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1.
PLoS Negl Trop Dis ; 14(8): e0008363, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32790716

RESUMO

Visceral leishmaniasis (VL) remains an important public health issue worldwide causing substantial morbidity and mortality. The Indian subcontinent accounted for up to 90% of the global VL burden in the past but made significant progress during recent years and is now moving towards elimination. However, to achieve and sustain elimination of VL, knowledge gaps on infection reservoirs and transmission need to be addressed urgently. Xenodiagnosis is the most direct way for testing the infectiousness of hosts to the vectors and can be used to investigate the dynamics and epidemiology of Leishmania donovani transmission. There are, however, several logistic and ethical issues with xenodiagnosis that need to be addressed before its application on human subjects. In the current Review, we discuss the critical knowledge gaps in VL transmission and the role of xenodiagnosis in disease transmission dynamics along with its technical challenges. Establishment of state of the art xenodiagnosis facilities is essential for the generation of much needed evidence in the VL elimination initiative.


Assuntos
Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/transmissão , Phlebotomus/parasitologia , Xenodiagnóstico , Animais , Ásia , Doenças Assintomáticas , Reservatórios de Doenças/parasitologia , Humanos , Leishmania donovani/fisiologia
2.
PLoS One ; 15(4): e0232116, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32343719

RESUMO

Myosin XXI (Myo21) is a novel class of myosin present in all kinetoplastid parasites, such as Trypanosoma and Leishmania. This protein in Leishmania promastigotes is predominantly localized to the proximal region of the flagellum, and is involved in the flagellum assembly, cell motility and intracellular vesicle transport. As Myo21 contains two ubiquitin associated (UBA)-like domains (UBLD) in its amino acid sequence, we considered it of interest to analyze the role of these domains in the intracellular distribution and functions of this protein in Leishmania cells. In this context, we created green fluorescent protein (GFP)-conjugates of Myo21 constructs lacking one of the two UBLDs at a time or both the UBLDs as well as GFP-conjugates of only the two UBLDs and Myo21 tail lacking the two UBLDs and separately expressed them in the Leishmania cells. Our results show that unlike Myo21-GFP, Myo21-GFP constructs lacking either one or both the UBLDs failed to concentrate and co-distribute with actin in the proximal region of the flagellum. Nevertheless, the GFP conjugate of only the two UBLDs was found to predominantly localize to the flagellum base. Additionally, the cells that expressed only one or both the UBLDs-deleted Myo21-GFP constructs possessed shorter flagellum and displayed slower motility, compared to Myo21-GFP expressing cells. Further, the intracellular vesicle transport and cell growth were severely impaired in the cells that expressed both the UBLDs deleted Myo21-GFP construct, but in contrast, virtually no effect was observed on the intracellular vesicle transport and growth in the cells that expressed single UBLD deleted mutant proteins. Moreover, the observed slower growth of both the UBLDs-deleted Myo21-GFP expressing cells was primarily due to delayed G2/M phase caused by aberrant nuclear and daughter cell segregation during their cell division process. These results taken together clearly reveal that the presence of UBLDs in Myo21 are essentially required for its predominant localization to the flagellum base, and perhaps also in its involvement in the flagellum assembly and cell division. Possible role of UBLDs in involvement of Myo21 during Leishmania flagellum assembly and cell cycle is discussed.


Assuntos
Flagelos/metabolismo , Leishmania donovani/fisiologia , Miosinas/química , Miosinas/metabolismo , Actinas/metabolismo , Ciclo Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/metabolismo , Miosinas/genética , Domínios Proteicos , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Vesículas Transportadoras/metabolismo , Ubiquitina/metabolismo
3.
PLoS Pathog ; 16(4): e1008456, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32282850

RESUMO

Leishmania donovani causes visceral leishmaniasis (VL), which is typically fatal without treatment. There is substantial variation between individuals in rates of disease progression, response to treatment and incidence of post-treatment sequelae, specifically post-kala-azar dermal leishmaniasis (PKDL). Nevertheless, the majority of infected people are asymptomatic carriers. Hamsters and mice are commonly used as models of fatal and non-fatal VL, respectively. Host and parasite genetics are likely to be important factors, but in general the reasons for heterogeneous disease presentation in humans and animal models are poorly understood. Host microbiota has become established as a factor in cutaneous forms of leishmaniasis but this has not been studied in VL. We induced intestinal dysbiosis in mice and hamsters by long-term treatment with broad-spectrum antibiotics in their drinking water. There were no significant differences in disease presentation in dysbiotic mice. In contrast, dysbiotic hamsters infected with L. donovani had delayed onset and progression of weight loss. Half of control hamsters had a rapid progression phenotype compared with none of the ABX-treated animals and the nine-month survival rate was significantly improved compared to untreated controls (40% vs. 10%). Antibiotic-treated hamsters also had significantly less severe hepatosplenomegaly, which was accompanied by a distinct cytokine gene expression profile. The protective effect was not explained by differences in parasite loads or haematological profiles. We further found evidence that the gut-liver axis is a key aspect of fatal VL progression in hamsters, including intestinal parasitism, bacterial translocation to the liver, malakoplakia and iron sequestration, none of which occurred in non-progressing murine VL. Diverse bacterial genera were cultured from VL affected livers, of which Rodentibacter was specifically absent from ABX-treated hamsters, indicating this pathobiont may play a role in promoting disease progression. The results provide experimental support for antibiotic prophylaxis against secondary bacterial infections as an adjunct therapy in human VL patients.


Assuntos
Antibacterianos/administração & dosagem , Infecções Bacterianas/prevenção & controle , Coinfecção/prevenção & controle , Enteropatias Parasitárias/parasitologia , Leishmaniose Visceral/parasitologia , Animais , Antibioticoprofilaxia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Fenômenos Fisiológicos Bacterianos , Coinfecção/microbiologia , Cricetinae , Progressão da Doença , Feminino , Microbioma Gastrointestinal , Humanos , Leishmania donovani/fisiologia , Leishmaniose Visceral/complicações , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Simbiose
4.
J Med Chem ; 63(8): 4306-4314, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32223141

RESUMO

We report for the first time a novel series of tellurides bearing sulfonamide as selective and potent inhibitors of the ß-class carbonic anhydrase (CA; EC 4.2.1.1) enzyme expressed in Leishmania donovani protozoa. Such derivatives showed high activity against axenic amastigotes, and among them, compound 5g (4-(((3,4,5-trimethoxyphenyl)tellanyl)methyl)benzenesulfonamide) showed an IC50 of 0.02 µM being highly selective for the parasites over THP-1 cells with a selectivity index of 300. The in vitro and in vivo toxicity experiments showed compound 5g to possess a safe profile and thus paving the way for tellurium-containing compounds as novel drug entities.


Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Leishmania donovani/efeitos dos fármacos , Sulfonamidas/farmacologia , Tripanossomicidas/farmacologia , Animais , Inibidores da Anidrase Carbônica/química , Relação Dose-Resposta a Droga , Feminino , Jejuno/efeitos dos fármacos , Jejuno/patologia , Leishmania donovani/fisiologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Wistar , Sulfonamidas/química , Tripanossomicidas/química
5.
Parasit Vectors ; 13(1): 132, 2020 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-32171322

RESUMO

BACKGROUND: Proving that specific genes are essential for the intracellular viability of Leishmania parasites within macrophages remains a challenge for the identification of suitable targets for drug development. This is especially evident in the absence of a robust inducible expression system or functioning RNAi machinery that works in all Leishmania species. Currently, if a target gene of interest in extracellular parasites can only be deleted from its genomic locus in the presence of ectopic expression from a wild type copy, it is assumed that this gene will also be essential for viability in disease-promoting intracellular parasites. However, functional essentiality must be proven independently in both life-cycle stages for robust validation of the gene of interest as a putative target for chemical intervention. METHODS: Here, we have used plasmid shuffle methods in vivo to provide supportive genetic evidence that N-myristoyltransferase (NMT) is essential for Leishmania viability throughout the parasite life-cycle. Following confirmation of NMT essentiality in vector-transmitted promastigotes, a range of mutant parasites were used to infect mice prior to negative selection pressure to test the hypothesis that NMT is also essential for parasite viability in an established infection. RESULTS: Ectopically-expressed NMT was only dispensable under negative selection in the presence of another copy. Total parasite burdens in animals subjected to negative selection were comparable to control groups only if an additional NMT copy, not affected by the negative selection, was expressed. CONCLUSIONS: NMT is an essential gene in all parasite life-cycle stages, confirming its role as a genetically-validated target for drug development.


Assuntos
Aciltransferases/genética , Genes Essenciais , Leishmania/genética , Leishmania/fisiologia , Estágios do Ciclo de Vida/genética , Proteínas de Protozoários/genética , Animais , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Genoma de Protozoário , Leishmania donovani/genética , Leishmania donovani/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Transcriptoma
6.
PLoS Negl Trop Dis ; 14(2): e0007991, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32023254

RESUMO

BACKGROUND: During infections involving intracellular pathogens, iron performs a double-edged function by providing the pathogen with nutrients, but also boosts the host's antimicrobial arsenal. Although the role of iron has been described in visceral leishmaniasis, information regarding its status in the dermal sequel, Post Kala-azar Dermal Leishmaniasis (PKDL) remains limited. Accordingly, this study aimed to establish the status of iron within monocytes/macrophages of PKDL cases. METHODOLOGY/PRINCIPAL FINDINGS: The intramonocytic labile iron pool (LIP), status of CD163 (hemoglobin-haptoglobin scavenging receptor) and CD71 (transferrin receptor, Tfr) were evaluated within CD14+ monocytes by flow cytometry, and soluble CD163 by ELISA. At the lesional sites, Fe3+ status was evaluated by Prussian blue staining, parasite load by qPCR, while the mRNA expression of Tfr (TfR1/CD71), CD163, divalent metal transporter-1 (DMT-1), Lipocalin-2 (Lcn-2), Heme-oxygenase-1 (HO-1), Ferritin, Natural resistance-associated macrophage protein (NRAMP-1) and Ferroportin (Fpn-1) was evaluated by droplet digital PCR. Circulating monocytes demonstrated elevated levels of CD71, CD163 and soluble CD163, which corroborated with an enhanced lesional mRNA expression of TfR, CD163, DMT1 and Lcn-2. Additionally, the LIP was raised along with an elevated mRNA expression of ferritin and HO-1, as also iron exporters NRAMP-1 and Fpn-1. CONCLUSIONS/SIGNIFICANCE: In monocytes/macrophages of PKDL cases, enhancement of the iron influx gateways (TfR, CD163, DMT-1 and Lcn-2) possibly accounted for the enhanced LIP. However, enhancement of the iron exporters (NRAMP-1 and Fpn-1) defied the classical Ferritinlow/Ferroportinhigh phenotype of alternatively activated macrophages. The creation of such a pro-parasitic environment suggests incorporation of chemotherapeutic strategies wherein the availability of iron to the parasite can be restricted.


Assuntos
Ferro/metabolismo , Leishmaniose Cutânea/metabolismo , Adolescente , Adulto , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Feminino , Humanos , Índia , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/fisiologia , Leishmaniose Cutânea/parasitologia , Lipocalina-2/genética , Lipocalina-2/metabolismo , Macrófagos/metabolismo , Masculino , Monócitos/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Adulto Jovem
7.
Parasit Vectors ; 13(1): 94, 2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-32085719

RESUMO

BACKGROUND: New therapeutic drugs are urgently needed against visceral leishmaniasis because current drugs, such as pentavalent antimonials and miltefosine, produce severe side effects and development of resistance. Whether cyclosporine A (CsA) and its derivatives can be used as therapeutic drugs for visceral leishmaniasis has been controversial for many years. METHODS: In this study, we evaluated the efficacy of CsA and its derivative, dihydrocyclosporin A (DHCsA-d), against promastigotes and intracellular amastigotes of Leishmania donovani. Sodium stibogluconate (SSG) was used as a positive control. RESULTS: Our results showed that DHCsA-d was able to inhibit the proliferation of L. donovani promastigotes (IC50: 21.24 µM and 12.14 µM at 24 h and 48 h, respectively) and intracellular amastigotes (IC50: 5.23 µM and 4.84 µM at 24 and 48 h, respectively) in vitro, but CsA treatment increased the number of amastigotes in host cells. Both DHCsA-d and CsA caused several alterations in the morphology and ultrastructure of L. donovani, especially in the mitochondria. However, DHCsA-d showed high cytotoxicity towards cells of the mouse macrophage cell line RAW264.7, with CC50 values of 7.98 µM (24 h) and 6.65 µM (48 h). Moreover, DHCsA-d could increase IL-12, TNF-α and IFN-γ production and decrease the levels of IL-10, IL-4, NO and H2O2 in infected macrophages. On the contrary, CsA decreased IL-12, TNF-α, and IFN-γ production and increased the levels of IL-10, IL-4, NO and H2O2 in infected macrophages. The expression of L. donovani cyclophilin A (LdCyPA) in promastigotes and intracellular amastigotes and the expression of cyclophilin A (CyPA) in RAW 264.7 cells were found to be significantly downregulated in the CsA-treated group compared to those in the untreated group. However, no significant changes in LdCyPA and CyPA levels were found after DHCsA-d or SSG treatment. CONCLUSIONS: Our findings initially resolved the dispute regarding the efficacy of CsA and DHCsA-d for visceral leishmaniasis treatment. CsA showed no significant inhibitory effect on intracellular amastigotes. DHCsA-d significantly inhibited promastigotes and intracellular amastigotes, but it was highly cytotoxic. Therefore, CsA and DHCsA-d are not recommended as antileishmanial drugs.


Assuntos
Antiprotozoários/farmacologia , Ciclosporina/farmacologia , Ciclosporinas/farmacologia , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/parasitologia , Animais , Avaliação Pré-Clínica de Medicamentos , Humanos , Interferon gama/imunologia , Interleucina-10/imunologia , Interleucina-2/imunologia , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/fisiologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/imunologia , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Células RAW 264.7
8.
Parasit Vectors ; 13(1): 96, 2020 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-32087758

RESUMO

BACKGROUND: Since the introduction of miltefosine (MIL) as first-line therapy in the kala-azar elimination programme in the Indian subcontinent, treatment failure rates have been increasing. Since parasite infectivity and virulence may become altered upon treatment relapse, this laboratory study assessed the phenotypic effects of repeated in vitro and in vivo MIL exposure. METHODS: Syngeneic Leishmania donovani lines either or not exposed to MIL were compared for drug susceptibility, rate of promastigote multiplication and metacyclogenesis, macrophage infectivity and behaviour in the sand fly vector, Lutzomyia longipalpis. RESULTS: Promastigotes of both in vitro and in vivo MIL-selected strains displayed a slightly reduced drug susceptibility that was associated with a reduced MIL-accumulation linked to a lower copy number (disomic state) of chromosome 13 harboring the miltefosine transporter (LdMT) gene. In vitro selected promastigotes showed a lower rate of metacyclogenesis whereas the in vivo derived promastigotes displayed a moderately increased growth rate. Repeated MIL exposure did neither influence the parasite load nor metacyclogenesis in the sand fly vector. CONCLUSIONS: Recurrent in vitro and in vivo MIL exposure evokes a number of very subtle phenotypic and genotypic changes which could make promastigotes less susceptible to MIL without attaining full resistance. These changes did not significantly impact on infection in the sand fly vector.


Assuntos
Antiprotozoários/farmacologia , Insetos Vetores/parasitologia , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/fisiologia , Fosforilcolina/análogos & derivados , Psychodidae/parasitologia , Aclimatação , Animais , Resistência a Medicamentos , Humanos , Leishmania donovani/patogenicidade , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/transmissão , Testes de Sensibilidade Parasitária , Fenótipo , Fosforilcolina/farmacologia , Virulência
9.
PLoS Negl Trop Dis ; 14(1): e0008020, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961866

RESUMO

Myeloid-related protein 14 (MRP14) belongs to the S100 calcium-binding protein family and is expressed in neutrophils and inflammatory macrophages. Increase in the number of MRP14+ cells or serum level of MRP14 is associated with various diseases such as autoimmune diseases and infectious diseases, suggesting the involvement of the molecule in pathogenesis of those diseases. In this study, to examine the pathological involvement of MRP14 during cutaneous and visceral leishmaniasis, wild-type (WT) and MRP14 knockout (MRP14KO) mice were infected with Leishmania major and L. donovani. Increase in the number of MRP14+ cells at the infection sites in wild-type mice was commonly found in the skin during L. major infection as well as the spleen and liver during L. donovani infection. In contrast, the influence of MRP14 to the pathology seemed different between the two infections. MRP14 depletion exacerbated the lesion development and ulcer formation in L. major infection. On the other hand, the depletion improved anemia and splenomegaly but not hepatomegaly at 24 weeks of L. donovani infection. These results suggest that, distinct from its protective role in CL, MRP14 is involved in exacerbation of some symptoms during VL.


Assuntos
Anemia/metabolismo , Anemia/patologia , Calgranulina B/metabolismo , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/patologia , Esplenomegalia/metabolismo , Esplenomegalia/patologia , Anemia/genética , Anemia/parasitologia , Animais , Calgranulina B/genética , Feminino , Humanos , Leishmania donovani/fisiologia , Leishmania major/fisiologia , Leishmaniose Visceral/genética , Leishmaniose Visceral/parasitologia , Fígado/metabolismo , Fígado/parasitologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Baço/metabolismo , Baço/parasitologia , Baço/patologia , Esplenomegalia/genética , Esplenomegalia/parasitologia
10.
J Immunol ; 204(3): 596-610, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31882519

RESUMO

Immune evasion strategies adopted by Leishmania donovani involve the exploitation of suppressor of cytokine signaling (SOCS) proteins that are well-known negative regulators of the JAK/STAT pathway. However, the cellular mechanism underpinning the induction of SOCS isoforms and their role in breaching the multilevel regulatory circuit connecting the innate and adaptive arms of immunity are still ambiguous during experimental visceral leishmaniasis. Using bone marrow-derived macrophages (BMMфs) and CD4+ T cells, we observed that L. donovani preferentially upregulates SOCS1 and SOCS3 expression in macrophages and T cells, respectively, whereas the SOCS1 level remains consistently high in BMMфs and SOCS3 expression is pronounced and long lasting in T cells. Consequently, this inhibits STAT1-mediated IL-12 induction in macrophages & STAT4-mediated IFN-γ synthesis in T cells. Mechanistically, PI3K/Akt-mediated SRF activation promotes nuclear translocation and binding of Egr2 to SOCS1 promoter for its early induction in infected BMMфs. Additionally, L. donovani activates IDO/kynurenine/AHR signaling in BMMфs to maintain prolonged SOCS1 expression. Later, PGE2, secreted from infected BMMфs induces cAMP-PKA pathway by binding to the EP2/EP4 receptor of CD4+ T cells, leading to SP1, CREB, and GATA1 activation and SOCS3 expression. Small interfering RNA-mediated silencing of SOCS1 and SOCS3 in macrophage and T cells, respectively, restored IL-12 and IFN-γ cytokine levels and BMMф-T cell interaction. Vivo morpholino-mediated silencing of SOCS1 and SOCS3 resulted in protective cytokine responses, thereby reducing organ parasite burden significantly in L. donovani-infected BALB/c mice. Collectively, our results imply that L. donovani orchestrates different SOCS isoforms to impair macrophage-T cell cross-talk and preserve its own niche.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Leishmania donovani/fisiologia , Leishmaniose Visceral/imunologia , Macrófagos/imunologia , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Animais , Comunicação Celular , Células Cultivadas , Modelos Animais de Doenças , Humanos , Evasão da Resposta Imune , Imunidade Celular , Interferon gama/metabolismo , Interleucina-12/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Isoformas de Proteínas/genética , RNA Interferente Pequeno/genética , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina/genética , Proteína 3 Supressora da Sinalização de Citocinas/genética , Regulação para Cima
11.
Front Immunol ; 10: 2749, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31849951

RESUMO

Visceral leishmaniasis (VL) caused by Leishmania donovani and L. infantum is a potentially fatal disease. To date there are no registered vaccines for disease prevention despite the fact that several vaccines are in preclinical development. Thus, new strategies are needed to improve vaccine efficacy based on a better understanding of the mechanisms mediating protective immunity and mechanisms of host immune responses subversion by immunopathogenic components of Leishmania. We found that mice vaccinated with CPA162-189-loaded p8-PLGA nanoparticles, an experimental nanovaccine, induced the differentiation of antigen-specific CD8+ T cells in spleen compared to control mice, characterized by increased dynamics of proliferation and high amounts of IFN-γ production after ex vivo re-stimulation with CPA162-189 antigen. Vaccination with CPA162-189-loaded p8-PLGA nanoparticles resulted in about 80% lower parasite load in spleen and liver at 4 weeks after challenge with L. infantum promastigotes as compared to control mice. However, 16 weeks after infection the parasite load in spleen was comparable in both mouse groups. Decreased protection levels in vaccinated mice were followed by up-regulation of the anti-inflammatory IL-10 production although at lower levels in comparison to control mice. Microarray analysis in spleen tissue at 4 weeks post challenge revealed different immune-related profiles among the two groups. Specifically, vaccinated mice were characterized by similar profile to naïve mice. On the other hand, the transcriptome of the non-vaccinated mice was dominated by increased expression of genes related to interferon type I, granulocyte chemotaxis, and immune cells suppression. This profile was significantly enriched at 16 weeks post challenge, a time-point which is relative to disease establishment, and was common for both groups, further suggesting that type I signaling and granulocyte influx has a significant role in disease establishment, pathogenesis and eventually in decreased vaccine efficacy for stimulating long-term protection. Overall, we put a spotlight on host immune networks during active VL as potential targets to improve and design more effective vaccines against disease.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Cisteína Proteases/imunologia , Leishmania donovani/fisiologia , Leishmania infantum/fisiologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/imunologia , Fígado/imunologia , Nanopartículas/administração & dosagem , Peptídeos/imunologia , Proteínas de Protozoários/imunologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Cisteína Proteases/química , Perfilação da Expressão Gênica , Humanos , Tolerância Imunológica , Interferon gama/metabolismo , Fígado/parasitologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Nanopartículas/química , Carga Parasitária , Peptídeos/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Proteínas de Protozoários/química
12.
PLoS Negl Trop Dis ; 13(11): e0007816, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31738750

RESUMO

Visceral leishmaniasis (VL) is caused by parasitic protozoa of the genus Leishmania and is characterized by clinical manifestations such as fever, hepatosplenomegaly and anemia. Hemophagocytosis, the phenomenon of phagocytosis of blood cells by macrophages, is found in VL patients. In a previous study we established an experimental model of VL, reproducing anemia in mice for the first time, and identified hemophagocytosis by heavily infected macrophages in the spleen as a possible cause of anemia. However, the mechanism for parasite-induced hemophagocytosis or its role in parasite survival remained unclear. Here, we established an in vitro model of Leishmania-induced hemophagocytosis to explore the molecules involved in this process. In contrast to naïve RAW264.7 cells (mouse macrophage cell line) which did not uptake freshly isolated erythrocytes, RAW264.7 cells infected with L. donovani showed enhanced phagocytosis of erythrocytes. Additionally, for hemophagocytes found both in vitro and in vivo, the expression of signal regulatory protein α (SIRPα), one of the receptors responsible for the 'don't-eat-me' signal was suppressed by post-transcriptional control. Furthermore, the overlapped phagocytosis of erythrocytes and Leishmania parasites within a given macrophage appeared to be beneficial to the parasites; the in vitro experiments showed a higher number of parasites within macrophages that had been induced to engulf erythrocytes. Together, these results suggest that Leishmania parasites may actively induce hemophagocytosis by manipulating the expression of SIRPα in macrophages/hemophagocytes, in order to secure their parasitism.


Assuntos
Leishmania donovani/fisiologia , Linfo-Histiocitose Hemofagocítica , Macrófagos/parasitologia , Fagocitose , Animais , Linhagem Celular , Modelos Animais de Doenças , Eritrócitos , Leishmania donovani/patogenicidade , Leishmaniose Visceral/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7 , Baço/parasitologia , Transcriptoma
13.
Front Immunol ; 10: 2273, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31608064

RESUMO

No vaccine exists against visceral leishmaniasis. Toward developing vaccines against VL, we have reported previously on the immunogenicity of live attenuated LdCen -/- parasites in animal models. Immunization with LdCen -/- parasites has been shown to induce durable protective immunity in pre-clinical animal models. Although the innate immune responses favoring a Th1 type immunity are produced following LdCen -/- immunization, the molecular determinants of such responses remain unknown. To identify early biomarkers of immunogenicity associated with live attenuated parasitic vaccines, we infected macrophages derived from healthy human blood donors with LdCen -/- or LdWT parasites ex vivo and compared the early gene expression profiles. In addition to altered expression of immune related genes, we identified several microRNAs that regulate important cytokine genes, significantly altered in LdCen -/- infection compared to LdWT infection. Importantly, we found that LdCen -/- infection suppresses the expression of microRNA-21 (miR-21) in human macrophages, which negatively regulates IL12, compared to LdWT infection. In murine DC experiments, LdCen -/- infection showed a reduced miR-21 expression with a concomitant induction of IL12. Silencing of miR-21 using specific inhibitors resulted in an augmented induction of IL12 in LdWT infected BMDCs, illustrating the role of miR-21 in LdWT mediated suppression of IL12. Further, exosomes isolated from LdCen -/- infected DCs contained significantly reduced levels of miR-21 compared to LdWT infection, that promoted proliferation of CD4+ T cells in vitro. Similar miR-21 mediated IL12 regulation was also observed in ex vivo human macrophage infection experiments indicating that miR-21 plays a role in early IL12 mediated immunity. Our studies demonstrate that LdCen -/- infection suppresses miR-21 expression, enables IL12 mediated induction of adaptive immunity including proliferation of antigen experienced CD4+ T cells and development of a Th1 immunity, and suggest that miR-21 could be an important biomarker for LdCen -/- vaccine immunity in human clinical trials. One Sentence Summary: Role of miR-21 in vaccine induced immunity.


Assuntos
Imunidade/imunologia , Leishmania donovani/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/imunologia , Macrófagos/imunologia , MicroRNAs/imunologia , Animais , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Humanos , Imunização/métodos , Leishmania donovani/genética , Leishmania donovani/fisiologia , Vacinas contra Leishmaniose/administração & dosagem , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/prevenção & controle , Macrófagos/parasitologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , MicroRNAs/genética , Células Th1/imunologia , Células Th1/metabolismo , Células Th1/parasitologia , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia
14.
BMC Res Notes ; 12(1): 566, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31511056

RESUMO

OBJECTIVES: Infection with the causative agent of visceral leishmaniasis (VL) may be either symptomatic or asymptomatic. In this study we aimed at investigating the prevalence of asymptomatic infections of leishmania in non-endemic villages in Gedaref state, Sudan. A descriptive cross-sectional study conducted during September and October 2014. Blood samples were collected for serological and molecular analysis. Sticky-traps, knockdown spray and CDC-miniature light traps were used for the collection of sandflies. RESULTS: Ninety-Five participants were included; 52 from Abukishma, 15 Algadamblia Tirfa, 25 Abualnaja and 3 were from Algadamblia Aljabal. Females constituted 56 (58.9%) of the study participants while males were 39 (41.1%). The most frequent age group was > 40-years (54.7%). Balanites/Acacia trees were the most planted tree inside the houses; 78 (82.1%). Also, 85 (89.5%) of the participants breed animals inside the house. DAT test revealed 5 positive participants (5.2%). 4/5 DAT positive were past VL infection. PCR detected 35 (36.8%) positive participants. A total of 31/35 was considered asymptomatic infections based on PCR. Households planted Balanites/Acacia trees or breed domestic animals were found in high percentages with L. donovani PCR positive participants (60.1%, 91.4%). No statistically significant was found for VL associated risk factors and VL asymptomatic participants.


Assuntos
Infecções Assintomáticas/epidemiologia , Leishmania donovani/genética , Leishmaniose Visceral/epidemiologia , Acacia/parasitologia , Adulto , Animais , Estudos Transversais , DNA de Protozoário/genética , Feminino , Geografia , Humanos , Leishmania donovani/fisiologia , Leishmaniose Visceral/sangue , Leishmaniose Visceral/parasitologia , Masculino , Phlebotomus/parasitologia , Reação em Cadeia da Polimerase , Prevalência , Sudão/epidemiologia
15.
PLoS Negl Trop Dis ; 13(9): e0007444, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31490933

RESUMO

Visceral leishmaniasis (VL) caused by Leishmania donovani remains of public health concern in rural India. Those at risk of VL are also at risk of other neglected tropical diseases (NTDs) including soil transmitted helminths. Intestinal helminths are potent regulators of host immune responses sometimes mediated through cross-talk with gut microbiota. We evaluate a meta-taxonomic approach to determine the composition of prokaryotic and eukaryotic gut microflora using amplicon-based sequencing of 16S ribosomal RNA (16S rRNA) and 18S rRNA gene regions. The most abundant bacterial taxa identified in faecal samples from Bihar State India were Prevotella (37.1%), Faecalibacterium (11.3%), Escherichia-Shigella (9.1%), Alloprevotella (4.5%), Bacteroides (4.1%), Ruminococcaceae UCG-002 (1.6%), and Bifidobacterium (1.5%). Eukaryotic taxa identified (excluding plant genera) included Blastocystis (57.9%; Order: Stramenopiles), Dientamoeba (12.1%; Family: Tritrichomonadea), Pentatrichomonas (10.1%; Family: Trichomonodea), Entamoeba (3.5%; Family: Entamoebida), Ascaridida (0.8%; Family: Chromodorea; concordant with Ascaris by microscopy), Rhabditida (0.8%; Family: Chromodorea; concordant with Strongyloides), and Cyclophyllidea (0.2%; Order: Eucestoda; concordant with Hymenolepis). Overall alpha (Shannon's, Faith's and Pielou's indices) and beta (Bray-Curtis dissimilarity statistic; weighted UniFrac distances) diversity of taxa did not differ significantly by age, sex, geographic subdistrict, or VL case (N = 23) versus endemic control (EC; N = 23) status. However, taxon-specific associations occurred: (i) Ruminococcaceae UCG- 014 and Gastranaerophilales_uncultured bacterium were enriched in EC compared to VL cases; (ii) Pentatrichomonas was more abundant in VL cases than in EC, whereas the reverse occurred for Entamoeba. Across the cohort, high Escherichia-Shigella was associated with reduced bacterial diversity, while high Blastocystis was associated with high bacterial diversity and low Escherichia-Shigella. Individuals with high Blastocystis had low Bacteroidaceae and high Clostridiales vadin BB60 whereas the reverse held true for low Blastocystis. This scoping study provides useful baseline data upon which to develop a broader analysis of pathogenic enteric microflora and their influence on gut microbial health and NTDs generally.


Assuntos
Bactérias/isolamento & purificação , Eucariotos/isolamento & purificação , Fezes/microbiologia , Fezes/parasitologia , Leishmaniose Visceral/microbiologia , Leishmaniose Visceral/parasitologia , Adolescente , Adulto , Bactérias/classificação , Bactérias/genética , Criança , Pré-Escolar , Estudos de Coortes , Eucariotos/classificação , Eucariotos/genética , Feminino , Microbioma Gastrointestinal , Humanos , Índia/epidemiologia , Leishmania donovani/fisiologia , Masculino , Pessoa de Meia-Idade , População Rural/estatística & dados numéricos , Adulto Jovem
16.
FASEB J ; 33(10): 10794-10807, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31284755

RESUMO

Visceral leishmaniasis is a deadly illness caused by Leishmania donovani that provokes liver and spleen inflammation and tissue destruction. In cutaneous leishmaniasis, the protein of L. major, named inhibitor of serine peptidases (ISP) 2, inactivates neutrophil elastase (NE) present at the macrophage surface, resulting in blockade of TLR4 activation, prevention of TNF-α and IFN-ß production, and parasite survival. We report poor intracellular growth of L. donovani in macrophages from knockout mice for NE (ela-/-), TLR4, or TLR2. NE and TLR4 colocalized with the parasite in the parasitophorous vacuole. Parasite load in the liver and spleen of ela-/- mice were reduced and accompanied by increased NO and decreased TGF-ß production. Expression of ISP2 was not detected in L. donovani, and a transgenic line constitutively expressing ISP2, displayed poor intracellular growth in macrophages and decreased burden in mice. Infected ela-/- macrophages displayed significantly lower IFN-ß mRNA than background mice macrophages, and the intracellular growth was fully restored by exogenous IFN-ß. We propose that L. donovani utilizes the host NE-TLR machinery to induce IFN-ß necessary for parasite survival and growth during early infection. Low or absent expression of parasite ISP2 in L. donovani is necessary to preserve the activation of the NE-TLR pathway.-Dias, B. T., Dias-Teixeira, K. L., Godinho, J. P., Faria, M. S., Calegari-Silva, T., Mukhtar, M. M., Lopes, U. G., Mottram, J. C., Lima, A. P. C. A. Neutrophil elastase promotes Leishmania donovani infection via interferon-ß.


Assuntos
Interferon beta/metabolismo , Leishmania donovani/patogenicidade , Leishmaniose Visceral/etiologia , Elastase de Leucócito/metabolismo , Animais , Animais Geneticamente Modificados , Leishmania donovani/genética , Leishmania donovani/fisiologia , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/parasitologia , Elastase de Leucócito/deficiência , Elastase de Leucócito/genética , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Protozoários/genética , Proteínas de Protozoários/fisiologia , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
17.
Curr Med Chem ; 26(36): 6572-6589, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31218950

RESUMO

Trypanosomatids are a group of flagellated unicellular eukaryotes, causing serious human diseases including Chagas disease (Trypanosoma cruzi), sleeping sickness (Trypanosoma brucei spp.) and Leishmaniasis (Leishmania spp.). The second messenger cAMP is involved in numerous and fundamental processes in these parasites including differentiation between stages, proliferation, osmoregulation, oxidative stress and quorum sensing. Interestingly, its signaling pathway is quite different from that of mammals, including structurally different adenylyl cyclases, the shortage of orthologous effector proteins and the absence of G-protein-coupled-receptors, among others. These characteristics make the proteins involved in these transduction pathways good candidates for therapeutic targets. However, the identification of new unknown druggable targets involves extensive research time and is economically very expensive, making difficult the transition from basic research to the clinical phase. Trypanosomatid PDEs have characteristic binding pockets that allow for a differential inhibition from their human orthologs. Modification in the approved drugs for human to convert them into trypanocidal treatments could lead to more effective therapies, shorter lab time and lower costs. In view of the fact that kinetoplastid PDEs are highly conserved with their mammalian counterparts, and since there are already numerous drugs on the market against human PDEs, the drug repositioning approach is highly promising. The development of new technologies, higher government and industrial involvement and more scientists committed to basic investigation, are the key to ultimately find an effective treatment and cure for the neglected tropical diseases.


Assuntos
Inibidores de Fosfodiesterase/farmacologia , Transdução de Sinais/efeitos dos fármacos , Adenilil Ciclases/fisiologia , Cálcio/fisiologia , Doença de Chagas/tratamento farmacológico , Reposicionamento de Medicamentos , Humanos , Leishmania donovani/enzimologia , Leishmania donovani/fisiologia , Proteínas Quinases/fisiologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/fisiologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/fisiologia
18.
J Med Chem ; 62(11): 5655-5671, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31124675

RESUMO

In our efforts to identify novel chemical scaffolds for the development of antileishmanial agents, a series of quinoline-metronidazole hybrid compounds was synthesized and tested against the murine model of visceral leishmaniasis. Among all synthesized derivatives, 15b and 15i showed significant antileishmanial efficacy against both extracellular promastigote (IC50 9.54 and 5.42 µM, respectively) and intracellular amastigote (IC50 9.81 and 3.75 µM, respectively) forms of Leishmania donovani with negligible cytotoxicity toward the host (J774 macrophages, Vero cells). However, compound 15i effectively inhibited the parasite burden in the liver and spleen (>80%) of infected BALB/c mice. Mechanistic studies revealed that 15i triggers oxidative stress which induces bioenergetic collapse and apoptosis of the parasite by decreasing ATP production and mitochondrial membrane potential. Structure-activity analyses and pharmacokinetic studies suggest 15i as a promising antileishmanial lead and emphasize the importance of quinoline-metronidazole series as a suitable platform for the future development of antileishmanial agents.


Assuntos
Antiprotozoários/síntese química , Antiprotozoários/farmacologia , Leishmaniose Visceral/tratamento farmacológico , Metronidazol/síntese química , Metronidazol/farmacologia , Quinolinas/química , Animais , Antiprotozoários/química , Antiprotozoários/farmacocinética , Técnicas de Química Sintética , Chlorocebus aethiops , Modelos Animais de Doenças , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/fisiologia , Metronidazol/química , Metronidazol/farmacocinética , Ratos , Relação Estrutura-Atividade , Distribuição Tecidual , Células Vero
19.
PLoS Negl Trop Dis ; 13(5): e0007227, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31071090

RESUMO

BACKGROUND: There is a pressing need for drug discovery against visceral leishmaniasis, a life-threatening protozoal infection, as the available chemotherapy is antiquated and not bereft of side effects. Plants as alternate drug resources has rewarded mankind in the past and aimed in this direction, we investigated the antileishmanial potential of Cinnamomum cassia. METHODOLOGY: Dichloromethane, ethanolic and aqueous fractions of C. cassia bark, prepared by sequential extraction, were appraised for their anti-promastigote activity along with apoptosis-inducing potential. The most potent, C. cassia dichloromethane fraction (CBD) was evaluated for anti-amastigote efficacy in infected macrophages and nitric oxide (NO) production studied. The in vivo antileishmanial efficacy was assessed in L. donovani infected BALB/c mice and hamsters and various correlates of host protective immunity ascertained. Toxicity profile of CBD was investigated in vitro against peritoneal macrophages and in vivo via alterations in liver and kidney functions. The plant secondary metabolites present in CBD were identified by gas chromatography-mass spectroscopy (GC-MS). PRINCIPAL FINDINGS: CBD displayed significant anti-promastigote activity with 50% inhibitory concentration (IC50) of 33.6 µg ml-1 that was mediated via apoptosis. This was evidenced by mitochondrial membrane depolarization, increased proportion of cells in sub-G0-G1 phase, ROS production, PS externalization and DNA fragmentation (TUNEL assay). CBD also inhibited intracellular amastigote proliferation (IC50 14.06 µg ml-1) independent of NO production. The in vivo protection achieved was 80.91% (liver) and 82.92% (spleen) in mice and 75.61% (liver) and 78.93% (spleen) in hamsters indicating its profound therapeutic efficacy. CBD exhibited direct antileishmanial activity, as it did not specifically induce a T helper type (Th)-1-polarized mileu in cured hosts. This was evidenced by insignificant modulation of NO production, lymphoproliferation, DTH (delayed type hypersensitivity), serum IgG2a and IgG1 levels and production of Th2 cytokines (IL-4 and IL-10) along with restoration of pro-inflammatory Th1 cytokines (INF-γ, IL-12p70) to the normal range. CBD was devoid of any toxicity in vitro as well as in vivo. The chemical constituents, cinnamaldehyde and its derivatives present in CBD may have imparted the observed antileishmanial effect. CONCLUSIONS: Our study highlights the profound antileishmanial efficacy of C. cassia bark DCM fraction and merits its further exploration as a source of safe and effective antieishmanial compounds.


Assuntos
Antiprotozoários/administração & dosagem , Cinnamomum aromaticum/química , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Extratos Vegetais/administração & dosagem , Animais , Antiprotozoários/isolamento & purificação , Cricetinae , Citocinas/genética , Citocinas/imunologia , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Leishmania donovani/fisiologia , Leishmaniose Visceral/genética , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Macrófagos Peritoneais/efeitos dos fármacos , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Casca de Planta/química , Extratos Vegetais/isolamento & purificação
20.
Acta Parasitol ; 64(4): 710-719, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30941668

RESUMO

BACKGROUND: Leishmania donovani (L. donovani) is one of the parasites that cause leishmaniasis. The mechanisms by which L. donovani fights against adverse environment and becomes resistant to drugs are not well understood yet. OBJECTIVE: The present study was designed to evaluate the effects of different regulators on the modulation of Transplasma Membrane Electron Transport (transPMET) systems of susceptible and resistant L. donovani cells. MATERIALS AND METHODS: Effects of UV, different buffers, and electron transport inhibitors and stimulators on the reduction of α-lipoic acid (ALA), 1,2-naphthoquinone-4-sulphonic acid (NQSA) and ferricyanide were determined. RESULTS AND DISCUSSION: ALA reductions were inhibited in susceptible, sodium antimony gluconate (SAG)-resistant and paromomycin (PMM)-resistant AG83 amastigote cells, and stimulated in susceptible and SAG-resistant AG83 promastigote cells upon UV exposure. The results indicate that UV irradiation almost oppositely affect ALA reductions in amastigotes and promastigotes. ALA reductions were stimulated in sensitive and inhibited in resistant GE1 amastigotes upon UV exposure. Susceptible amastigotes and promastigotes inhibited, and resistant amastigotes and promastigotes stimulated NQSA reduction under UV irradiation. Thus, susceptible and drug-resistant amastigotes and promastigotes are different in the reduction of ALA. Susceptible and resistant AG83 amastigotes and promastigotes inhibited the ferricyanide reductions upon UV exposure, which indicates, there is no such difference in ferricyanide reductions among susceptible as well as resistant AG83 amastigotes and promastigotes. The reductions of extracellular electron excerptors in susceptible promastigotes requires the availability of Na+ and Cl- ions for maximal activity but susceptible amastigotes are mostly not dependent on the availability of Na+ and Cl- ions. Both in promastigotes and amastigotes, reductions of electron acceptors were strongly inhibited by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. Furthermore, antimycin A, rotenone and capsaicin markedly inhibited the reductions of electron acceptors in promastigotes, but not in amastigotes. CONCLUSION: Results of this study suggest that the transPMET system is functionally different in wild and resistant strains of L. donovani.


Assuntos
Resistência a Medicamentos , Transporte de Elétrons , Leishmania donovani/fisiologia , Ferricianetos , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/efeitos da radiação , Estágios do Ciclo de Vida , Naftoquinonas/farmacologia , Oxirredução , Paromomicina/farmacologia , Ácidos Sulfônicos/farmacologia , Ácido Tióctico/metabolismo , Raios Ultravioleta
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