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1.
Parasite Immunol ; 42(9): e12732, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32418227

RESUMO

Visceral leishmaniasis (VL) is caused by a protozoan parasite Leishmania donovani mainly influencing the population of tropical and subtropical regions across the globe. The arsenal of drugs available is limited, and prolonged use of such drugs makes parasite to become resistant. Therefore, it is very imperative to develop a safe, cost-effective and inexpensive vaccine against VL. Although in recent years, many strategies have been pursued by researchers, so far only some of the vaccine candidates reached for clinical trial and more than half of them are still in pipeline. There is now a broad consent among Leishmania researchers that the perseverance of parasite is very essential for eliciting a protective immune response and may perhaps be attained by live attenuated parasite vaccination. For making a live attenuated parasite, it is very essential to ensure that the parasite is deficient of virulence and should further study genetically modified parasites to perceive the mechanism of pathogenesis. So it is believed that in the near future, a complete understanding of the Leishmania genome will explore clear strategies to discover a novel vaccine. This review describes the need for a genetically modified live attenuated vaccine against VL, and obstacles associated with its development.


Assuntos
Leishmania donovani/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/prevenção & controle , Animais , Humanos , Leishmania donovani/patogenicidade , Leishmaniose Visceral/imunologia , Vacinas Atenuadas/imunologia
2.
PLoS Negl Trop Dis ; 14(4): e0008167, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32275661

RESUMO

Leishmania donovani, an intracellular protozoan parasite upon infection, encounters a range of antimicrobial factors within the host cells. Consequently, the parasite has evolved mechanisms to evade this hostile defense system through inhibition of macrophage activation that, in turn, enables parasite replication and survival. There is growing evidence that epigenetic down-regulation of the host genome by intracellular pathogens leads to acute infection. Epigenetic modification is mediated by chromatin remodeling, histone modifications, or DNA methylation. Histone deacetylases (HDACs) removes acetyl groups from lysine residues on histones, thereby leading to chromatin remodeling and gene silencing. Here, using L. donovani infected macrophages differentiated from THP-1 human monocytic cells, we report a link between host chromatin modifications, transcription of defense genes and intracellular infection with L. donovani. Infection with L. donovani led to the silencing of host defense gene expression. Histone deacetylase 1 (HDAC1) transcript levels, protein expression, and enzyme activity showed a significant increase upon infection. HDAC1 occupancy at the promoters of the defense genes significantly increased upon infection, which in turn resulted in decreased histone H3 acetylation in infected cells, resulting in the down-regulation of mRNA expression of host defense genes. Small molecule mediated inhibition and siRNA mediated down-regulation of HDAC1 increased the expression levels of host defense genes. Interestingly, in this study, we demonstrate that the silencing of HDAC1 by both siRNA and pharmacological inhibitors resulted in decreased intracellular parasite survival. The present data not only demonstrate that up-regulation of HDAC1 and epigenetic silencing of host cell defense genes is essential for L. donovani infection but also provides novel therapeutic strategies against leishmaniasis.


Assuntos
Citoplasma/metabolismo , Epigênese Genética , Histona Desacetilase 1/genética , Leishmania donovani/patogenicidade , Leishmaniose/genética , Macrófagos/parasitologia , Linhagem Celular , Montagem e Desmontagem da Cromatina , Citoplasma/parasitologia , Metilação de DNA , Regulação para Baixo , Regulação da Expressão Gênica , Inativação Gênica , Histona Desacetilase 1/metabolismo , Histonas/genética , Histonas/metabolismo , Interações Hospedeiro-Parasita/genética , Humanos , Monócitos/metabolismo , Monócitos/parasitologia , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Células THP-1
3.
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
4.
Molecules ; 25(3)2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31979089

RESUMO

A chemically diverse range of novel tetraoxanes was synthesized and evaluated in vitro against intramacrophage amastigote forms of Leishmania donovani. All 15 tested tetraoxanes displayed activity, with IC50 values ranging from 2 to 45 µm. The most active tetraoxane, compound LC140, exhibited an IC50 value of 2.52 ± 0.65 µm on L. donovani intramacrophage amastigotes, with a selectivity index of 13.5. This compound reduced the liver parasite burden of L. donovani-infected mice by 37% after an intraperitoneal treatment at 10 mg/kg/day for five consecutive days, whereas miltefosine, an antileishmanial drug in use, reduced it by 66%. These results provide a relevant basis for the development of further tetraoxanes as effective, safe, and cheap drugs against leishmaniasis.


Assuntos
Antiprotozoários/química , Antiprotozoários/uso terapêutico , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/patogenicidade , Tetraoxanos/química , Tetraoxanos/uso terapêutico , Animais , Leishmaniose/tratamento farmacológico , Leishmaniose/parasitologia , Camundongos , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Fosforilcolina/uso terapêutico
5.
Parasite Immunol ; 42(1): e12678, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31610026

RESUMO

Dominant-negative mutation of LdeK1 gene, an eIF2α kinase from Leishmania donovani, revealed its role in translation regulation in response to nutrient starvation earlier. However, whether the kinase influences the infectivity of the parasites which naturally encounters nutrient deprivation during its life cycle was interesting to investigate. Both in vitro and in vivo experiments resulted in decrease of the parasite burden in peritoneal macrophages and in splenic/ hepatic load, respectively. An insight into the immune response of mice infected with mutant parasite showed enhanced pro-inflammatory cytokines and nitric oxide levels but reduced TH 2 and Treg population. The significantly reduced loss of infectivity of the parasites lacking a functional LdeK1 by modulating the immune response towards host protection makes it a potential vaccine candidate against Leishmaniasis.


Assuntos
Leishmania donovani/genética , Leishmania donovani/patogenicidade , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , eIF-2 Quinase/genética , Animais , Citocinas/imunologia , Feminino , Imunidade Celular , Leishmania donovani/imunologia , Fígado/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/metabolismo , Carga Parasitária , Baço/imunologia , Baço/parasitologia , Linfócitos T/imunologia , Virulência
6.
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
7.
Cell Death Dis ; 10(11): 808, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31649242

RESUMO

The importance of autophagy in parasites with a digenetic life cycle like Leishmania spp. is significant. The parasite survives as promastigotes in the insect gut and as immotile amastigotes in mammals. This study demonstrates increased autophagy in Leishmania parasite during progression of in vitro life cycle and upon exposure to stress stimuli like starvation, oxidative stress, and drugs. Autophagy inhibition during stress exposure increased cell death, indicating the importance of autophagy in cellular defense against adverse conditions. Atg8 protein, a homolog of mammalian autophagy protein LC3 is expressed in Leishmania parasite but its function remains unknown. Overexpression of Atg8 (Atg8-OE) rendered the parasites resistant to stress and capable of infecting macrophages in substantial numbers; however, disruption of the Atg8 gene (ΔAtg8) resulting in suppression of Atg8 protein expression, increased susceptibility to stress and reduced the capability to cause infection. A critical event in the Leishmania parasite lifecycle is the differentiation of promastigote forms to the disease causing amastigote forms. The failure of ΔAtg8 parasites lacking Atg8 protein to differentiate into amastigotes, unlike the Atg8-OE and vector-transfected parasites, clearly indicated Atg8 involvement in a crucial event. The inability of ΔAtg8 parasites to infect macrophages in vitro was verified in an in vivo mouse model of leishmaniases where infection could not be induced by the ΔAtg8 parasites. Autophagy is known to be involved in the remodeling of damaged organelles. The accumulation of Atg8 around damaged mitochondria suggested increase of autophagy in the vicinity of the organelle. This buildup was prevented when mitochondria generated reactive oxygen species that were quenched, suggesting them as possible signaling molecules for sensing mitochondrial instability. In summary, our study provides new evidences for a crucial role of Atg8 protein in sustaining Leishmania parasite survival during life cycle and stress exposure, differentiation to amastigotes, and their infective abilities.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/genética , Autofagia/genética , Leishmania donovani/genética , Leishmaniose/genética , Animais , Modelos Animais de Doenças , Humanos , Leishmania donovani/patogenicidade , Leishmaniose/parasitologia , Estágios do Ciclo de Vida/genética , Camundongos , Mitocôndrias/genética , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo
8.
ACS Infect Dis ; 5(12): 2087-2095, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31618572

RESUMO

Visceral leishmaniasis, one of the fatal forms of the disease, is caused by Leishmania donovani and presents morbid clinical manifestations. The parasite evades pro-inflammatory immune responses by several reported mechanisms and modulates the host immune system to cause fatal symptoms. A plethora of reports related to the role of BLIMP-1 and its involvement in suppressing the immune response in various infectious diseases have been documented. Higher parasitic burden due to increased BLIMP-1 production has been reported earlier for malaria and leishmaniasis with no detailed information. We report for the first time the role of BLIMP-1 in suppressing macrophage pyroptosis during L. donovani infection and thereby tweaking the tight regulation of the NFκß-NLRP3 signaling pathway. Expression analyses of BLIMP-1 and NFκß have been measured using real-time PCR and Western blotting. The importance of BLIMP-1 has been validated using a siRNA-mediated experiment along with caspase 1 activity, LDH release assay, and infectivity index analyses. An inverse relationship between BLIMP-1 and NFκß expression has been highlighted during L. donovani infection, which is reversed in blimp-1 deficient cells infected with promastigotes. The above fact has been further validated with caspase 1 activity assay, and LDH release along with IFNγ and TNF-α release assay. Finally, resumption of pyroptosis has been concluded in infected blimp-1 deficient cells in contrast to wild type infected cells. We conjecture that parasites modulate the NFκß-NLRP3 signaling pathway by taking advantage of BLIMP-1 dependent IL-10 production and finally disrupting an inflammation-mediated pyroptosis cell death pathway in infected cells.


Assuntos
Leishmania donovani/patogenicidade , Macrófagos/parasitologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Animais , Linhagem Celular , Humanos , Interleucina-10/metabolismo , Macrófagos/fisiologia , Camundongos , Modelos Biológicos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Piroptose , Transdução de Sinais , Células THP-1 , Regulação para Cima
9.
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
10.
Biochem Biophys Res Commun ; 516(3): 770-776, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31253400

RESUMO

Protein phosphorylation, governed by kinases and phosphatases, plays a pivotal role in enormous cellular signaling pathways. Although PPP family of serine/threonine phosphatases have been involved in multiplication and growth of trypanosomatid parasites, but comprehensive knowledge is still very limited. In the present study, protein phosphatase 1 from Leishmania donovani (LdPP1) was purified to homogeneity and its structural attributes were explored employing CD and fluorescence spectroscopy as well as bioinformatics methods. The CD analysis revealed an appropriate secondary structure with α-helices content outnumbering the ß-sheets, whereas intrinsic fluorescence study depicted about the buried positioning of tryptophan residues. The three-dimensional structure of LdPP1, determined by homology modeling, displayed all the characteristic features including similar position of metal as well as inhibitor binding site corresponding to the known PP1 structures. Furthermore, ELISA and qRT-PCR results showed that LdPP1 elicit the pro-inflammatory cytokines TNF-α and IL-6 at translated and transcriptional levels in THP1 macrophages. Subsequently, immune effector molecule nitric oxide and transcription factor NF-κB production was also found to be increased upon LdPP1 stimulation. Altogether, this is the first report on PPP phosphatase of trypanosomatid parasite that represents the structural highlights along with protein-mediated immunomodulation in human macrophages.


Assuntos
Leishmania donovani/imunologia , Macrófagos/imunologia , Proteína Fosfatase 1/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Domínio Catalítico , Dicroísmo Circular , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Expressão Gênica/imunologia , Humanos , Leishmania donovani/genética , Leishmania donovani/patogenicidade , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos , NF-kappa B/imunologia , NF-kappa B/metabolismo , Óxido Nítrico/imunologia , Óxido Nítrico/metabolismo , Conformação Proteica , Proteína Fosfatase 1/química , Proteína Fosfatase 1/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Células RAW 264.7 , Células THP-1
11.
Biomed Res Int ; 2019: 4093603, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31111052

RESUMO

Sri Lanka reports a large epidemic of cutaneous leishmaniasis (CL) caused by an atypical L. donovani while regional leishmaniasis elimination drive aims at achieving its targets in 2020. Visceralization, mucotrophism, and CL associated poor treatment response were recently reported. Long-term clinico-epidemiological trends (2001-2013) in this focus were examined for the first time. Both constant and changing features were observed. Sociodemographic patient characteristics that differ significantly from those of country profile, microchanges within CL profile, spatial expansion, constant biannual seasonal variation, and nondependency of clinical profile on age or gender were evident. Classical CL remains the main clinical entity without clinical evidence for subsequent visceralization indicating presence of parasite strain variation. These observations make a scientific platform for disease control preferably timed based on seasonal variation and highlights the importance of periodic and continued surveillance of clinic-epidemiological and other characteristics.


Assuntos
Leishmania donovani/patogenicidade , Leishmaniose Cutânea/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Medula Óssea , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Leishmaniose Cutânea/parasitologia , Masculino , Pessoa de Meia-Idade , Estações do Ano , Pele , Sri Lanka/epidemiologia , Adulto Jovem
12.
PLoS One ; 14(4): e0214193, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30939131

RESUMO

Cocos nucifera (C. nucifera) (the coconut palm tree) has been traditionally used to fight a number of human diseases, but only a few studies have tested its components against parasites such as those that cause malaria. In this study, C. nucifera samples were collected from a private natural reserve in Punta Patiño, Darien, Panama. The husk, leaves, pulp, and milk of C. nucifera were extracted and evaluated against the parasites that cause Chagas' disease or American trypanosomiasis (Trypanosoma cruzi), leishmaniasis (Leishmania donovani) and malaria (Plasmodium falciparum), as well as against a line of breast cancer cells. While there was no activity in the rest of the tests, five and fifteen-minute aqueous decoctions of leaves showed antiplasmodial activity at 10% v/v concentration. Removal of some HPLC fractions resulted in loss of activity, pointing to the presence of synergy between the components of the decoction. Chemical molecules were separated and identified using an ultra-performance liquid chromatography (UPLC) approach coupled to tandem mass spectrometry (LC-MS/MS) using atmospheric pressure chemical ionization quadrupole-time of flight mass spectrometry (APCI-Q-TOF-MS) and molecular networking analysis, revealing the presence of compounds including polyphenol, flavone, sterol, fatty acid and chlorophyll families, among others.


Assuntos
Antiparasitários/farmacologia , Cocos/química , Leishmaniose/tratamento farmacológico , Malária Falciparum/tratamento farmacológico , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Antiparasitários/química , Arecaceae/química , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Humanos , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/patogenicidade , Leishmaniose/parasitologia , Malária Falciparum/parasitologia , Panamá , Folhas de Planta/química , Espectrometria de Massas em Tandem , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/patogenicidade
13.
Front Immunol ; 10: 670, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31024534

RESUMO

Leishmaniasis is a parasitic disease of humans, highly prevalent in parts of the tropics, subtropics, and southern Europe. The disease mainly occurs in three different clinical forms namely cutaneous, mucocutaneous, and visceral leishmaniasis (VL). The VL affects several internal organs and is the deadliest form of the disease. Epidemiology and clinical manifestations of VL are variable based on the vector, parasite (e.g., species, strains, and antigen diversity), host (e.g., genetic background, nutrition, diversity in antigen presentation and immunity) and the environment (e.g., temperature, humidity, and hygiene). Chemotherapy of VL is limited to a few drugs which is expensive and associated with profound toxicity, and could become ineffective due to the parasites developing resistance. Till date, there are no licensed vaccines for humans against leishmaniasis. Recently, immunotherapy has become an attractive strategy as it is cost-effective, causes limited side-effects and do not suffer from the downside of pathogens developing resistance. Among various immunotherapeutic approaches, cytokines (produced by helper T-lymphocytes) based immunotherapy has received great attention especially for drug refractive cases of human VL. Therefore, a comprehensive knowledge on the molecular interactions of immune cells or components and on cytokines interplay in the host defense or pathogenesis is important to determine appropriate immunotherapies for leishmaniasis. Here, we summarized the current understanding of a wide-spectrum of cytokines and their interaction with immune cells that determine the clinical outcome of leishmaniasis. We have also highlighted opportunities for the development of novel diagnostics and intervention therapies for VL.


Assuntos
Citocinas/imunologia , Imunoterapia/métodos , Leishmania donovani/imunologia , Leishmania donovani/patogenicidade , Leishmaniose Visceral/imunologia , Animais , Progressão da Doença , Resistência à Doença , Vetores de Doenças , Humanos , Imunidade Celular , Leishmaniose Visceral/diagnóstico por imagem , Leishmaniose Visceral/terapia , Doenças Negligenciadas , Pele/patologia
14.
J Cell Sci ; 132(9)2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30910830

RESUMO

Previously, we documented the role of the programmed death-1 (PD-1, also known as PDCD1) pathway in macrophage apoptosis and the downregulation of this signaling during infection by the intra-macrophage parasite Leishmania donovani However, we also found that, during the late phase of infection, PD-1 expression was significantly increased without activating host cell apoptosis; here we show that inhibition of PD-1 led to markedly decreased parasite survival, along with increased production of TNFα, IL-12, reactive oxygen species (ROS) and nitric oxide (NO). Increased PD-1 led to inactivation of AKT proteins resulting in nuclear sequestration of FOXO-1. Transfecting infected cells with constitutively active FOXO-1 (CA-FOXO) led to increased cell death, thereby suggesting that nuclear FOXO-1 might be inactivated. Infection significantly induced the expression of SIRT1, which inactivated FOXO-1 through deacetylation, and its knockdown led to increased apoptosis. SIRT1 knockdown also significantly decreased parasite survival along with increased production of TNFα, ROS and NO. Administration of the SIRT1 inhibitor sirtinol (10 mg/kg body weight) in infected mice decreased spleen parasite burden and a synergistic effect was found with PD-1 inhibitor. Collectively, our study shows that Leishmania utilizes the SIRT1/FOXO-1 axis for differentially regulating PD-1 signaling and, although they are interconnected, both pathways independently contribute to intracellular parasite survival.This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteína Forkhead Box O1/metabolismo , Interações Hospedeiro-Parasita , Leishmania donovani , Receptor de Morte Celular Programada 1/metabolismo , Sirtuína 1/metabolismo , Animais , Apoptose , Benzamidas/farmacologia , Linhagem Celular , Citocinas/metabolismo , Progressão da Doença , Interações Hospedeiro-Parasita/imunologia , Interações Hospedeiro-Parasita/fisiologia , Evasão da Resposta Imune/fisiologia , Leishmania donovani/parasitologia , Leishmania donovani/patogenicidade , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Naftóis/farmacologia , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Sirtuína 1/efeitos dos fármacos , Baço/parasitologia , Fator de Necrose Tumoral alfa/metabolismo
15.
Sci Rep ; 9(1): 5074, 2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30911045

RESUMO

Leishmania parasites are thought to control protein activity at the post-translational level, e.g. by protein phosphorylation. In the pathogenic amastigote, the mammalian stage of Leishmania parasites, heat shock proteins show increased phosphorylation, indicating a role in stage-specific signal transduction. Here we investigate the impact of phosphosites in the L. donovani heat shock protein 90. Using a chemical knock-down/genetic complementation approach, we mutated 11 confirmed or presumed phosphorylation sites and assessed the impact on overall fitness, morphology and in vitro infectivity. Most phosphosite mutations affected the growth and morphology of promastigotes in vitro, but with one exception, none of the phosphorylation site mutants had a selective impact on the in vitro infection of macrophages. Surprisingly, aspartate replacements mimicking the negative charge of phosphorylated serines or threonines had mostly negative impacts on viability and infectivity. HSP90 is a substrate for casein kinase 1.2-catalysed phosphorylation in vitro. While several putative phosphosite mutations abrogated casein kinase 1.2 activity on HSP90, only Ser289 could be identified as casein kinase target by mass spectrometry. In summary, our data show HSP90 as a downstream client of phosphorylation-mediated signalling in an organism that depends on post-transcriptional gene regulation.


Assuntos
Caseína Quinases/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Leishmania donovani/metabolismo , Leishmania donovani/patogenicidade , Sequência de Aminoácidos , Caseína Quinases/genética , Proteínas de Choque Térmico HSP90/genética , Leishmania donovani/genética , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutagênese , Mutação , Fosforilação , Transdução de Sinais/genética
16.
Chem Biol Drug Des ; 93(6): 1050-1060, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30891955

RESUMO

Visceral leishmaniasis affects millions of people worldwide in areas where Leishmania donovani is endemic. The protozoan species serves a greater threat as it has gradually evolved drug resistance whereby requiring newer approaches to treat the infection. State-of-art techniques are mostly directed toward finding better targets extracted from the available proteome data. In light of recent computational advancements, we ascertain and validate one such target, adenylosuccinate lyase (ADSL) by implementation of in-silico methods which led to the identification of critical amino acid residues that affects its functional attributes. Our target selection was based on comprehensive topological analysis of a knowledge-based protein-protein interaction network. Subsequently, mutations were incorporated and the dynamic behavior of mutated and native proteins was traced using MD simulations for a total time span of 600 ns. Comparative analysis of the native and mutated structures exhibited perceptible changes in the ligand-bound catalytic region with respect to time. The unfavorable changes in the orientations of specific catalytic residues, His118 and His196, induced by generated mutations reduce the enzyme specificity. In summary, this integrative approach is able to select a target against pathogen, identify crucial residues, and challenge its functionality through the selected mutations.


Assuntos
Adenilossuccinato Liase/metabolismo , Leishmaniose Visceral/enzimologia , Simulação de Dinâmica Molecular , Mutação , Biologia de Sistemas , Animais , Leishmania donovani/patogenicidade , Leishmaniose Visceral/parasitologia , Ligação Proteica
17.
Nanomedicine (Lond) ; 14(4): 387-406, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30688557

RESUMO

AIM: The present study evaluates the efficacy of paromomycin (PM)-loaded mannosylated thiomeric nanoparticles for the targeted delivery to pathological organs for the oral therapy of visceral leishmaniasis. MATERIALS & METHODS: Mannosylated thiolated chitosan (MTC)-coated PM-loaded PLGA nanoparticles (MTC-PLGA-PM) were synthesized and evaluated for morphology, drug release, permeation enhancing and antileishmanial potential. RESULTS: MTC-PLGA-PM were spherical in shape with a size of 391.24 ± 6.91 nm and an encapsulation efficiency of 67.16 ± 14%. Ex vivo permeation indicated 12.73-fold higher permeation of PM with MTC-PLGA-PM against the free PM. Flow cytometry indicated enhanced macrophage uptake and parasite killing in Leishmania donovani infected macrophage model. In vitro antileishmanial activity indicated 36-fold lower IC50 for MTC-PLGA-PM as compared with PM. The in vivo studies indicated 3.6-fold reduced parasitic burden in the L. donovani infected BALB/c mice model. CONCLUSION: The results encouraged the concept of MTC-PLGA-PM nanoparticles as promising strategy for visceral leishmaniasis.


Assuntos
Leishmaniose Visceral/tratamento farmacológico , Nanopartículas/química , Paromomicina/química , Paromomicina/uso terapêutico , Animais , Antiprotozoários/administração & dosagem , Antiprotozoários/química , Antiprotozoários/uso terapêutico , Células Cultivadas , Citometria de Fluxo , Lectinas Tipo C/metabolismo , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/patogenicidade , Leishmaniose Visceral/metabolismo , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Paromomicina/administração & dosagem , Receptores de Superfície Celular/metabolismo
18.
Sci Rep ; 9(1): 438, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30679614

RESUMO

The limited success of recent phenotypic anti-leishmanial drug screening campaigns calls for new screening strategies for the discovery of clinically relevant hits. Here we present such a novel strategy based on physiologically relevant, ex vivo biology. We established high content phenotypic assays that combine primary murine macrophages and lesion-derived, virulent L. donovani and L. amazonensis amastigotes, which we applied to validate previously identified, anti-leishmanial hit compounds referred to as 'GSK Leish-Box'. Together with secondary screens using cultured promastigotes, our pipeline distinguished stage- and/or species-specific compounds, including 20 hits with broad activity at 10 µM against intracellular amastigotes of both viscerotropic and dermotropic Leishmania. Even though the GSK Leish-Box hits were identified by phenotypic screening using THP-1 macrophage-like cells hosting culture-derived L. donovani LdBob parasites, our ex vivo assays only validated anti-leishmanial activity at 10 µM on intra-macrophagic L. donovani for 23 out of the 188 GSK Leish-Box hits. In conclusion, our comparative approach allowed the identification of hits with broad anti-leishmanial activity that represent interesting novel candidates to be tested in animal models. Physiologically more relevant screening approaches such as described here may reduce the very high attrition rate observed during pre-clinical and clinical phases of the drug development process.


Assuntos
Antiprotozoários/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Leishmania donovani/efeitos dos fármacos , Leishmania mexicana/efeitos dos fármacos , Leishmaniose Cutânea/prevenção & controle , Leishmaniose Visceral/prevenção & controle , Animais , Antiprotozoários/química , Células Cultivadas , Humanos , Leishmania donovani/patogenicidade , Leishmania mexicana/patogenicidade , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos Endogâmicos BALB C , Estrutura Molecular , Fenótipo , Especificidade da Espécie , Células THP-1 , Virulência/efeitos dos fármacos
19.
Sci Rep ; 9(1): 762, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30679687

RESUMO

Post Kala-azar dermal leishmaniasis (PKDL), caused by Leishmania donovani is the dermal sequel of Visceral Leishmaniasis and importantly, is the proposed disease reservoir. The survival of Leishmania parasites within monocytes/macrophages hinges on its ability to effectively nullify immune activation mechanisms. Thus, delineating the disease-promoting immune mechanisms can facilitate development of immunotherapeutic strategies. Accordingly, in the absence of an animal model, this study aimed to delineate the status of CD8+ T-cells in patients with PKDL. At disease presentation, the absence of CD4+ T-cells at lesional sites was concomitant with an overwhelming infiltration of CD8+ T-cells that demonstrated an absence of Perforin, Granzyme and Zap-70, along with an enhanced expression of Programmed Death-1 (PD-1) and the skin-homing CCL17. Additionally, the lesional CCR4+CD8+ population was associated with an enhanced expression of IL-10 and IL-5. In circulation, the enhanced CD8+CCR4+ T-cell population and raised levels of CCL17/22 was associated with an increased frequency of PD-1, while CD127 was decreased. Taken together, in PKDL, the enhanced plasma and lesional CCL17 accounted for the dermal homing of CD8+CCR4+ T-cells, that along with a concomitant upregulation of PD-1 and IL-10 mediated immune inactivation, emphasizing the need for designing immunotherapies capable of reinvigorating T-cell potency.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Interleucina-10/genética , Leishmania donovani/imunologia , Leishmaniose Visceral/genética , Receptor de Morte Celular Programada 1/genética , Adolescente , Adulto , Linfócitos T CD8-Positivos/parasitologia , Quimiocina CCL17/genética , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , Interleucina-10/imunologia , Subunidade alfa de Receptor de Interleucina-7 , Leishmania donovani/patogenicidade , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/patologia , Macrófagos/imunologia , Masculino , Monócitos/imunologia , Perforina/genética , Receptores CCR4/genética , Adulto Jovem , Proteína-Tirosina Quinase ZAP-70/genética
20.
Clin Infect Dis ; 68(9): 1530-1538, 2019 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-30188978

RESUMO

BACKGROUND: Convenient, safe, and effective treatments for visceral leishmaniasis in Eastern African children are lacking. Miltefosine, the only oral treatment, failed to achieve adequate efficacy, particularly in children, in whom linear dosing (2.5 mg/kg/day for 28 days) resulted in a 59% cure rate, with lower systemic exposure than in adults. METHODS: We conducted a Phase II trial in 30 children with visceral leishmaniasis, aged 4-12 years, to test whether 28 days of allometric miltefosine dosing safely achieves a higher systemic exposure than linear dosing. RESULTS: Miltefosine accumulated during treatment. Median areas under the concentration time curve from days 0-210 and plasma maximum concentration values were slightly higher than those reported previously for children on linear dosing, but not dose-proportionally. Miltefosine exposure at the start of treatment was increased, with higher median plasma concentrations on day 7 (5.88 versus 2.67 µg/mL). Concentration-time curves were less variable, avoiding the low levels of exposure observed with linear dosing. The 210-day cure rate was 90% (95% confidence interval, 73-98%), similar to that previously described in adults. There were 19 treatment-related adverse events (AEs), but none caused treatment discontinuation. There were 2 serious AEs: both were unrelated to treatment and both patients were fully recovered. CONCLUSIONS: Allometric miltefosine dosing achieved increased and less-variable exposure than linear dosing, though not reaching the expected exposure levels. The new dosing regimen safely increased the efficacy of miltefosine for Eastern African children with visceral leishmaniasis. Further development of miltefosine should adopt allometric dosing in pediatric patients. CLINICAL TRIALS REGISTRATION: NCT02431143.


Assuntos
Antiprotozoários/farmacocinética , Leishmaniose Visceral/tratamento farmacológico , Fosforilcolina/análogos & derivados , África Oriental , Antiprotozoários/sangue , Antiprotozoários/farmacologia , Área Sob a Curva , Criança , Pré-Escolar , Esquema de Medicação , Feminino , Humanos , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/patogenicidade , Leishmaniose Visceral/sangue , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/patologia , Masculino , Segurança do Paciente , Fosforilcolina/sangue , Fosforilcolina/farmacocinética , Fosforilcolina/farmacologia , Resultado do Tratamento
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