brucei with IC50 values in the range between 0.9 and 13.4 μM, and one compound also showed activity against L. infantum (IC50 = 3.13 μM). Several molecules presented a selectivity index higher than 10. The most active compound against booth parasites is derivative 4c, with IC50 = 0.94 μM (SI > 107) against T. brucei and IC50 = 3.13 μM (SI > 32) against L. infantum. This data enabled the identification of a new promising molecular scaffold for developing a novel class of antitrypanosomal and antileishmanial agents.
Fish rely on their innate immune responses to cope with the challenging aquatic environment, with antimicrobial peptides (AMPs) being one of the first line of defenses. Piscidins are a group of fish ...specific AMPs isolated in several species. However, in the European sea bass (
), the piscidin family remains poorly understood. We identified six different piscidins in sea bass, performed an in-depth molecular characterization and evaluated their antimicrobial activities against several bacterial and parasitic pathogens. Sea bass piscidins present variable amino acid sequences and antimicrobial activities, and can be divided in different sub groups: group 1, formed by piscidins 1 and 4; group 2, constituted by piscidins 2 and 5, and group 3, formed by piscidins 6 and 7. Additionally, we demonstrate that piscidins 1 to 5 possess a broad effect on multiple microorganisms, including mammalian parasites, while piscidins 6 and 7 have poor antibacterial and antiparasitic activities. These results raise questions on the functions of these peptides, particularly piscidins 6 and 7. Considering their limited antimicrobial activity, these piscidins might have other functional roles, but further studies are necessary to better understand what roles might those be.
Canine leishmaniasis is a major veterinary issue and also a public health challenge due to its zoonotic potential. In this context, serological evaluation is essential for Canine leishmaniasis ...management. Several serological alternatives, such as rapid diagnostic tests, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence antibody test (IFAT), are well established. In fact, the capacity of distinct tests and antigens, evaluated by their sensitivity and specificity, to detect disease is normally considered sufficient for diagnosing Canine leishmaniasis. In this context, we evaluated the seropositivity using 8 different serological tests (ELISA with Leishmania recombinant proteins (rK39, LicTXNPx); soluble promastigote Leishmania antigens (SPLA); commercial ELISA test) in 82 clinically suspect animals from Northern Portugal. The obtained serological data originated 50% of inconclusive serological information with a mixture of seropositive and seronegative results for individual animals. Cut-off independent risk groups were then generated from the serological data to evaluate the clustering of the samples. This analysis originated risk groups that correlated with the most seropositive samples, suggesting that this method might be used, in a cut-off independent manner, to improve conventional serological evaluation. Ultimately, given that no test prioritization exists, the use of any single serological test increases the potential for misdiagnosis, along with all associated risks for the dog as well as public health. The use of a cut-off independent analysis has the potential to improve the predictive values of these tests, enabling a more accurate evaluation of the dog's condition.
The genus
(Trypanosomatida: Trypanosomatidae) currently comprises just over 50 species, of which about 20 cause several syndromes in humans, collectively known as leishmaniasis or "leishmaniases" ....
The ability to manipulate the Leishmania genome to create genetically modified parasites by introducing or eliminating genes is considered a powerful alternative for developing a new generation ...vaccine against leishmaniasis. Previously, we showed that the deletion of one allele of the Leishmania infantum silent information regulatory 2 (LiSIR2) locus was sufficient to dramatically affect amastigote axenic proliferation. Furthermore, LiSIR2 single knockout (LiSIR2(+/-)) amastigotes were unable to replicate in vitro inside macrophages. Because this L. infantum mutant persisted in BALB/c mice for up to 6 wk but failed to establish an infection, we tested its ability to provide protection toward a virulent L. infantum challenge. Strikingly, vaccination with a single i.p. injection of LiSIR2(+/-) single knockout elicits complete protection. Thus, vaccinated BALB/c mice showed a reversal of T cell anergy with specific anti-Leishmania cytotoxic activity and high levels of NO production. Moreover, vaccinated mice simultaneously generated specific anti-Leishmania IgG Ab subclasses suggestive of both type 1 and type 2 responses. A strong correlation was found between the elimination of the parasites and an increased Leishmania-specific IFN-gamma/IL-10 ratio. Therefore, we propose that the polarization to a high IFN-gamma/low IL-10 ratio after challenge is a clear indicator of vaccine success. Furthermore these mutants, which presented attenuated virulence, represent a good model to understand the correlatives of protection in visceral leishmaniasis.
Leishmaniasis, a vector-borne parasitic protozoan disease, is among the most important neglected tropical diseases. In the absence of vaccines, disease management is challenging. The available ...chemotherapy is suboptimal, and there are growing concerns about the emergence of drug resistance. Thus, a better understanding of parasite biology is essential to generate new strategies for disease control. In this context, in vitro parasite exoproteome characterization enabled the identification of proteins involved in parasite survival, pathogenesis, and other biologically relevant processes. After 2005, with the availability of genomic information, these studies became increasingly feasible and revealed the true complexity of the parasite exoproteome. After the discovery of
extracellular vesicles (EVs), most exoproteome studies shifted to the characterization of EVs. The non-EV portion of the exoproteome, named the vesicle-depleted exoproteome (VDE), has been mostly ignored even if it accounts for a significant portion of the total exoproteome proteins. Herein, we summarize the importance of total exoproteome studies followed by a special emphasis on the available information and the biological relevance of the VDE. Finally, we report on how VDE can be studied and disclose how it might contribute to providing biologically relevant targets for diagnosis, drug, and vaccine development.
In Europe, up to 70% of visceral leishmaniasis (VL) cases occurring in adults living with HIV. People living with HIV with VL co-infection often display persistent parasitemia, requiring chronic ...intermittent anti-Leishmania therapies. Consequently, frequent VL relapses and higher mortality rates are common in these individuals. As such, it is of paramount importance to understand the reasons for parasite persistence to improve infection management.
To outline possible causes for treatment failure in the context of HIV-VL, we followed a person living with HIV-VL co-infection for nine years in a 12-month period. We characterized: HIV-related clinicopathological alterations (CD4+ T counts and viremia) and Leishmania-specific seroreactivity, parasitemia, quantification of pro-inflammatory cytokines upon stimulation and studied a Leishmania clinical isolate recovered during this period.
The subject presented controlled viremia and low CD4+ counts. The subject remained PCR positive for Leishmania and also seropositive. The cellular response to parasite antigens was erratic. The isolate was identified as the first Leishmania infantum case with evidence of decreased miltefosine susceptibility in Portugal.
Treatment failure is a multifactorial process driven by host and parasite determinants. Still, the real-time determination of drug susceptibility profiles in clinical isolates is an unexplored resource in the monitoring of VL.
Leishmaniasis is an important vector-borne neglected tropical disease caused by Leishmania parasites. Current anti-Leishmania chemotherapy is unsatisfactory, justifying the continued search for ...alternative treatment options. Herein, we demonstrate that luciferase-expressing Leishmania infantum axenic amastigotes, unlike promastigotes, are highly infectious to BALB/c mice and thus generate a robust bioluminescent signal in target organs, such as the liver and the spleen, as early as two weeks after infection. Treatment with the reference drugs amphotericin B and miltefosine was effective at reducing parasite burdens. This model allows the assessment of treatment efficacy using whole-mouse bioluminescence imaging without the need to wait several weeks for spleen infections to be detectable by this non-invasive method. In conclusion, we propose the use of this model in an initial approach to evaluate the treatment efficacy of promising chemical entities without having to sacrifice large numbers of animals or to wait several days for a readout.
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