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1.
PLoS Negl Trop Dis ; 15(2): e0009196, 2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33617566

RESUMO

Leishmaniasis is a major infectious disease with hundreds of thousands of new cases and over 20,000 deaths each year. The current drugs to treat this life-threatening infection have several drawbacks such as toxicity and long treatment regimens. A library of 1.8 million compounds, from which the hits reported here are publicly available, was screened against Leishmania infantum as part of an optimization program; a compound was found with a 2-aminobenzimidazole functionality presenting moderate potency, low metabolic stability and high lipophilicity. Several rounds of synthesis were performed to incorporate chemical groups capable of reducing lipophilicity and clearance, leading to the identification of compounds that are active against different parasite strains and have improved in vitro properties. As a result of this optimization program, a group of compounds was further tested in anticipation of in vivo evaluation. In vivo tests were carried out with compounds 29 (L. infantum IC50: 4.1 µM) and 39 (L. infantum IC50: 0.5 µM) in an acute L. infantum VL mouse model, which showed problems of poor exposure and lack of efficacy, despite the good in vitro potency.

2.
Eur J Med Chem ; 212: 113101, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33385837

RESUMO

The kinetoplastid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are the causative agents of neglected tropical diseases with a serious burden in several parts of the world. These parasites are incapable of synthesizing purines de novo, and therefore rely on ingenious purine salvage pathways to acquire and process purines from their host. Purine nucleoside analogs that may interfere with these pathways therefore constitute a privileged source of new antikinetoplastid agents. In this study, we synthetized a collection of C-nucleosides employing five different heterocyclic nucleobase surrogates. C-nucleosides are chemically and enzymatically stable and allow for extensive structural modification. Inspired by earlier 7-deazaadenosine nucleosides and known antileishmanial C-nucleosides, we introduced different modifications tailored towards antikinetoplastid activity. Both adenosine and inosine analogs were synthesized with the aim of discovering new antikinetoplastid hits and expanding knowledge of structure-activity relationships. Several promising hits with potent activity against Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum were discovered, and the nature of the nucleobase surrogate was found to have a profound influence on the selectivity profile of the compounds.

3.
Commun Biol ; 4(1): 67, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33452445

RESUMO

Deployment of Wolbachia to mitigate dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) transmission is ongoing in 12 countries. One way to assess the efficacy of Wolbachia releases is to determine invasion rates within the wild population of Aedes aegypti following their release. Herein we evaluated the accuracy, sensitivity and specificity of the Near Infrared Spectroscopy (NIRS) in estimating the time post death, ZIKV-, CHIKV-, and Wolbachia-infection in trapped dead female Ae. aegypti mosquitoes over a period of 7 days. Regardless of the infection type, time post-death of mosquitoes was accurately predicted into four categories (fresh, 1 day old, 2-4 days old and 5-7 days old). Overall accuracies of 93.2, 97 and 90.3% were observed when NIRS was used to detect ZIKV, CHIKV and Wolbachia in dead Ae. aegypti female mosquitoes indicating NIRS could be potentially applied as a rapid and cost-effective arbovirus surveillance tool. However, field data is required to demonstrate the full capacity of NIRS for detecting these infections under field conditions.

4.
Antimicrob Agents Chemother ; 65(3)2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33361300

RESUMO

The phosphodiesterase inhibitor tetrahydrophthalazinone NPD-008 was explored by phenotypic in vitro screening, target validation, and ultrastructural approaches against Trypanosoma cruzi NPD-008 displayed activity against different forms and strains of T. cruzi (50% effective concentration [EC50], 6.6 to 39.5 µM). NPD-008 increased cAMP levels of T. cruzi and its combination with benznidazole gave synergistic interaction. It was also moderately active against intracellular amastigotes of Leishmania amazonensis and Leishmania infantum, confirming a potential activity profile as an antitrypanosomatid drug candidate.

5.
Eur J Med Chem ; : 112914, 2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-33268145

RESUMO

Previous investigation of the potent antileishmanial properties of antitubercular 7-substituted 2-nitroimidazo[2,1-b][1,3]oxazines with biaryl side chains led to our development of a new clinical candidate for visceral leishmaniasis (DNDI-0690). Within a collaborative backup program, a racemic monoaryl lead (3) possessing comparable activity in mice but a greater hERG liability formed the starting point for our pursuit of efficacious second generation analogues having good solubility and safety. Asymmetric synthesis and appraisal of its enantiomers first established that chiral preferences for in vivo efficacy were species dependent and that neither form afforded a reduced hERG risk. However, in line with our findings in a structurally related series, less lipophilic heteroaryl ethers provided significant solubility enhancements (up to 16-fold) and concomitantly attenuated hERG inhibition. One promising pyridine derivative (49) displayed 100% oral bioavailability in mice and delivered a 96% parasite burden reduction when dosed at 50 mg/kg in a Leishmania donovani mouse model of visceral leishmaniasis.

6.
Molecules ; 25(21)2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-33139647

RESUMO

Neglected parasitic diseases remain a major public health issue worldwide, especially in tropical and subtropical areas. Human parasite diversity is very large, ranging from protozoa to worms. In most cases, more effective and new drugs are urgently needed. Previous studies indicated that the gold(I) drug auranofin (Ridaura®) is effective against several parasites. Among new gold(I) complexes, the phosphole-containing gold(I) complex {1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl (abbreviated as GoPI) is an irreversible inhibitor of both purified human glutathione and thioredoxin reductases. GoPI-sugar is a novel 1-thio-ß-d-glucopyranose 2,3,4,6-tetraacetato-S-derivative that is a chimera of the structures of GoPI and auranofin, designed to improve stability and bioavailability of GoPI. These metal-ligand complexes are of particular interest because of their combined abilities to irreversibly target the essential dithiol/selenol catalytic pair of selenium-dependent thioredoxin reductase activity, and to kill cells from breast and brain tumors. In this work, screening of various parasites-protozoans, trematodes, and nematodes-was undertaken to determine the in vitro killing activity of GoPI-sugar compared to auranofin. GoPI-sugar was found to efficiently kill intramacrophagic Leishmania donovani amastigotes and adult filarial and trematode worms.

7.
J Ethnopharmacol ; 267: 113624, 2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33246123

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Terminalia albida (Combretaceae), widely used in Guinean traditional medicine, showed promising activity against Plasmodium falciparum and Candida albicans in previous studies. Bioassay-guided fractionation was carried out in order to isolate the compounds responsible for these activities. MATERIALS AND METHODS: Fractionation and isolation were performed by flash chromatography, followed by semi-preparative HPLC-DAD-MS. The structural elucidation of the isolated compounds was carried out by 1D and 2D NMR as well as HR-ESI-MS. Isolated compounds were evaluated against Plasmodium falciparum, Candida albicans, Staphylococcus aureus and Escherichia coli, and their cytotoxicity against MRC-5 cells was determined. RESULTS: Bioassay-guided fractionation of Terminalia albida root resulted in the isolation of 14 compounds (1-14), and their antimicrobial properties were evaluated. Pantolactone (1) (IC50 0.60 ± 0.03 µM) demonstrated significant activity against P. falciparum. Other compounds, including 3,4,3'-tri-O-methyl-ellagic acid (3), the triterpenes arjunolic acid (5), arjungenin (6), arjunic acid (7) and arjunglucoside II (10), and the phenol glycoside calophymembranside-B (14), were less active and showed IC50 values in the range 5-15 µM. None of the tested compound showed antibacterial or antifungal activity. CONCLUSION: These results may explain at least in part the activity of the root extract of T. albida against P. falciparum.

8.
ChemMedChem ; 2020 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-33078573

RESUMO

Neglected tropical diseases remain among the most critical public health concerns in Africa and South America. The drug treatments for these diseases are limited, which invariably leads to fatal cases. Hence, there is an urgent need for new antitrypanosomal drugs. To address this issue, a large number of diverse heterocyclic compounds were prepared. Straightforward synthetic approaches tolerated pre-functionalized structures, giving rise to a structurally diverse set of analogs. We report on a set of 57 heterocyclic compounds with selective activity potential against kinetoplastid parasites. In general, 29 and 19 compounds of the total set could be defined as active against Trypanosoma cruzi and T. brucei brucei, respectively (antitrypanosomal activities <10 µM). The present work discusses the structure-activity relationships of new fused-ring scaffolds based on imidazopyridine/pyrimidine and furopyridine cores. This library of compounds shows significant potential for anti-trypanosomiases drug discovery.

9.
Molecules ; 25(19)2020 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-33007887

RESUMO

For the development of new and potent antimalarial drugs, we designed the virtual library with three points of randomization of novel [1,2,4]triazolo[4,3-a]pyridines bearing a sulfonamide fragment. The library of 1561 compounds has been investigated by both virtual screening and molecular docking methods using falcipain-2 as a target enzyme. 25 chosen hits were synthesized and evaluated for their antimalarial activity in vitro against Plasmodium falciparum. 3-Ethyl-N-(3-fluorobenzyl)-N-(4-methoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine-6-sulfonamide and 2-(3-chlorobenzyl)-8-(piperidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one showed in vitro good antimalarial activity with inhibitory concentration IC50 = 2.24 and 4.98 µM, respectively. This new series of compounds may serve as a starting point for future antimalarial drug discovery programs.

10.
AAPS PharmSciTech ; 21(7): 275, 2020 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033847

RESUMO

In the published manuscript, co-author Sarah Hendrickx name was misspelled and co-author Guy Caljon's last and first names were inadvertently switched.

11.
Sci Rep ; 10(1): 17268, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33057006

RESUMO

Dry eye syndrome (DES), a multifactorial disorder which leads to ocular discomfort, visual disturbance and tear film instability, has a rising prevalence and limited treatment options. In this study, a newly developed trypsin-like serine protease inhibitor (UAMC-00050) in a tear drop formulation was evaluated to treat ocular inflammation. A surgical animal model of dry eye was employed to investigate the potential of UAMC-00050 on dry eye pathology. Animals treated with UAMC-00050 displayed a significant reduction in ocular surface damage after evaluation with sodium fluorescein, compared to untreated, vehicle treated and cyclosporine-treated animals. The concentrations of IL-1α and TNF-α were also significantly reduced in tear fluid from UAMC-00050-treated rats. Additionally, inflammatory cell infiltration in the palpebral conjunctiva (CD3 and CD45), was substantially reduced. An accumulation of pro-MMP-9 and a decrease in active MMP-9 were found in tear fluid from animals treated with UAMC-00050, suggesting that trypsin-like serine proteases play a role in activating MMP-9 in ocular inflammation in this animal model. Comparative qRT-PCR analyses on ocular tissue indicated the upregulation of tryptase, urokinase plasminogen activator receptor (uPAR) and protease-activated receptor 2 (PAR2). The developed UAMC-00050 formulation was stable up to 6 months at room temperature in the absence of light, non-irritating and sterile with compatible pH and osmolarity. These results provide a proof-of-concept for the in vivo modifying potential of UAMC-00050 on dry eye pathology and suggest a central role of trypsin-like serine proteases and PAR2 in dry eye derived ocular inflammation.

12.
J Ethnopharmacol ; 263: 113232, 2020 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-32768641

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: In Guinea, medicinal plants play an important role in the management of infectious diseases including urinary disorders, skin diseases and oral diseases. This study was carried out to collect medicinal plant species employed for the treatment of these diseases and to investigate their antimicrobial potential. MATERIALS AND METHODS: Based on an ethnobotanical investigation carried out in three Guinean regions, 74 traditional healers and 28 herbalists were interviewed and medicinal plants were collected. The most quoted plant species were evaluated for their antimicrobial activities against Staphylococcus aureus, Escherichia coli, Candida albicans, and in addition against Plasmodium falciparum. RESULTS: A total of 112 plant species belonging to 102 genera distributed over 42 botanical families were inventoried. Among the selected plant species, promising activities against C. albicans were obtained for the methanolic extracts of the stem bark of Terminalia albida (IC50 1.2 µg/ml), the leaves of Tetracera alnifolia (IC50 1.6 µg/ml) and the root bark of Swartzia madagascariensis (IC50 7.8 µg/ml). The highest activity against S. aureus was obtained for the dichloromethane extracts of the leaves of Pavetta crassipes (IC50 8.5 µg/ml) and the root of Swartzia madagascariensis (IC50 12.8 µg/ml). Twenty one extracts, obtained from twelve plant species, were strongly active against Plasmodium falciparum, including the dichloromethane extracts of the root and stem bark of Terminalia albida root (IC50 0.6 and 0.8 µg/ml), the leaves of Landolphia heudelotii (IC50 0.5 µg/ml), the stem bark of Combretum paniculatum (IC50 0.4 µg/ml) and the leaves of Gardenia ternifolia (IC50 1.3 µg/ml). CONCLUSION: The present study provides a comprehensive overview of medicinal plants employed by Guinean traditional healers for the treatment of various microbial diseases, including urinary disorders, skin diseases and oral diseases. Some of the studied plant species showed promising antimicrobial activity and could be considered as a potential source for the development of new antifungal and/or antimalarial agents.

13.
Trends Parasitol ; 36(9): 785-795, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32713762

RESUMO

Leishmania parasites have the capacity to rapidly adapt to changing environments in their digenetic life cycle which alternates between a vertebrate and an invertebrate host. Emergence of resistance following drug exposure can evoke phenotypic alterations that affect several aspects of parasite fitness in both hosts. Current studies of the impact of resistance are mostly limited to interactions with the mammalian host and characterization of in vitro parasite growth and differentiation. Development in the vector and transmission capacity have been largely ignored. This review reflects on the impact of drug resistance on its spreading potential with specific focus on the use of the sand fly infection model to evaluate parasite development in the vector and the ensuing transmission potential of drug-resistant phenotypes.


Assuntos
Resistência a Medicamentos , Insetos Vetores/parasitologia , Leishmaniose/transmissão , Psychodidae/parasitologia , Animais , Antiparasitários/farmacologia , Humanos , Leishmania/efeitos dos fármacos , Leishmania/crescimento & desenvolvimento , Leishmaniose/parasitologia , Estágios do Ciclo de Vida/fisiologia
14.
AAPS PharmSciTech ; 21(5): 185, 2020 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-32632542

RESUMO

The present study aimed to develop, characterize and evaluate the amphotericin B-loaded nanostructured lipid carriers (AmB-NLCs) for topical treatment of cutaneous leishmaniasis (CL) and vulvovaginal candidiasis (VVC). AmB-NLCs were characterized for particle size, zeta potential, encapsulation efficiency and surface morphology. Prepared NLCs were also characterized for in vitro drug release, ex vivo skin permeation and deposition before evaluating their in vitro and in vivo efficacy. Cytotoxicity of NLCs was assessed on MRC-5 cells, whereas skin irritation potential was evaluated in vivo using rats. Significant accumulation of drug in to the skin supported the topical application potential of drug-loaded NLCs. Encapsulation of AmB in NLCs resulted in enhanced in vitro potency against promastigotes and intracellular amastigotes of L. major JISH 118 (IC50 ± SEM = 0.02 ± 0.1 µM for both) compared with free drug (IC50 ± SEM = 0.15 ± 0.2 & 0.14 ± 0.0, respectively). Similar improved potency of AmB-NLCs was also observed for other Leishmania and fungal strains compared with drug solution. Topical application of AmB-NLCs on L. major-infected BALB/c mice caused a significant reduction in parasite burden per mg of lesion (65 × 108 ± 13) compared with the control group (> 167.8 × 108 ± 11). Topical AmB-NLCs gel demonstrated superior efficacy in the vaginal C. albicans rat model for VVC as compared with plain AmB gel. Moreover, results of in vitro cytotoxicity assay and in vivo skin irritation test confirmed AmB-NLCs to be non-toxic and safe for topical use. In conclusion, NLCs may have promising potential as carrier for topical treatment of various conditions of skin and mucosa.


Assuntos
Anfotericina B/administração & dosagem , Candidíase Vulvovaginal/tratamento farmacológico , Leishmaniose Cutânea/tratamento farmacológico , Nanoestruturas/administração & dosagem , Administração Tópica , Animais , Candidíase Vulvovaginal/metabolismo , Portadores de Fármacos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Liberação Controlada de Fármacos , Feminino , Géis/metabolismo , Humanos , Lipídeos/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Tamanho da Partícula , Ratos , Pele/metabolismo , Absorção Cutânea
15.
Microorganisms ; 8(6)2020 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-32599761

RESUMO

Kinetoplastids are the causative agents of leishmaniasis, human African trypanosomiasis, and American trypanosomiasis. They are responsible for high mortality and morbidity in (sub)tropical regions. Adequate treatment options are limited and have several drawbacks, such as toxicity, need for parenteral administration, and occurrence of treatment failure and drug resistance. Therefore, there is an urgency for the development of new drugs. Phenotypic screening already allowed the identification of promising new chemical entities with anti-kinetoplastid activity potential, but knowledge on their mode-of-action (MoA) is lacking due to the generally applied whole-cell based approach. However, identification of the drug target is essential to steer further drug discovery and development. Multiple complementary techniques have indeed been used for MoA elucidation. In this review, the different 'omics' approaches employed to define the MoA or mode-of-resistance of current reference drugs and some new anti-kinetoplastid compounds are discussed.

16.
Artigo em Inglês | MEDLINE | ID: mdl-32601163

RESUMO

Pyrazolones are heterocyclic compounds with interesting biological properties. Some derivatives inhibit phosphodiesterases (PDEs) and thereby increase the cellular concentration of cyclic AMP (cAMP), which plays a vital role in the control of metabolism in eukaryotic cells, including the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease (CD), a major neglected tropical disease. In vitro phenotypic screening identified a 4-bromophenyl-dihydropyrazole dimer as an anti-T. cruzi hit and 17 novel pyrazolone analogues with variations on the phenyl ring were investigated in a panel of phenotypic laboratory models. Potent activity against the intracellular forms (Tulahuen and Y strains) was obtained with 50% effective concentration (EC50) values within the 0.17 to 3.3 µM range. Although most were not active against bloodstream trypomastigotes, an altered morphology and loss of infectivity were observed. Pretreatment of the mammalian host cells with pyrazolones did not interfere with infection and proliferation, showing that the drug activity was not the result of changes to host cell metabolism. The pyrazolone NPD-227 increased the intracellular cAMP levels and was able to sterilize T. cruzi-infected cell cultures. Thus, due to its high potency and selectivity in vitro, and its additive interaction with benznidazole (Bz), NPD-227 was next assessed in the acute mouse model. Oral dosing for 5 days of NPD-227 at 10 mg/kg + Bz at 10 mg/kg not only reduced parasitemia (>87%) but also protected against mortality (>83% survival), hence demonstrating superiority to the monotherapy schemes. These data support these pyrazolone molecules as potential novel therapeutic alternatives for Chagas disease.

17.
Parasit Vectors ; 13(1): 276, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32487217

RESUMO

BACKGROUND: In eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection of Leishmania in field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts. METHODS: Field-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target for Leishmania detection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve. RESULTS: The qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (ΔCq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10-3 and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents. CONCLUSIONS: This study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay is inexpensive, sensitive and pan-Leishmania specific, and accordingly an excellent assay for high-throughput screening in entomological research.

18.
ACS Infect Dis ; 6(8): 2045-2056, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32568511

RESUMO

Human African trypanosomiasis is a neglected tropical disease caused by Trypanosoma brucei parasites. These protists are unable to produce the purine ring, making them vulnerable to the effects of purine nucleoside analogues. Starting from 3'-deoxytubercidin (5), a lead compound with activity against central-nervous-stage human African trypanosomiasis, we investigate the structure-activity relationships of the purine and ribofuranose rings. The purine ring tolerated only modifications at C7, while from the many alterations of the 3'-deoxyribofuranosyl moiety only the arabino analogue 48 showed pronounced antitrypanosomal activity. Profiling of the most potent analogues against resistant T. brucei strains (resistant to pentamidine, diminazene, and isometamidium) showed reduced dependence on uptake mediated by the P2 aminopurine transporter relative to 5. The introduction of a 7-substituent confers up to 10-fold increased affinity for the P1 nucleoside transporter while generally retaining high affinity for P2. Four of the most promising analogues were found to be metabolically stable, earmarking them as suitable backup analogues for lead 5.

19.
Front Immunol ; 11: 1113, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32582193

RESUMO

Type I interferons (IFNs) induced by an endogenous Leishmania RNA virus or exogenous viral infections have been shown to exacerbate infections with New World Cutaneous Leishmania parasites, however, the impact of type I IFNs in visceral Leishmania infections and implicated mechanisms remain to be unraveled. This study assessed the impact of type I IFN on macrophage infection with L. infantum and L. donovani and the implication of sialoadhesin (Siglec-1/CD169, Sn) as an IFN-inducible surface receptor. Stimulation of bone marrow-derived macrophages with type I IFN (IFN-α) significantly enhanced susceptibility to infection of reference laboratory strains and a set of recent clinical isolates. IFN-α particularly enhanced promastigote uptake. Enhanced macrophage susceptibility was linked to upregulated Sn surface expression as a major contributing factor to the infection exacerbating effect of IFN-α. Stimulation experiments in Sn-deficient macrophages, macrophage pretreatment with a monoclonal anti-Sn antibody or a novel bivalent anti-Sn nanobody and blocking of parasites with soluble Sn restored normal susceptibility levels. Infection of Sn-deficient mice with bioluminescent L. infantum promastigotes revealed a moderate, strain-dependent role for Sn during visceral infection under the used experimental conditions. These data indicate that IFN-responsive Sn expression can enhance the susceptibility of macrophages to infection with visceral Leishmania promastigotes and that targeting of Sn may have some protective effects in early infection.

20.
J Microbiol Methods ; 173: 105935, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32376283

RESUMO

BACKGROUND: Molecular detection techniques using peripheral blood are preferred over invasive tissue aspiration for the diagnosis and post-treatment follow-up of visceral leishmaniasis (VL) patients. This study aims to identify suitable stabilizing reagents to prevent DNA and RNA degradation during storage and transport to specialized laboratories where molecular diagnosis is performed. METHODOLOGY: The stabilizing capacities of different commercially available reagents were compared using promastigote-spiked human blood and peripheral blood of Syrian golden hamsters subjected to experimental infection, treatment (miltefosine or aminopyrazole DNDi-1044) and immunosuppression. The impact of various storage temperature conditions was tested in combination with an established kinetoplast DNA (kDNA) qPCR and a recently developed spliced leader RNA (SL-RNA) assay for Leishmania detection. PRINCIPAL FINDINGS: Irrespective of the blood type and stabilizer used, threshold (cT) values obtained with the SL-RNA qPCR were systematically lower than those obtained with the kDNA assay, confirming the advantage of the SL-RNA assay over the widely used kDNA assay for low-level Leishmania detection. Peripheral blood parasite levels correlated relatively well with hepatic burdens. RNA protect cell reagent provided the most optimal simultaneous DNA and RNA stabilization in both human and hamster blood. However, this stabilizer requires an erythrocyte lysis step, which can be challenging under field conditions. DNA/RNA shield provides a good alternative for downstream kDNA and SL-RNA assays, especially if sample storage capacity at 4 °C can be guaranteed. CONCLUSIONS/SIGNIFICANCE: The recommended stabilizing reagents are compatible with RNA- and DNA-based Leishmania detection in peripheral blood in the VL hamster model and spiked human blood. Since molecular detection techniques using peripheral blood are less invasive than microscopic assessment of tissue aspirates, the findings of this study may be applied to human VL clinical studies.

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