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1.
Mem Inst Oswaldo Cruz ; 115: e190408, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32321156

RESUMO

BACKGROUND: The mechanism of resistance to SbIII in Leishmania is complex, multifactorial and involves not only biochemical mechanisms, but also other elements, such as the immune system of the host. OBJECTIVES: In this study, putative changes in the immunological profile of human monocytes infected with wild-type (WT) and antimony (SbIII)-resistant Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum lines were evaluated. METHODS: Susceptibility assays WT and SbIII-resistant L. braziliensis and L. infantum were performed using lines THP-1 human monocytic lineage. Phagocytic capacity, cytokine profile, intracellular nitric oxide (NO) production and surface carbohydrate residues profile were performed in peripheral blood monocytes by flow cytometry. FINDINGS: The phagocytic capacity and intracellular NO production by classical (CD14++CD16-) and proinflammatory (CD14++CD16+) monocytes were higher in the presence of L. infantum lines compared to L. braziliensis lines. The results also highlight proinflammatory monocytes as the cellular subpopulation of major relevance in a phagocytosis event and NO expression. It is important to note that L. infantum induced a proinflammatory cytokine profile characterised by higher levels of TNF-α in culture supernatant than L. braziliensis. Conversely, both Leishmania lines induce high levels of IL-6 in culture supernatant. Analysis of the expression profile of surface carbohydrates showed that L. braziliensis presents 4.3-fold higher expression of galactose(ß1,4)N-acetylglucosamine than L. infantum line. Interestingly, the expression level of α-N-acetylgalactosamine residues was 2-fold lower in the SbIII-resistant L. braziliensis line than its counterpart WT line, indicating differences in surface glycoconjugates between these lines. MAIN CONCLUSIONS: Our results showed that L. braziliensis and L. infantum induce different innate immune responses and a highly inflammatory profile, which is characteristic of infection by L. infantum, the species associated with visceral disease.


Assuntos
Antimônio/farmacologia , Antiprotozoários/farmacologia , Leishmania braziliensis/imunologia , Leishmania infantum/imunologia , Monócitos/parasitologia , Óxido Nítrico/biossíntese , Fagocitose/imunologia , Adulto , Resistência a Medicamentos , Feminino , Citometria de Fluxo , Humanos , Imunidade Inata , Leishmania braziliensis/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Masculino , Monócitos/imunologia , Adulto Jovem
2.
PLoS Negl Trop Dis ; 14(3): e0008014, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32126078

RESUMO

BACKGROUND: Leishmaniasis is a vector-borne neglected disease. Inside the natural sand fly vector, the promastigote forms of Leishmania undergo a series of extracellular developmental stages to reach the infectious stage, the metacyclic promastigote. There is limited information regarding the expression profile of L. infantum developmental stages inside the sand fly vector, and molecular markers that can distinguish the different parasite stages are lacking. METHODOLOGY/PRINCIPAL FINDINGS: We performed RNAseq on unaltered midguts of the sand fly Lutzomyia longipalpis after infection with L. infantum parasites. RNAseq was carried out at various time points throughout parasite development. Principal component analysis separated the transcripts corresponding to the different Leishmania promastigote stages, the procyclic, nectomonad, leptomonad and metacyclics. Importantly, there were a significant number of differentially expressed genes when comparing the sequential development of the various Leishmania stages in the sand fly. There were 836 differentially expressed (DE) genes between procyclic and long nectomonad promastigotes; 113 DE genes between nectomonad and leptomonad promastigotes; and 302 DE genes between leptomonad and metacyclic promastigotes. Most of the DE genes do not overlap across stages, highlighting the uniqueness of each Leishmania stage. Furthermore, the different stages of Leishmania parasites exhibited specific transcriptional enrichment across chromosomes. Using the transcriptional signatures exhibited by distinct Leishmania stages during their development in the sand fly midgut, we determined the genes predominantly enriched in each stage, identifying multiple potential stage-specific markers for L. infantum. CONCLUSIONS: Overall, these findings demonstrate the transcriptional plasticity of the Leishmania parasite inside the sand fly vector and provide a repertoire of potential stage-specific markers for further development as molecular tools for epidemiological studies.


Assuntos
Expressão Gênica , Marcadores Genéticos , Leishmania infantum/crescimento & desenvolvimento , Leishmania infantum/genética , Psychodidae/parasitologia , Animais , Feminino , Trato Gastrointestinal/parasitologia , Perfilação da Expressão Gênica , Análise de Sequência de RNA
3.
PLoS Negl Trop Dis ; 14(3): e0008077, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32214313

RESUMO

BACKGROUND: Phlebotomus (Larroussius) perniciosus and Canis familiaris are respectively the only confirmed vector and reservoir for the transmission of Leishmania (L.) infantum MON-1 in Tunisia. However, the vector and reservoir hosts of the two other zymodemes, MON-24 and MON-80, are still unknown. The aim of this study was to analyze the L. infantum life cycle in a Tunisian leishmaniasis focus. For this purpose, we have focused on: i) the detection, quantification and identification of Leishmania among this sand fly population, and ii) the analysis of the blood meal preferences of Larroussius (Lar.) subgenus sand flies to identify the potential reservoirs. METHODOLOGY AND FINDINGS: A total of 3,831 sand flies were collected in seven locations from the center of Tunisia affected by human visceral leishmaniasis. The collected sand flies belonged to two genus Phlebotomus (Ph.) (five species) and Sergentomyia (four species). From the collected 1,029 Lar. subgenus female sand flies, 8.26% was positive to Leishmania by ITS1 nested PCR. Three Leishmania spp. were identified: L. infantum 28% (24/85), L. killicki 13% (11/85), and L. major 22% (19/85). To identify the blood meal sources in Ph. Lar. subgenus sand flies, engorged females were analyzed by PCR-sequencing targeting the vertebrate cytochrome b gene. Among the 177 analyzed blood-fed females, 169 samples were positive. Sequencing results showed seven blood sources: cattle, human, sheep, chicken, goat, donkey, and turkey. In addition, mixed blood meals were detected in twelve cases. Leishmania DNA was found in 21 engorged females, with a wide range of blood meal sources: cattle, chicken, goat, chicken/cattle, chicken/sheep, chicken/turkey and human/cattle. The parasite load was quantified in fed and unfed infected sand flies using a real time PCR targeting kinetoplast DNA. The average parasite load was 1,174 parasites/reaction and 90 parasites/reaction in unfed and fed flies, respectively. CONCLUSION: Our results support the role of Ph. longicuspis, Ph. perfiliewi, and Ph. perniciosus in L. infantum transmission. Furthermore, these species could be involved in L. major and L. killicki life cycles. The combination of the parasite detection and the blood meal analysis in this study highlights the incrimination of the identified vertebrate in Leishmania transmission. In addition, we quantify for the first time the parasite load in naturally infected sand flies caught in Tunisia. These findings are relevant for a better understanding of L. infantum transmission cycle in the country. Further investigations and control measures are needed to manage L. infantum transmission and its spreading.


Assuntos
DNA/análise , Comportamento Alimentar , Conteúdo Gastrointestinal/química , Conteúdo Gastrointestinal/parasitologia , Especificidade de Hospedeiro , Leishmania infantum/isolamento & purificação , Phlebotomus/fisiologia , Animais , DNA/genética , Impressões Digitais de DNA , DNA Espaçador Ribossômico/genética , Transmissão de Doença Infecciosa , Feminino , Humanos , Leishmania infantum/genética , Masculino , Phlebotomus/parasitologia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Tunísia
4.
Rev Soc Bras Med Trop ; 53: e20190446, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32130324

RESUMO

INTRODUCTION: Visceral leishmaniasis (VL) represents a public health concern in several areas of the world. In the American continent, VL transmission is typically zoonotic, but humans with active VL caused by Leishmania infantum are able to infect sandflies. Thus, individuals with cutaneous parasitic infections may act as reservoirs and allow interhuman transmission. Additionally, the skin may be responsible for reactivation of the disease after therapy. This study's objective was to evaluate cutaneous parasitism in humans with VL in an American endemic area. METHODS: A cross-sectional hospital-based study was conducted in northeast Brazil from October 2016 to April 2017. Biopsies of healthy skin for histopathology and immunohistochemistry were performed prior to treatment in all study patients. RESULTS: Twenty-two patients between the ages of five months to 78 years were included in the study. Seven patients (31.8%) tested positive for HIV. Only one patient had cutaneous parasitism, as confirmed by immunohistochemistry prior to treatment. Parasitism was not detected after treatment. CONCLUSIONS: Cutaneous parasitism in the healthy skin of humans with visceral leishmaniasis, although unusual, may be a source of infection for phlebotomine sandflies.


Assuntos
Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/parasitologia , Pele/parasitologia , Adolescente , Adulto , Idoso , Biópsia , Criança , Pré-Escolar , Estudos Transversais , Escolaridade , Doenças Endêmicas , Feminino , Humanos , Imuno-Histoquímica , Lactente , Leishmaniose Visceral/patologia , Masculino , Pessoa de Meia-Idade , Pele/patologia , Adulto Jovem
5.
Rev Bras Parasitol Vet ; 29(1): e016319, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32049143

RESUMO

Leishmania infantum is a trypanosomatid that causes parasitic dermatopathy in dogs. Trypanosoma caninum is another trypanosomatid, which infects the skin of dogs, although cutaneous abnormalities are absent. This study aimed to investigate the occurrence of T. caninum infection and its associated cutaneous and histological changes and compare it with the occurrence of L. infantum infection in dogs. The study included 150 dogs, of which T. caninum infection was identified in 3 (2%) and L. infantum infection in 15 (10%) of them, with no association (p>0.05) of these infections with the breed, gender, age, or cutaneous abnormalities. The cutaneous abnormalities were based on 1 (4.8%) and 12 (57.1%) dogs infected by T. caninum and L. infantum, respectively. The dermatohistopathological abnormalities in the dogs infected with T. caninum included mild perivascular lymphohistioplasmacytic infiltrates in the clinically asymptomatic ones, while in those with dermatological abnormalities, acanthosis, epidermal orthokeratotic hyperkeratosis, melanomacrophages, and co-infection with Microsporum sp. and Trichophyton sp. were observed. InL. infantum infected, the histopathological findings included chronic granulomatous inflammatory infiltrates and structures compatible with amastigotes. Despite the low frequency of T. caninum infection, our findings suggest that this trypanosomatid, unlike L. infantum, does not cause any macroscopic skin abnormalities.


Assuntos
Doenças do Cão/patologia , Leishmania infantum/genética , Leishmaniose Visceral/veterinária , Trypanosoma/genética , Tripanossomíase/veterinária , Animais , Brasil/epidemiologia , Coinfecção , DNA de Protozoário/genética , Doenças do Cão/epidemiologia , Cães , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/patologia , Reação em Cadeia da Polimerase , Prevalência , Tripanossomíase/epidemiologia , Tripanossomíase/patologia
6.
Am J Trop Med Hyg ; 102(3): 593-597, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31971146

RESUMO

Endemic cutaneous leishmaniasis (CL) in northern Argentina has traditionally been caused by Leishmania braziliensis. This study aims to describe an outbreak of Leishmania infantum-caused human CL in the Department Capital of Corrientes Province, Argentina. We retrospectively analyzed the reported cases of CL in this area from May 2015 to December 2016. Eighty cases of CL were clinically and analytically diagnosed, and there was one case of visceral leishmaniasis in a boy who also had CL. Patients' median age was 33.6 years (range 1-89 years), and 18.5% were younger than 15 years; the male:female ratio was 3.5:1. Cases lived mostly in the municipality of Corrientes (72.8%), whereas 27.2% resided in Riachuelo. Although 67.9% had a single lesion, 32.1% had several. Molecular analyses showed that L. infantum was the causative species in all cases. Our results show that for the first time, there was an outbreak of CL by L. infantum in an urban area of Argentina.


Assuntos
Surtos de Doenças , Leishmania infantum , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/parasitologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Argentina/epidemiologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , População Urbana , Adulto Jovem
7.
PLoS Negl Trop Dis ; 14(1): e0008021, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961868

RESUMO

Domestic dogs are the main reservoir of Leishmania infantum, a causative agent of visceral leishmaniasis (VL). The number of human disease cases is associated with the rate of canine infection. Currently available drugs are not efficient at treating canine leishmaniasis (CanL) and months after the treatment most dogs show disease relapse, therefore the development of new drugs or new therapeutic strategies should be sought. In CanL, dogs lack the ability to mount a specific cellular immune response suitable for combating the parasite and manipulation of cytokine signaling pathway has the potential to form part of effective immunotherapeutic methods. In this study, recombinant canine cytokines (rcaIL-12, rcaIL-2, rcaIL-15 and rcaIL-7) and soluble receptor IL-10R1 (rcasIL-10R1), with antagonistic activity, were evaluated for the first time in combination (rcaIL-12/rcaIL-2, rcaIL-12/rcaIL-15, rcaIL-12/rcasIL-10R1, rcaIL-15/rcaIL-7) or alone (rcasIL-10R1) to evaluate their immunomodulatory capacity in peripheral blood mononuclear cells (PBMCs) from dogs with leishmaniasis. All the combinations of recombinant proteins tested were shown to improve lymphoproliferative response. Further, the combinations rcaIL-12/rcaIL-2 and rcaIL-12/rcaIL-15 promoted a decrease in programmed cell death protein 1 (PD-1) expression in lymphocytes. These same combinations of cytokines and rcaIL-12/rcasIL-10R1 induced IFN-γ and TNF-α production in PBMCs. Furthermore, the combination IL-12/IL-15 led to an increased in T-bet expression in lymphocytes. These findings are encouraging and indicate the use of rcaIL-12 and rcaIL-15 in future in vivo studies aimed at achieving polarization of cellular immune responses in dogs with leishmaniasis, which may contribute to the development of an effective treatment against CanL.


Assuntos
Doenças do Cão/tratamento farmacológico , Doenças do Cão/imunologia , Interleucina-12/administração & dosagem , Interleucina-15/administração & dosagem , Leishmaniose Visceral/imunologia , Animais , Doenças do Cão/genética , Doenças do Cão/parasitologia , Cães , Imunidade Celular , Interferon gama/genética , Interferon gama/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-15/genética , Interleucina-15/imunologia , Leishmania infantum/fisiologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/veterinária , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/parasitologia , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Receptores de Citocinas/genética , Receptores de Citocinas/imunologia
8.
PLoS Negl Trop Dis ; 14(1): e0007949, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961876

RESUMO

Leishmaniasis is caused by intracellular parasites transmitted to vertebrates by sandfly bites. Clinical manifestations include cutaneous, mucosal or visceral involvement depending upon the host immune response and the parasite species. To assure their survival inside macrophages, these parasites developed a plethora of highly successful strategies to manipulate various immune system pathways. Considering that inflammasome activation is critical for the establishment of a protective immune response in many parasite infections, in this study we determined the transcriptome of THP-1 cells after infection with L. infantum, with a particular focus on the inflammasome components. To this end, the human cell line THP-1, previously differentiated into macrophages by PMA treatment, was infected with L. infantum promastigotes. Differentiated THP-1 cells were also stimulated with LPS to be used as a comparative parameter. The gene expression signature was determined 8 hours after by RNA-seq technique. Infected or uninfected THP-1 cells were stimulated with nigericin (NIG) to measure active caspase-1 and TNF-α, IL-6 and IL-1ß levels in culture supernatants after 8, 24 and 48 hours. L. infantum triggered a gene expression pattern more similar to non-infected THP-1 cells and very distinct from LPS-stimulated cells. Some of the most up-regulated genes in L. infantum-infected cells were CDC20, CSF1, RPS6KA1, CD36, DUSP2, DUSP5, DUSP7 and TNFAIP3. Some up-regulated GO terms in infected cells included cell coagulation, regulation of MAPK cascade, response to peptide hormone stimulus, negative regulation of transcription from RNA polymerase II promoter and nerve growth factor receptor signaling pathway. Infection was not able to induce the expression of genes associated with the inflammasome signaling pathway. This finding was confirmed by the absence of caspase-1 activation and IL-1ß production after 8, 24 and 48 hours of infection. Our results indicate that L. infantum was unable to activate the inflammasomes during the initial interaction with THP-1 cells.


Assuntos
Inflamassomos/imunologia , Leishmania infantum/fisiologia , Leishmaniose/genética , Monócitos/imunologia , Monócitos/parasitologia , Caspase 1/genética , Caspase 1/imunologia , Fosfatases de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/imunologia , Humanos , Inflamassomos/genética , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Leishmaniose/imunologia , Leishmaniose/parasitologia , Células THP-1 , Transcriptoma , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
9.
Chem Biol Interact ; 315: 108899, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31738906

RESUMO

Parasitic diseases still represent serious public health problems, since the high and steady emergence of resistant strains is evident. Because parasitic infections are distributed predominantly in developing countries, less toxic, more efficient, safer and more accessible drugs have become desirable in the treatment of the infected population. This is the case of leishmaniasis, an infectious disease caused by a protozoan of the genus Leishmania sp., responsible for triggering pathological processes from the simplest to the most severe forms leading to high rates of morbidity and mortality throughout the world. In the search for new leishmanicidal drugs, the thiosemicarbazones and the indole fragments have been identified as promising structures for leishmanicidal activity. The present study proposes the synthesis and structural characterization of new indole-thiosemicarbazone derivatives (2a-j), in addition to performing in vitro evaluations through cytotoxicity assays using macrophages (J774) activity against forms of Leishmania infantum and Leishmania amazonensis promastigote as well as ultrastructural analyzes in promastigotes of L. infantum. Results show that the indole-thiosemicarbazone derivatives were obtained with yield values varying from 32.09 to 94.64%. In the evaluation of cytotoxicity, the indole-thiosemicarbazone compounds presented CC50 values between 53.23 and 357.97 µM. Concerning the evaluation against L. amazonensis promastigote forms, IC50 values ranged between 12.31 and  > 481.52 µM, while the activity against L. infantum promastigotes obtained IC50 values between 4.36 and 23.35 µM. The compounds 2d and 2i tested against L. infantum were the most promising in the series, as they showed the lowest IC50 values: 5.60 and 4.36 respectively. The parasites treated with the compounds 2d and 2i showed several structural alterations, such as shrinkage of the cell body, shortening and loss of the flagellum, intense mitochondrial swelling and vacuolization of the cytoplasm leading the parasite to cellular unviability. Therefore, the indole-thiosemicarbazone compounds are promising because they yield considerable synthesis, have low cytotoxicity to mammalian cells and act as leishmanicidal agents.


Assuntos
Antiprotozoários/farmacologia , Indóis/farmacologia , Leishmania infantum/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Tiossemicarbazonas/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Leishmaniose/parasitologia , Macrófagos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos
10.
Vet Parasitol ; 277: 109015, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31874403

RESUMO

Canine leishmaniosis (CanL)-associated chronic kidney disease is a leading cause of morbidity and mortality in Mediterranean countries. Novel renal biomarkers, such as serum symmetric dimethylarginine (sSDMA), may be useful surrogates for the detection of renal functional impairment. The objectives of this study were to investigate sSDMA concentrations in dogs with CanL, with and without azotemia, and to establish any potential association with the prevalence and severity of proteinuria, with the prevalence of decreased urine specific gravity and with the LeishVet clinical stages of CanL. Serum samples from 68 dogs with CanL (50 nonazotemic and 18 azotemic) and 17 healthy dogs were retrospectively examined. Increased sSDMA was documented in 26 % of dogs with CanL without azotemia and in 83.3 % of dogs with azotemia. Serum SDMA was significantly higher in azotemic compared to nonazotemic dogs and was associated with the presence and severity of proteinuria, the decreased urine specific gravity and the advanced clinical stages of CanL. The results of the present study indicate that sSDMA may be a useful adjunct to serum creatinine and urine protein/creatinine ratio for the detection of CanL-associated nephropathy, but it is of limited value for distinguishing among the LeishVet clinical stages of CanL.


Assuntos
Arginina/análogos & derivados , Doenças do Cão/diagnóstico , Leishmaniose Visceral/complicações , Leishmaniose Visceral/diagnóstico , Insuficiência Renal Crônica/etiologia , Animais , Arginina/sangue , Azotemia/veterinária , Biomarcadores/sangue , Doenças do Cão/sangue , Cães , Leishmania infantum/fisiologia , Insuficiência Renal Crônica/sangue , Estudos Retrospectivos
11.
Acta Trop ; 201: 105178, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31606374

RESUMO

Leishmaniasis is a complex disease caused by Leishmania species belonging to subgenera Leishmania and Viannia. In South America, L. (L.) infantum is considered the most important causative agent of visceral leishmaniasis, while L. (L.) amazonensis and Viannia subgenus species are responsible for the different cutaneous or mucocutaneous forms. In our previous work, we developed a diagnostic approach for Leishmania species discrimination based on two qPCRs (qPCR-ML and qPCR-ama) targeting the minicircle kDNA followed by melting analysis. This approach allowed to (i) differentiate the subgenera Leishmania and Viannia, and (ii) distinguish between L. (L.) infantum and L. (L.) amazonensis. The aim of this work was to demonstrate the applicability of the approach previously described, using human and canine clinical samples and strains from a Brazilian region, where L. (L.) infantum, L. (L.) amazonensis and Viannia subgenus species coexist. After validation on New World strains, the diagnostic approach was applied blindly to 36 canine clinical samples (peripheral blood and bone marrow) and 11 human clinical samples (peripheral blood and bone marrow). The sensitivity was 95.6% (95% confidence interval 77.3-100%) and 100% (95% confidence interval 76.9-100%) in the canine bone marrow samples and human (peripheral blood and bone marrow) samples, respectively, compared to conventional PCR assays. Concerning the Leishmania species identification, the conventional and qPCR-based methods showed kappa value of 0.876 (95% confidence interval 0.638-1.000), indicating good agreement. Therefore, this approach proved to be useful in both veterinary and human clinical context in regions co-endemic for L. (L.) infantum, L. (L.) amazonensis, and Viannia subgenus, helping to provide rapid diagnosis and to allow studies of species distribution.


Assuntos
Leishmania infantum/genética , Leishmania mexicana/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Brasil , Cães , Humanos
12.
Acta Trop ; 201: 105217, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31605692

RESUMO

Glycosomes of trypanosomatids are peroxisome-like organelles comprising unique metabolic features, among which the lack of the hallmark peroxisomal enzyme catalase. The absence of this highly efficient peroxidase from glycosomes is presumably compensated by other antioxidants, peroxidases of the peroxiredoxin (PRX) family being the most promising candidates for this function. Here, we follow on this premise and investigate the product of a Leishmania infantum gene coding for a putative glycosomal PRX (LigPRX). First, we demonstrate that LigPRX localizes to glycosomes, resorting to indirect immunofluorescence analysis. Second, we prove that purified recombinant LigPRX is an active peroxidase in vitro. Third, we generate viable LigPRX-depleted L. infantum promastigotes by classical homologous recombination. Surprisingly, phenotypic analysis of these knockout parasites revealed that promastigote survival, replication, and protection from oxidative and nitrosative insults can proceed normally in the absence of LigPRX. Noticeably, we also witness that LigPRX-depleted parasites can infect and thrive in mice to the same extent as wild type parasites. Overall, by disclosing the dispensable character of the glycosomal peroxiredoxin in L. infantum, this work excludes this enzyme from being a key component of the glycosomal hydroperoxide metabolism and contemplates alternative players for this function.


Assuntos
Leishmania infantum/genética , Leishmania infantum/metabolismo , Microcorpos/metabolismo , Oxirredutases/metabolismo , Peroxirredoxinas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Animais , Camundongos , Microcorpos/genética , Oxirredutases/genética , Peroxirredoxinas/genética
13.
PLoS Negl Trop Dis ; 13(12): e0007885, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31790397

RESUMO

Monitoring the drug susceptibility of Leishmania isolates still largely relies on standard in vitro cell-based susceptibility assays using (patient-isolated) promastigotes for infection. Although this assay is widely used, no fully standardized/harmonized protocol is yet available hence resulting in the application of a wide variety of host cells (primary cells and cell lines), different drug exposure times, detection methods and endpoint criteria. Advocacy for standardization to decrease inter-laboratory variation and improve interpretation of results has already repeatedly been made, unfortunately still with unsatisfactory progress. As a logical next step, it would be useful to reach at least some agreement on the type of host cell and basic experimental design for routine amastigote susceptibility determination. The present laboratory study using different L. infantum strains as a model for visceral leishmaniasis species compared primary cells (mouse peritoneal exudate (PEC), mouse bone marrow derived macrophages and human peripheral blood monocyte derived macrophages) and commercially available cell lines (THP-1, J774, RAW) for either their susceptibility to infection, their role in supporting intracellular amastigote multiplication and overall feasibility/accessibility of experimental assay protocol. The major findings were that primary cells are better than cell lines in supporting infection and intracellular parasite multiplication, with PECs to be preferred for technical reasons. Cell lines require drug exposure of >96h with THP-1 to be preferred but subject to a variable response to PMA stimulation. The fast dividing J774 and RAW cells out-compete parasite-infected cells precluding proper assay read-out. Some findings could possibly also be applicable to cutaneous Leishmania strains, but this still needs cross-checking. Besides inherent limitations in a clinical setting, susceptibility testing of clinical isolates may remain problematic because of the reliance on patient-derived promastigotes which may exhibit variable degrees of metacyclogenesis and infectivity.


Assuntos
Antiprotozoários/farmacologia , Leishmania infantum/efeitos dos fármacos , Testes de Sensibilidade Parasitária/métodos , Animais , Células Cultivadas , Feminino , Humanos , Leishmania infantum/crescimento & desenvolvimento , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/parasitologia , Camundongos , Testes de Sensibilidade Parasitária/normas
14.
Parasit Vectors ; 12(1): 593, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852505

RESUMO

BACKGROUND: Kinesin-related gene diversity among strains and species of Leishmania may impact the sensitivity and specificity of serodiagnostic tests for visceral leishmaniasis (VL). METHODS: In this study, we report on the recombinant expression of this novel Iranian Leishmania infantum (MCAN14/47) homologue of rK39 (Li-rK39), in L. tarentolae. The diagnostic potential of the Li-rK39 antigen was evaluated in an ELISA, using sera from 100 VL patients, 190 healthy endemic controls, 46 non-endemic healthy controls and 47 patients with other infections. RESULTS: The results showed a sensitivity of 96% and a specificity of 93.8%. A commercial rK39 immunochromatographic test (ICT) was 90% sensitive and 100% specific on the same cohort. CONCLUSIONS: Here, we show that the K39 gene from an Iranian L. infantum isolate is heterozygous as compared to the sequence of the Brazilian L. infantum (former L. chagasi), whose antigen is incorporated in most rK39-based immunochromatographic tests. Therefore, Li-rK39 has the potential to be used as an alternative for VL diagnosis in Iran.


Assuntos
Antígenos de Protozoários/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Leishmania infantum/genética , Leishmania/genética , Leishmaniose Visceral/diagnóstico , Proteínas de Protozoários/sangue , Adolescente , Adulto , Animais , Antígenos de Protozoários/genética , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Expressão Gênica , Humanos , Lactente , Irã (Geográfico) , Leishmania/metabolismo , Leishmania infantum/imunologia , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/sangue , Leishmaniose Visceral/parasitologia , Masculino , Proteínas de Protozoários/genética , Testes Sorológicos/métodos , Adulto Jovem
15.
Molecules ; 24(24)2019 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-31847066

RESUMO

Banana inflorescences are a byproduct of banana cultivation consumed in various regions of Brazil as a non-conventional food. This byproduct represents an alternative food supply that can contribute to the resolution of nutritional problems and hunger. This product is also used in Asia as a traditional remedy for the treatment of various illnesses such as bronchitis and dysentery. However, there is a lack of chemical and pharmacological data to support its consumption as a functional food. Therefore, this work aimed to study the anti-inflammatory action of Musa acuminata blossom by quantifying the cytokine levels (NOx, IL-1ß, TNF-α, and IL-6) in peritoneal neutrophils, and to study its antiparasitic activities using the intracellular forms of T. cruzi, L. amazonensis, and L. infantum. This work also aimed to establish the chemical profile of the inflorescence using UPLC-ESI-MS analysis. Flowers and the crude bract extracts were partitioned in dichloromethane and n-butanol to afford four fractions (FDCM, FNBU, BDCM, and BNBU). FDCM showed moderate trypanocidal activity and promising anti-inflammatory properties by inhibiting IL-1ß, TNF-α, and IL-6. BDCM significantly inhibited the secretion of TNF-α, while BNBU was active against IL-6 and NOx. LCMS data of these fractions revealed an unprecedented presence of arylpropanoid sucroses alongside flavonoids, triterpenes, benzofurans, stilbenes, and iridoids. The obtained results revealed that banana inflorescences could be used as an anti-inflammatory food ingredient to control inflammatory diseases.


Assuntos
1-Butanol/farmacologia , Anti-Inflamatórios/farmacologia , Cloreto de Metileno/farmacologia , Musa/química , Tripanossomicidas/farmacologia , 1-Butanol/química , Animais , Anti-Inflamatórios/química , Sobrevivência Celular/efeitos dos fármacos , Flores/química , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Leishmania/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Cloreto de Metileno/química , Camundongos , NADPH Oxidases/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Células THP-1 , Tripanossomicidas/química , Fator de Necrose Tumoral alfa/metabolismo
16.
Turkiye Parazitol Derg ; 43(4): 158-164, 2019 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-31865648

RESUMO

Objective: Current in-silico research was designed and administered for the screening of 20000 Food and Drug Administration-approved drug compounds with the goal of finding promising drugs against lipophosphoglycan (LPG) and γ-glutamylcysteine synthetase (γ-GCS) of Leishmania infantum. Methods: After the protein sequence of both targets was taken, the 3D structures of protein of interest were predicted and validated. Molecular docking was done among the two putative targets (LPG and γ-GCS) and approved compounds were selected using AutoDock 4.2 program to predict ligand-receptor interactions. Results: After docking experiment was done on 20000 drug compounds, a total number of seven ligands, two for γ-GCS receptor and five for LPG receptor, were assigned as novel, potent anti-leishmanial drugs based on their binding affinity and energy. Of those, five ligands possessed cytotoxic and anti-cancer characteristics and showed good binding capacity to LPG receptor with ΔGbinding up to 8.5 kcal/mol more negative; while two compounds showed good binding capacity to glutamyl receptor with ΔGbinding up to 7.8 kcal/mol more negative. Conclusion: The latest software-based methods are powerful tools for scanning and predicting new peptide templates specific to biological targets in organisms for new drug discovery. However, the use of in vitro and in vivo techniques is a requirement for better evaluation of the potential of projected ligands with the help of in-silico approaches, identifying molecular mechanism of action of the more active compounds is possible. This can help in defining the most likely molecular target, so that the subsequent optimization using in vitro and in vivo techniques can be undertaken.


Assuntos
Antiprotozoários/farmacologia , Glutamato-Cisteína Ligase/antagonistas & inibidores , Glicoesfingolipídeos/antagonistas & inibidores , Leishmania infantum/efeitos dos fármacos , Sequência de Aminoácidos , Anfotericina B/farmacologia , Simulação por Computador , Aprovação de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala , Humanos , Leishmania infantum/química , Leishmania infantum/enzimologia , Ligantes , Antimoniato de Meglumina/farmacologia , Simulação de Acoplamento Molecular , Projetos de Pesquisa , Software
17.
Genes (Basel) ; 11(1)2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31861501

RESUMO

Pathogen fitness landscapes change when transmission cycles establish in non-native environments or spill over into new vectors and hosts. The introduction of Leishmania infantum in the Americas into the Neotropics during European colonization represents a unique case study to investigate the mechanisms of ecological adaptation of this important parasite. Defining the evolutionary trajectories that drive L. infantum fitness in this new environment are of great public health importance as they will allow unique insight into pathways of host/pathogen co-evolution and their consequences for region-specific changes in disease manifestation. This review summarizes current knowledge on L. infantum genetic and phenotypic diversity in the Americas and its possible role in the unique epidemiology of visceral leishmaniasis (VL) in the New World. We highlight the importance of appreciating adaptive molecular mechanisms in L. infantum to understand the parasites' successful establishment on the continent.


Assuntos
Leishmania infantum/classificação , Leishmaniose Visceral/transmissão , Oceano Atlântico , Evolução Molecular , Aptidão Genética , Humanos , Leishmania infantum/genética , Fenótipo
18.
Iran J Immunol ; 16(4): 311-320, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31885008

RESUMO

BACKGROUND: Visceral leishmaniasis (VL) can lead to death in more than 95% of cases if left untreated. Accurate and early diagnosis has an important role in reducing mortality rate of this disease. OBJECTIVE: To express recombinant H2B antigen from an Iranian isolate of Leishmania Infantum and evaluate its efficacy in the diagnosis of VL. METHODS: The recombinant H2B antigen was produced in a prokaryotic system, and its efficacy for VL diagnosis was evaluated by ELISA. The serum samples from 80 VL patients, 100 individuals from endemic and non-endemic regions of VL, and 58 non-VL patients were collected. VL cases were confirmed based on the clinical sign, positive IFAT (>64), real time PCR, and response to treatment. RESULTS: The H2B gene sequence of the Iranian L. infantum isolate had about 4% diversity in comparison with the H2B gene of the L. infantum counterpart. ELISA, using the produced H2B recombinant antigen, showed sensitivity of 71.25% (95% CI: 60.05%-80.82%) and specificity of 69.62% (95% CI: 61.81%-76.68%) regarding VL diagnosis. CONCLUSION: Recombinant H2B antigen expressed in the prokaryotic system had suboptimal performance for the serological diagnosis of VL. It seems that the production and expression of recombinant H2B antigen in a eukaryotic system may enhance the performance of this antigen in the diagnosis of VL in Iran.


Assuntos
Anticorpos Antiprotozoários/sangue , Leishmania infantum/química , Proteínas de Protozoários/química , Testes Sorológicos , Adolescente , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Lactente , Irã (Geográfico) , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico , Masculino , Proteínas Recombinantes/química
19.
PLoS One ; 14(12): e0226336, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31841533

RESUMO

INTRODUCTION: In southern European countries, multicentric lymphoma and leishmaniosis are the main differential diagnoses in dogs presented with generalized lymphadenomegaly. The cytological examination is in some cases inconclusive and polymerase chain reaction (PCR) for antigen receptor rearrangement (PARR) has become a common method to confirm or rule out a lymphoproliferative neoplasia. According to the literature, leishmaniosis may lead to clonal arrangements and therefore to a false diagnosis of lymphoma, but this assumption is made from a single leishmania infected dog. Therefore, the objective of this study was to prospectively evaluate results from PARR in dogs with lymphadenomegaly due to clinical leishmaniosis at the moment of diagnosis. MATERIALS AND METHODS: 31 dogs with a diagnosis of leishmaniosis based on the LeishVet guidelines were included in the study. Samples from enlarged lymph nodes were taken for cytological examination, clonality testing and Leishmania infantum PCR. RESULTS: All 31 dogs had medium to high positive antibody titers against Leishmania spp. and 30/31 had a positive Leishmania PCR from the lymph node. A polyclonal arrangement for B cells (immunoglobulin heavy chain gene) and T cells (T-cell receptor gamma chain gene) antigen receptors was found in 28/31 dogs. Two out of 31 dogs showed a monoclonal arrangement for Ig with high (1:2) and low (1:7) polyclonal background respectively; and one of the 31 dogs showed a monoclonal arrangement for T cell receptor with low (1:3) polyclonal background. CONCLUSION: Infections with Leishmania infantum resulted in clonal rearrangement, and therefore in a possible false diagnosis of lymphoma, in 3 out of 31 dogs (9.7%). Although, PARR is a useful method to differentiate lymphoma from reactive lymphoid hyperplasia in dogs with leishmaniosis, mono-/biclonal results should be interpreted carefully, especially in the presence of any degree of polyclonal background, and together with other clinicopathological findings.


Assuntos
Evolução Clonal , Doenças do Cão/imunologia , Leishmania infantum/imunologia , Leishmaniose Visceral/diagnóstico , Linfonodos/metabolismo , Linfadenopatia/diagnóstico , Animais , Evolução Clonal/genética , Evolução Clonal/imunologia , Diagnóstico Diferencial , Doenças do Cão/diagnóstico , Doenças do Cão/genética , Doenças do Cão/patologia , Cães , Feminino , Rearranjo Gênico do Linfócito B , Rearranjo Gênico do Linfócito T , Testes Genéticos/métodos , Testes Genéticos/veterinária , Leishmania infantum/genética , Leishmaniose Visceral/genética , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/patologia , Linfonodos/imunologia , Linfadenopatia/genética , Linfadenopatia/imunologia , Linfadenopatia/parasitologia , Linfoma/diagnóstico , Linfoma/genética , Linfoma/veterinária , Masculino , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterinária , Estudos Prospectivos , Esplenomegalia/diagnóstico , Esplenomegalia/veterinária
20.
Rev Soc Bras Med Trop ; 53: e20190169, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31859944

RESUMO

INTRODUCTION: Leishmania infantum was considered to be absent from Amapá until 2017 when canine infection was detected. However, there is a lack of knowledge about which reservoir species are involved in transmission in this region. METHODS: Between 2014 and 2016, 86 samples from wild mammals and 74 from domestic dogs were collected in Wajãpi Indigenous Territory and were tested for the presence of deoxyribonucleic acid (DNA) of Leishmania. RESULTS: The DNA of Le. infantum was detected in two rodent samples, Dasyprocta sp. and Proechimys cuvieri. CONCLUSIONS: This is the first evidence characterizing a sylvatic transmission cycle of Le. infantum in the State of Amapá.


Assuntos
Reservatórios de Doenças/veterinária , Doenças do Cão/epidemiologia , Leishmania infantum/genética , Leishmaniose Visceral/epidemiologia , Roedores/parasitologia , Animais , Brasil/epidemiologia , DNA de Protozoário , Doenças do Cão/parasitologia , Cães , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/transmissão
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