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1.
Cad Saude Publica ; 37(3): e00041320, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33852659

RESUMO

Dogs are the main reservoirs in the domestic transmission cycle of visceral leishmaniasis, and the diagnosis is essential for the effectiveness of the control measures recommended by the Brazilian Ministry of Health. We assessed the diagnostic performance of the ELISA-Vetlisa/BIOCLIN prototype with serum samples from 200 dogs, in triplicate, including symptomatic, oligosymptomatic, asymptomatic, and healthy dogs, originated by two distinct panels (A and B) characterized by parasitological tests as the reference standard. In this study, the prototype kit showed a 99% sensitivity (95%CI: 94.5-100.0) and a 100% specificity (95%CI: 96.4-100.0). The sensitivity of the prototype kit did not vary significantly with the clinical status of the dogs. Considering the final result classification (positive or negative), agreement between the results of repeated tests was almost perfect (kappa = 0.99; 95%CI: 0.98-1.00). ELISA-Vetlisa/BIOCLIN is a promising option for the serological diagnosis of canine visceral leishmaniasis in Brazil.


Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Animais , Brasil , Doenças do Cão/diagnóstico , Cães , Ensaio de Imunoadsorção Enzimática , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/veterinária , Sensibilidade e Especificidade
2.
Parasite ; 28: 38, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33851916

RESUMO

Current treatments of visceral leishmaniasis face limitations due to drug side effects and/or high cost, along with the emergence of parasite resistance. Novel and low-cost antileishmanial agents are therefore required. We report herein the antileishmanial activity of ß-acetyl-digitoxin (b-AD), a cardenolide isolated from Digitalis lanata leaves, assayed in vitro and in vivo against Leishmania infantum. Results showed direct action of b-AD against parasites, as well as efficacy for the treatment of Leishmania-infected macrophages. In vivo experiments using b-AD-containing Pluronic® F127 polymeric micelles (b-AD/Mic) to treat L. infantum-infected mice showed that this composition reduced the parasite load in distinct organs in more significant levels. It also induced the development of anti-parasite Th1-type immunity, attested by high levels of IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and specific IgG2a antibodies, in addition to low IL-4 and IL-10 contents, along with higher IFN-γ-producing CD4+ and CD8+ T-cell frequency. Furthermore, low toxicity was found in the organs of the treated animals. Comparing the therapeutic effect between the treatments, b-AD/Mic was the most effective in protecting animals against infection, when compared to the other groups including miltefosine used as a drug control. Data found 15 days after treatment were similar to those obtained one day post-therapy. In conclusion, the results obtained suggest that b-AD/Mic is a promising antileishmanial agent and deserves further studies to investigate its potential to treat visceral leishmaniasis.


Assuntos
Antiprotozoários , Digitalis , Leishmania infantum , Leishmaniose Visceral , Animais , Antiprotozoários/uso terapêutico , Cardenolídeos/uso terapêutico , Digitoxina/uso terapêutico , Leishmaniose Visceral/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C
3.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 52(2): 194-201, 2021 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-33829691

RESUMO

Objective: To construct eukaryotic and prokaryotic recombinant vectors containing Pepck- Gp63 and to achieve protein expression by selecting the dominant epitope genes of Pepck and Gp63 of Leishmania infantum. Methods: The secondary structure and HLA epitopes of phosphoenolpyruvate carboxylase (PEPCK) were predicted by in silico analysis, and the dominant epitopes were picked out. According to the analysis results of glycoprotein of 63×10 3(GP63) epitopes identified by the same method in our laboratory, the dominant epitope genes of Pepck and Gp63 were used to construct pET32a- Pepck- Gp63 and pVAX1- Pepck- Gp63 by overlapping PCR and enzyme reaction. Then, for protein expression, the prokaryotic vectors were transfected into E.coil while the eukaryotic vectors were transfected into NIH3T3 cells by liposome transfection. Results: There were multiple dominant epitopes in Pepckand there were Pepck-Gp63 sequences in the polyclonal site of expression vector. The expression of Pepck-Gp63 in E.coil appeared in inclusion form and led to 74 kDa band in SDS-PAGE. The immunofluorescence results of NIH3T3 cells transfected by pVAX1- Pepck-Gp63 were positive. Conclusion: The recombinant prokaryotic expression plasmids pET32a- Pepck-Gp63 and eukaryotic expression plasmids pVAX1- P epck -Gp63 were successfully constructed, and it was shown that the recombinant plasmids were able to express the corresponding target proteins in E. coli and NIH3T3 cells, respectively, providing a preliminary experimental basis for the subsequent study of immunization strategies.


Assuntos
Leishmania infantum , Animais , Epitopos/genética , Escherichia coli/genética , Eucariotos , Vetores Genéticos/genética , Leishmania infantum/genética , Camundongos , Células NIH 3T3 , Fosfoenolpiruvato Carboxilase , Plasmídeos
4.
Mem Inst Oswaldo Cruz ; 116: e200428, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33729396

RESUMO

BACKGROUND: Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE: We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmania-derived recombinant antigens. METHODS: Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS: For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS: For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.


Assuntos
Anticorpos Antiprotozoários/sangue , Doenças do Cão/diagnóstico , Ensaio de Imunoadsorção Enzimática/normas , Ensaio de Imunoadsorção Enzimática/veterinária , Leishmania infantum/imunologia , Leishmaniose Visceral/diagnóstico , Animais , Antígenos de Protozoários/biossíntese , Brasil , Cães , Ensaio de Imunoadsorção Enzimática/métodos , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/sangue , Leishmaniose Visceral/veterinária , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Testes Sorológicos
5.
Rev Soc Bras Med Trop ; 54: e05862020, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33656152

RESUMO

INTRODUCTION: Visceral leishmaniasis (VL) transmission has been associated with two different populations of the Lutzomyia longipalpis complex in São Paulo state. METHODS: In a recent focus of VL, we captured and dissected sand flies and investigated Leishmania infantum infection by parasitological, PCR, and sequencing analysis. RESULTS: Flagellates were observed in 2 of 47 (4.2%) cembrene-1 Lu. longipalpis females. The sequences obtained matched those of Le. infantum. CONCLUSIONS: We found that the transmission of Le. infantum by cembrene-1 females may occur at a high rate in this focus of VL and presented new data on the vector capacity of this population.


Assuntos
Leishmania infantum , Leishmaniose Visceral , Psychodidae , Animais , Brasil , Feminino , Insetos Vetores
6.
Mem Inst Oswaldo Cruz ; 116: e200571, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33681890

RESUMO

Leishmania infantum chagasi is the causative agent and Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the Americas. We investigated the expression of Leishmania genes within L. longipalpis after artificial infection. mRNAs from genes involved in sugar and amino acid metabolism were upregulated at times of high parasite proliferation inside the insect. mRNAs from genes involved in metacyclogenesis had higher expression in late stages of infection. Other modulated genes of interest were involved in immunomodulation, purine salvage pathway and protein recycling. These data reveal aspects of the adaptation of the parasite to the microenvironment of the vector gut and reflect the preparation for infection in the vertebrate.


Assuntos
Insetos Vetores/parasitologia , Leishmania infantum/genética , Leishmania/isolamento & purificação , Leishmaniose Visceral/transmissão , Psychodidae/parasitologia , Animais , Brasil , Expressão Gênica , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Estágios do Ciclo de Vida , Psychodidae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
7.
Arch Oral Biol ; 124: 105077, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33601301

RESUMO

OBJECTIVE: The objective of this study was to perform a histopathological, morphometric and proteomic study of the parotid gland of dogs naturally infected with Leishmania infantum treated and not treated with Allopurinol. DESIGN: Parotid glands from 14 dogs were used, divided into two groups: untreated and treated with oral allopurinol (20 mg / kg, once daily for 90 days). After adequate dissection, the organs were submitted to histopathological, histomorphometric and immunohistochemical techniques, using the monoclonal anti-ß-catenin antibody. RESULTS: Histopathological evaluation of treated and untreated groups showed acinar hypertrophy, structural disorganization of the nucleus and cytoplasm. There was an increase in the area and perimeter of the parotid acini in the experimental groups. The immunostaining of the ß-catenin protein in the membrane was severely reduced in the treated and untreated groups. CONCLUSIONS: These findings suggest that Leishmania infantum infection and treatment with Allopurinol alter the tissue structure of the parotid gland in dogs, promoting an increase in the acinar volume and a decrease in the expression of ß-catenin in cell membranes.


Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Animais , Cães , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/veterinária , Glândula Parótida , Proteômica
8.
Artigo em Inglês | MEDLINE | ID: mdl-33533815

RESUMO

Visceral leishmaniasis (VL) is a neglected tropical disease caused by the Leishmania infantum parasite. The protozoan is able to infect several domestic and wild mammals. Since the first report on Leishmania spp. infection in horses in South America, leishmaniasis in equids has been highlighted in Brazil. A molecular epidemiological survey was carried out to verify the occurrence of Leishmania spp. DNA in horses and donkeys, in leishmaniases endemic areas in Sao Paulo State, Brazil. To this end, blood samples were obtained from 107 horses and 36 donkeys and subjected to DNA extraction followed by PCR targeting the ITS-1 region. Among the horses and donkeys, 1.87% (2/107) and 8.33% (3/36) were positive by PCR, respectively. The DNA sequencing of the ITS-1 amplification products confirmed L. infantum DNA in these animals. Our results suggest that horses and donkeys from non-VL and VL endemic areas of São Paulo State may be infected by the parasite.


Assuntos
Equidae/sangue , Cavalos/sangue , Leishmania infantum/genética , Leishmaniose Visceral/diagnóstico , Animais , Brasil , DNA , Leishmaniose Visceral/veterinária , Reação em Cadeia da Polimerase
9.
Parasitol Res ; 120(2): 679-692, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33415401

RESUMO

Owing to the importance and clinical diversity of Leishmania infantum, studying its virulence factors is promising for understanding the relationship between parasites and hosts. In the present study, differentially abundant proteins from strains with different degrees of virulence in promastigote and amastigote forms were compared using two quantitative proteomics techniques, differential gel electrophoresis and isobaric mass tag labeling, followed by identification by mass spectrometry. A total of 142 proteins were identified: 96 upregulated and 46 downregulated proteins in the most virulent strain compared to less virulent. The interaction between the proteins identified in each evolutionary form was predicted. The results showed that in the amastigote form of the most virulent strain, there was a large group of proteins related to glycolysis, heat shock, and ribosomal proteins, whereas in the promastigote form, the group consisted of stress response, heat shock, and ribosomal proteins. In addition, biological processes related to metabolic pathways, ribosomes, and oxidative phosphorylation were enriched in the most virulent strain (BH400). Finally, we noted several proteins previously found to play important roles in L. infantum infection, which showed increased abundance in the virulent strain, such as ribosomal proteins, HSP70, enolase, fructose 1,6-biphosphate aldolase, peroxidoxin, and tryparedoxin peroxidase, many of which interact with each other.


Assuntos
Leishmania infantum/metabolismo , Leishmania infantum/patogenicidade , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Leishmania infantum/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Proteômica , Virulência , Fatores de Virulência/metabolismo
10.
PLoS One ; 16(1): e0239171, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33465107

RESUMO

rIL-10 plays a major role in restricting exaggerated inflammatory and immune responses, thus preventing tissue damage. However, the restriction of inflammatory and immune responses by IL-10 can also favor the development and/or persistence of chronic infections or neoplasms. Dogs that succumb to canine leishmaniasis (CanL) caused by L. infantum develop exhaustion of T lymphocytes and are unable to mount appropriate cellular immune responses to control the infection. These animals fail to mount specific lymphoproliferative responses and produce interferon gamma and TNF-alpha that would activate macrophages and promote destruction of intracellular parasites. Blocking IL-10 signaling may contribute to the treatment of CanL. In order to obtain a tool for this blockage, the present work endeavored to identify the canine casIL-10R1 amino acid sequence, generate a recombinant baculovirus chromosome encoding this molecule, which was expressed in insect cells and subsequently purified to obtain rcasIL-10R1. In addition, rcasIL-10R1 was able to bind to homologous IL-10 and block IL-10 signaling pathway, as well as to promote lymphoproliferation in dogs with leishmaniasis caused by L. infantum.


Assuntos
Interleucina-10/metabolismo , Leishmaniose/tratamento farmacológico , Receptores de Interleucina-10/metabolismo , Animais , Linhagem Celular , Citocinas/metabolismo , Doenças do Cão/genética , Cães , Feminino , Imunidade Celular/imunologia , Imunidade Celular/fisiologia , Interferon gama/genética , Interleucina-10/agonistas , Interleucina-12/genética , Leishmania infantum/imunologia , Leishmania infantum/patogenicidade , Leishmaniose/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Receptores de Interleucina-10/efeitos dos fármacos , Transdução de Sinais , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa
11.
Eur J Med Chem ; 212: 113101, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33385837

RESUMO

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


Assuntos
Antiprotozoários/farmacologia , Leishmania infantum/efeitos dos fármacos , Nucleosídeos/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Antiprotozoários/síntese química , Antiprotozoários/química , Relação Dose-Resposta a Droga , Estrutura Molecular , Nucleosídeos/síntese química , Nucleosídeos/química , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade
12.
Acta Trop ; 215: 105804, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33385362

RESUMO

Immunomagnetic Separation (IMS) assay has been used for isolation of viable whole organisms. The objective of our work is to produce anti-Leishmania magnetic beads and to assess the efficiency of the IMS technique on Leishmania promastigote capture in culture media. Polyclonal anti-Leishmania antibodies were produced by intravenous injection of viable metacyclic promastigotes of Leishmania (L.) major to rabbit. Purified anti-Leishmania IgG was assessed for their reactivity against both L. major and L. infantum promastigotes then covalently conjugated to magnetic beads and used for IMS. This latter was applied on either L. major promastigote cultures of known concentrations or early stage (24h, 48h, 72h) Novy-MacNeal-Nicolle (NNN) cultures of tissue fluid obtained from cutaneous leishmaniasis (CL) lesions. Promastigotes capture was assessed by either microscopy or qPCR after sample boiling. Indirect immunofluorescence assay showed that polyclonal antibodies reacted against both L. major and L. infantum promastigotes. In 50 µL solution, immunomagnetic beads were able to capture 5 live promastigotes out of 20 and 1050 out of 2500, giving an estimated efficiency of 25-42%. The efficiency of the IMS was lower for a lower number of parasites but still repeatable. On the other hand, IMS-qPCR applied to 14 NNN cultures of confirmed Leishmania lesions showed a higher sensitivity to detect live parasites than routine microscopy observation of promastigotes growth (93% positivity at 72h versus 50% positivity within 2-4 weeks incubation). The estimated number of captured parasites at 72h ranged from 1 to more than 100 parasites / 50 µL liquid phase of culture. These preliminary results open the way for interesting perspectives in the use of cultures for leishmaniasis diagnosis and also for other applications such as Leishmania detection in cultures taken from reservoir animals or sandflies.


Assuntos
Separação Imunomagnética/métodos , Leishmania infantum/isolamento & purificação , Leishmania major/isolamento & purificação , Animais , Feminino , Humanos , Leishmania infantum/imunologia , Leishmania major/imunologia , Coelhos
13.
Exp Parasitol ; 222: 108065, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33428893

RESUMO

Visceral leishmaniasis (VL) is a protozoan disease caused by Leishmania infantum in the Mediterranean region including Iran. In 95% of cases, the disease can be fatal if not rapidly diagnosed and left untreated. We aimed to identify immunoreactive proteins of L. infantum (Iranian strain), and to design and evaluate a recombinant multi-epitope antigen for serodiagnosis of human VL. To detect the immunoreactive proteins of L. infantum promastigotes, 2DE immunoblotting technique was performed using different pooled sera of VL patients. The candidate immunoreactive proteins were identified using MALDI-TOF/TOF mass spectrophotometry. Among 125 immunoreactive spots detected in 2-DE gels, glucose-regulated protein 78 (GRP78), ubiquitin-conjugating enzyme E2, calreticulin, mitochondrial heat shock 70-related protein 1 (mtHSP70), heat shock protein 70-related protein, i/6 autoantigen-like protein, ATPase beta subunit, and proteasome alpha subunit 5 were identified. The potent epitopes from candidate immunodominant proteins including GRP78, mtHSP70 and ubiquitin-conjugating enzyme E2 were then selected to design a recombinant antigenic protein (GRP-UBI-HSP). The recombinant antigen was evaluated by ELISA and compared to direct agglutination test for detection of anti L. infantum human antibodies. We screened 34 sera of VL patients from endemic areas and 107 sera of individuals without L. infantum infection from non-endemic area of VL. The recombinant protein-based ELISA provided a sensitivity of 70.6% and a specificity of 84.1%. These results showed that GRP78, ubiquitin-conjugating enzyme E2, and mtHSP70 proteins are potential immunodominant targets of the host immune system in response to the parasite and they can be considered as potential candidate markers for diagnosis purposes.


Assuntos
Epitopos Imunodominantes/isolamento & purificação , Leishmania infantum/imunologia , Leishmaniose Visceral/diagnóstico , Proteômica/métodos , Sequência de Aminoácidos , Antígenos de Protozoários/isolamento & purificação , Western Blotting , Biologia Computacional/métodos , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Epitopos/isolamento & purificação , Humanos , Immunoblotting , Leishmaniose Visceral/imunologia , Conformação Molecular , Estrutura Secundária de Proteína , Proteômica/normas , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Testes Sorológicos/métodos , Testes Sorológicos/normas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
14.
Parasit Vectors ; 14(1): 36, 2021 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-33422141

RESUMO

BACKGROUND: Zoonotic visceral leishmaniasis by Leishmania infantum is a first-order pathology in canine veterinary clinics in endemic areas. Moreover, canine infections are considered the main reservoir for human disease; despite their importance in the control of the disease within a One Health approach, no scientometric study has been published. Aims of the study included analyzing the impact of canine leishmaniasis (CanL) on the scientific literature, drugs or combinations used, trends in the period from 2000 to 2020 and efficacy criteria employed. METHODS: A Web of Science (WOS)-based analysis of publications on CanL and chemotherapy of the disease in the period 2000-2020 was carried out using a stepwise methodology. Data were analyzed by year, geographical origin, chemical groups, drugs and combinations, and efficacy criteria. RESULTS: Reports on CanL (n = 3324) represented < 16% of all publications on leishmaniasis (n = 20,968), and of these around 18% (n = 596) were related to chemotherapy. Publication records on CanL followed the distribution of the infection by L. infantum in endemic areas although Mediterranean countries were overrepresented in the reports on chemotherapy of CanL. Publications on the main antileishmanial drugs used in clinical practice showed a sustained tendency in the period analyzed. Pentavalent antimonials (SbV), alone or in combination with allopurinol, represented > 50% of all publications on chemotherapy of CanL despite the availability of more recently marketed drugs. CONCLUSIONS: Chemotherapy of CanL still relies on SbV and combinations and to a lesser extent on miltefosine (MIL). Reports on chemotherapy are scarce and mostly publicly funded, and the variability of experimental conditions hampers the direct comparison of the efficacy of drugs, combinations and schedules. The vast majority of reports on efficacy do not include any information on supportive therapy; this reduces the actual value of the studies if intended for the practical management of the disease. Complete reports on the chemotherapy (etiological + symptomatic) would add value to the trials performed.


Assuntos
Antiprotozoários/uso terapêutico , Doenças do Cão/tratamento farmacológico , Tratamento Farmacológico/métodos , Leishmaniose/tratamento farmacológico , Alopurinol/uso terapêutico , Anfotericina B/uso terapêutico , Animais , Doenças do Cão/epidemiologia , Cães , Combinação de Medicamentos , Humanos , Leishmania infantum , Leishmaniose/epidemiologia , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/terapia , Fosforilcolina/análogos & derivados , Publicações
15.
Vaccine ; 39(2): 282-291, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33309484

RESUMO

Visceral leishmaniasis (VL) is a serious neglected tropical disease that affects humans and dogs in urban areas. There are no vaccines against human VL, and few licensed canine VL vaccines are currently available, which instigates the search for new antigens and vaccine formulations with prophylactic potential against VL in these hosts. In this study, we evaluated the immunization using the native and recombinant Leishmania infantum chagasi (L. chagasi) lipophosphoglycan-3 (LPG3) and the adjuvants saponin (SAP) and incomplete Freund adjuvant (IFA) against L. chagasi infection in BALB/c mice. The native LPG3 vaccine was immunogenic, inducing splenic IFN-γ and IL-10 production, and mixed Th1/Th2 response when associated with IFA. However, only mice vaccinated with LPG3-IFA presented a reduction in the splenic parasite load (96% in comparison to the PBS control group), but without a significant reduction in the hepatic parasitism. On the other hand, mice immunized with the LPG3-SAP vaccine presented a reduction of approximately 98% in both splenic and hepatic parasite load, accompanied by a Th1/Th17 response and IL-10 production by L. chagasi antigen (AgLc)-stimulated splenic cells. Importantly, vaccination with recombinant LPG3 (rLPG3)-SAP presented similar results to the native LPG3-SAP vaccine. Therefore, the rLPG3-SAP vaccine is qualified to be used in future tests in canine and human models, considering the technical and economic advantages of the recombinant protein production compared to the native protein and the results obtained in the murine model.


Assuntos
Leishmania infantum , Vacinas contra Leishmaniose , Leishmaniose Visceral , Saponinas , Animais , Anticorpos Antiprotozoários , Antígenos de Protozoários , Cães , Glicoesfingolipídeos , Leishmaniose Visceral/prevenção & controle , Leishmaniose Visceral/veterinária , Camundongos , Camundongos Endogâmicos BALB C
16.
Acta Trop ; 215: 105801, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33352169

RESUMO

Compounds 1 and 2 (selenocyanate and diselenide derivatives, respectively) were evaluated for their potential use in vivo against visceral leishmaniasis (VL). Both entities showed low cytoxicity in vitro in Vero and Caco-2 cell lines. However, the compounds were not suitable for their oral administration, since they exhibited poor values of intestinal permeability in vitro. Microsomal stability assays did not show any metabolite for compound 1 after 120 min, whereas 2 was highly metabolized by the enzyme CYP450. Thus, the in vivo efficacy of compound 1 was assessed in a murine model of L. infantum VL. The daily i.v. administration of 1 mg/kg of compound 1 during 5 consecutive days reduced parasite load in liver, spleen and bone marrow (99.2%, 91.7% and 61.4%, respectively) compared to non-treated mice. To the best of our knowledge, this is the first time that a selenium compound has been tested in vivo against VL. Thus, this work evidences the possible usefulness of selenocyanate derivatives for the treatment of this disease.


Assuntos
Cianatos/uso terapêutico , Leishmania infantum , Leishmaniose Visceral/tratamento farmacológico , Compostos de Selênio/uso terapêutico , Animais , Células Cultivadas , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Wistar
17.
Exp Parasitol ; 221: 108059, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33338468

RESUMO

Treatment for visceral leishmaniasis (VL) is hindered mainly by the toxicity and/or high cost of therapeutic drugs. In addition, parasite resistance has been registered. Thus, there is an urgent need for the identification of novel, effective and low-cost antileishmanial agents. Since drug discovery is a long and expensive process, drug repositioning for treatment of leishmaniasis should be considered. In the present study, Ivermectin (IVE), a broad-spectrum drug used for treatment of parasitic diseases, was evaluated in vitro and in vivo against Leishmania infantum species. Results in vitro showed that IVE presented 50% Leishmania and macrophage inhibitory concentrations (IC50 and CC50, respectively) of 3.64 ± 0.48 µM and 427.50 ± 17.60 µM, respectively, with a selectivity index (SI) of 117.45; whereas Amphotericin B (AmpB), which was used as control, showed IC50 and CC50 values of 0.12 ± 0.05 µM and 1.06 ± 0.23 µM, respectively, with a corresponding SI of 8.90. Treatment with IVE effectively reduced the infection percentage and parasite burden in infected and treated macrophages and displayed a prophylactic activity by inhibiting macrophage infection with pre-treated parasites. Furthermore, preliminary studies suggested that IVE targets the parasite's mitochondria. Activity of IVE in its free format or incorporated into Pluronic® F127-based polymeric micelles (IVE/Mic) was also evaluated in vivo as a treating drug for L. infantum-infected BALB/c mice. Miltefosine was used as a control. Results showed that Miltefosine, IVE and IVE/Mic-treated animals presented significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, as well as development of an antileishmanial Th1-type immune response one and 15 days after treatment. Notably, IVE/Mic showed a better parasitological and immunological response in comparison to other alternative treatments. In conclusion, results suggest that IVE/Mic could be considered in future studies as a therapeutic alternative to treat VL.


Assuntos
Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Ivermectina/farmacologia , Ivermectina/uso terapêutico , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Anfotericina B/farmacologia , Animais , Antiprotozoários/toxicidade , Eritrócitos/efeitos dos fármacos , Feminino , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Concentração Inibidora 50 , Ivermectina/toxicidade , Macrófagos Peritoneais/efeitos dos fármacos , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos BALB C , Espécies Reativas de Oxigênio/metabolismo , Baço/parasitologia
18.
PLoS Negl Trop Dis ; 14(12): e0008947, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33338041

RESUMO

Leishmaniasis is among the world's most neglected diseases. Dogs are the main reservoirs/hosts of Leishmania infantum, causative agent of both canine and human visceral leishmaniosis. Canine leishmaniasis (CanL) represents a public health problem as one of the most prevalent zoonotic diseases worldwide. Current therapeutics present drawbacks; thus, there is a need for more effective, safer, and cheaper drugs. The aim of this study was to evaluate and to compare the efficacy of oral administration of artesunate or meglumine antimoniate/allopurinol in dogs with clinical leishmaniasis. Forty-two dogs with naturally occurring clinical leishmaniasis were included in this open-label, simple randomized positive-control clinical field trial with 6 months of follow-up. Dogs received meglumine antimoniate 100 mg/kg/day and allopurinol 30 mg/kg/day for 28 days (control group, n = 26) or artesunate 25 mg/kg/day for 6 days (test group, n = 16). The animals were evaluated for their clinical evolution, parasite load (by qPCR) and humoral response at different time points: 0, 30, 90, and 180 days after treatment. Data analyses showed a significant improvement in both groups in clinical scores, parasitemia and antibody titers after treatment. Compared to the control group, the artesunate group showed significantly lower clinical score (P = 0.0001), lower parasitemia (P = 0.0001) and antibody titers after 6 months of follow-up. Compared to baseline values, a rapid, significant reduction (P < 0.012) in antibody levels, 2.28- versus 3.04-fold for the control versus artesunate groups, respectively, was observed 30 days after treatment. Antibody levels continued to decrease further in the artesunate group, where 58% of cases became seronegative at the 6-month follow-up. All qPCR-positive dogs were negative after treatment with artesunate, while 14.3% remained positive with the appearance of two new cases in the control group. Artesunate was well tolerated, and no side effects were recorded. Treatment failures were similar in both groups with 27.27% (6/22), including 18.18% (4/22) mortality in the control group, versus 26.66% (4/15), including 13.33% (2/15) mortality in the artesunate group. This is the first report showing the potential of artesunate in the treatment of dogs with clinical leishmaniasis. Artesunate showed higher efficacy than the current first-line treatment for CanL without any adverse effects. It could be a good alternative chemotherapy for CanL, and may be considered for further studies in human leishmaniases. Further clinical trials are needed to confirm these findings, to determine if there are relapses after treatment and if dogs remain infective to sandflies, to define the ideal therapeutic dosage and duration of treatment with artesunate.


Assuntos
Alopurinol/uso terapêutico , Antiprotozoários/uso terapêutico , Artesunato/uso terapêutico , Doenças do Cão/tratamento farmacológico , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/veterinária , Antimoniato de Meglumina/uso terapêutico , Animais , Doenças do Cão/parasitologia , Cães , Feminino , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Masculino , Carga Parasitária/veterinária , Parasitemia/tratamento farmacológico , Zoonoses
19.
Mol Immunol ; 127: 95-106, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32949849

RESUMO

Leishmania infantum infection may cause visceral leishmaniasis (VL), a fatal disease having worldwide distribution, that may be silent or asymptomatic. The latter indicates that immunity is naturally developed in some individuals, and, therefore, a vaccine against VL would be possible. Molecular mechanisms of gene expression are being understood in Leishmania, and this knowledge may be useful for vaccine development. The aim of this study was developing an attenuated strain by regulating the expression of toxic proteins in a stage specific manner. For that purpose, the 3' UTR of an amastin gene, known by its increased expression in the amastigote phase, was selected for direct the expression of exogenous proteins. This construct (pFL-AMA), firstly, was proved effective for the expression of mCherry specifically in the intracellular form of L. infantum, as demonstrated by fluorescence microscopy, flow cytometry and Western blotting. Afterwards, mCherry coding sequence was replaced, in the pFL-AMA plasmid, by either egg avidin or the active form of bovine trypsin. Viability of transfected parasites was evaluated in promastigote axenic cultures and in in vitro infection of macrophages. Both lines of transfected parasites showed a limited capacity to multiply inside macrophages. BALB/c mice were inoculated intraperitoneally (i.p.) with a single dose consisting of 2 × 106L. infantum promastigotes transfected with plasmids bearing the toxic genes. After 10 weeks post-inoculation, no parasites were recovered by limiting dilution in either liver or spleen, but a specific immunological response was detected. The immunization with transfected parasites induced cellular and humoral immune responses with activation of TCD4+, TCD8+ and B cells, having a TH1-type response with increased levels of pro-inflammatory cytokines such as IFN-γ, TNF-α and IL-6. In parallel groups of mice, a challenge consisting on 1 × 106 virulent parasites of either L. infantum (inoculated i.p.) or L. amazonensis subcutaneously (s.c.) was performed. Vaccinated mice, challenged with L. infantum, showed lower parasite burdens in liver, spleen and bone marrow than infected mice with WT L. infantum (non-vaccinated); similarly, vaccinated mice developed smaller footpad inflammation than control group. These data support this strategy as an efficient immunization system aimed to the development of vaccines against different forms of leishmaniasis.


Assuntos
Leishmania infantum/fisiologia , Leishmania/fisiologia , Leishmaniose/prevenção & controle , Leishmaniose/parasitologia , Plasmídeos/metabolismo , Toxinas Biológicas/metabolismo , Transfecção , Animais , Feminino , Regulação da Expressão Gênica , Humanos , Imunidade Celular , Imunidade Humoral , Imunização , Leishmania/patogenicidade , Leishmania infantum/crescimento & desenvolvimento , Leishmania infantum/patogenicidade , Leishmaniose/imunologia , Estágios do Ciclo de Vida , Camundongos Endogâmicos BALB C , Parasitos/metabolismo , Parasitos/patogenicidade , Proteínas de Protozoários/metabolismo , Virulência
20.
Mem Inst Oswaldo Cruz ; 115: e200140, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32965329

RESUMO

Although Leishmania infantum is well-known as the aethiological agent of visceral leishmaniasis (VL), in some Central American countries it may cause atypical non-ulcerated cutaneous leishmaniasis (NUCL). However, the mechanisms favoring its establishment in the skin are still unknown. Lipophosphoglycan (LPG) is the major Leishmania multivirulence factor involved in parasite-host interaction. In the case of viscerotropic L. infantum, it causes an immunosuppression during the interaction with macrophages. Here, we investigated the biochemical and functional roles of LPGs from four dermotropic L. infantum strains from Honduras during in vitro interaction with murine macrophages. LPGs were extracted, purified and their repeat units analysed. They did not have side chains consisting of Gal(ß1,4)Man(α1)-PO4 common to all LPGs. Peritoneal macrophages from BALB/c and C57BL/6 were exposed to LPG for nitric oxide (NO) and cytokine (TNF-α and, IL-6) production. LPGs from dermotropic strains from Honduras triggered higher NO and cytokine levels compared to those from viscerotropic strains. In conclusion, LPGs from dermotropic strains are devoid of side-chains and exhibit high pro-inflammatory activity.


Assuntos
Glicoesfingolipídeos , Leishmania infantum/fisiologia , Animais , América Central , Honduras , Humanos , Macrófagos/imunologia , Masculino , Camundongos
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