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1.
Acta Trop ; 218: 105890, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33744245

RESUMO

Leishmania (Viannia) braziliensis is one of the main etiological agents of tegumentary leishmaniasis in Latin America. The establishment of a successful infection in host cells requires several key events including phagocytosis, phagolysosomal maturation impairment, and parasite replication. Autophagy is accountable for the physiological turnover of cellular organelles, degradation of macromolecular structures, and pathogen elimination. In many cases, autophagy control leads to a successful infection, both impairing pathogen elimination or providing nutrients. Here, we have investigated the relationship between autophagy and L. braziliensis infection. We observed that BECLIN1 expression was upregulated early on infection in both in vitro macrophage cultures and biopsies of cutaneous lesions from L. braziliensis infected patients. On the other hand, LC3B expression was downregulated in cutaneous lesions biopsies. A transient pattern of LC3+ cells was observed along L. braziliensis infection, but the number of LC3 puncta did not vary. Additionally, autophagy induction, with rapamycin treatment or through starvation, reduced infection. As expected, rapamycin increased the percentage of LC3+ cells and the number of puncta, but the presence of parasite restricted this effect, indicating LC3-associated autophagy impairment by L. braziliensis. Finally, silencing LC3B but not BECLIN1 promoted infection, confirming BECLIN1 independent and LC3B-related control by the parasite. Taken together, these data indicate macrophage autophagic machinery manipulation by L. braziliensis, resulting in successful establishment and survival into the host cell.


Assuntos
Autofagia , Leishmania braziliensis/fisiologia , Leishmaniose Cutânea/imunologia , Macrófagos/citologia , Macrófagos/parasitologia , Animais , Proteína Beclina-1/metabolismo , Feminino , Humanos , Leishmaniose Cutânea/metabolismo , Macrófagos/imunologia , Proteínas Associadas aos Microtúbulos/metabolismo , Fagocitose
2.
PLoS Pathog ; 16(8): e1008810, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32817704

RESUMO

Sterol 14-α-demethylase (C14DM) is a key enzyme in the biosynthesis of sterols and the primary target of azoles. In Leishmania major, genetic or chemical inactivation of C14DM leads to accumulation of 14-methylated sterol intermediates and profound plasma membrane abnormalities including increased fluidity and failure to maintain ordered membrane microdomains. These defects likely contribute to the hypersensitivity to heat and severely reduced virulence displayed by the C14DM-null mutants (c14dm‾). In addition to plasma membrane, sterols are present in intracellular organelles. In this study, we investigated the impact of C14DM ablation on mitochondria. Our results demonstrate that c14dm‾ mutants have significantly higher mitochondrial membrane potential than wild type parasites. Such high potential leads to the buildup of reactive oxygen species in the mitochondria, especially under nutrient-limiting conditions. Consistent with these mitochondrial alterations, c14dm‾ mutants show impairment in respiration and are heavily dependent on glucose uptake and glycolysis to generate energy. Consequently, these mutants are extremely sensitive to glucose deprivation and such vulnerability can be rescued through the supplementation of glucose or glycerol. In addition, the accumulation of oxidants may also contribute to the heat sensitivity exhibited by c14dm‾. Finally, genetic or chemical ablation of C14DM causes increased susceptibility to pentamidine, an antimicrobial agent with activity against trypanosomatids. In summary, our investigation reveals that alteration of sterol synthesis can negatively affect multiple cellular processes in Leishmania parasites and make them vulnerable to clinically relevant stress conditions.


Assuntos
Leishmania major/enzimologia , Leishmania major/fisiologia , Leishmaniose Cutânea/patologia , Mitocôndrias/fisiologia , Proteínas de Protozoários/metabolismo , Esterol 14-Desmetilase/metabolismo , Esteróis/metabolismo , Humanos , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/parasitologia , Potencial da Membrana Mitocondrial , Proteínas de Protozoários/genética , Espécies Reativas de Oxigênio/metabolismo , Esterol 14-Desmetilase/genética
3.
Eur J Pharm Sci ; 145: 105256, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-32032778

RESUMO

The present study evaluates the efficacy of sodium stibogluconate (SSG) co-loaded with ketoconazole (KTZ) in nano-elastic liposomes (NELs) for the topical treatment of cutaneous leishmaniasis (CL). SSG-KTZ co-loaded NELs were developed and assessed for various physicochemical properties and anti-leishmanial potential. The optimized nano-vesicles have an average size of 212.8 ± 3.1 nm and entrapment efficiency of 61.2 ± 2.9%. SSG-KTZ co-loaded NELs displayed 5.37-fold higher skin permeation of SSG as compared to drug solution. SSG and KTZ displayed a synergistic interaction and flow cytometry revealed enhanced killing of DsRed Leishmania mexicana in infected macrophages. In-vitro and in-vivo anti-leishmanial studies indicated a 10.67-fold lower IC50 value and a 35.33-fold reduced parasitic burden as compared with plain SSG solution, respectively. SSG-KTZ co-loaded NELs were found to be a promising approach for the topical treatment of CL.


Assuntos
Gluconato de Antimônio e Sódio/administração & dosagem , Antiprotozoários/administração & dosagem , Elasticidade , Cetoconazol/administração & dosagem , Leishmaniose Cutânea/tratamento farmacológico , Nanopartículas/administração & dosagem , Administração Tópica , Animais , Gluconato de Antimônio e Sódio/metabolismo , Antiprotozoários/metabolismo , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/metabolismo , Feminino , Cetoconazol/metabolismo , Leishmaniose Cutânea/metabolismo , Lipossomos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/metabolismo , Técnicas de Cultura de Órgãos , Distribuição Aleatória , Pele/efeitos dos fármacos , Pele/metabolismo
4.
PLoS Negl Trop Dis ; 14(2): e0008029, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32023240

RESUMO

Interleukin-32 is a novel inflammatory mediator that has been described to be important in the immunopathogenesis and control of infections caused by Leishmania parasites. By performing experiments with primary human cells in vitro, we demonstrate that the expression of IL-32 isoforms is dependent on the time exposed to L. amazonensis and L. braziliensis antigens. Moreover, for the first time we show the functional consequences of three different genetic variations in the IL32 (rs4786370, rs4349147, rs1555001) modulating IL-32γ expression, influencing innate and adaptive cytokine production after Leishmania exposure. Using a Brazilian cohort of 107 American Tegumentary Leishmaniasis patients and a control cohort of 245 healthy individuals, the IL32 rs4786370 genetic variant was associated with protection against ATL, whereas the IL32 rs4349147 was associated with susceptibility to the development of localized cutaneous and mucosal leishmaniasis. These novel insights may help improve therapeutic strategies and lead to benefits for patients suffering from Leishmania infections.


Assuntos
Predisposição Genética para Doença , Variação Genética , Interleucinas/genética , Leishmania/classificação , Leishmaniose Cutânea/genética , Adulto , Idoso , Brasil/epidemiologia , Citocinas/genética , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/metabolismo , Masculino , Pessoa de Meia-Idade , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Isoformas de Proteínas , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
5.
PLoS Negl Trop Dis ; 14(2): e0007991, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32023254

RESUMO

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


Assuntos
Ferro/metabolismo , Leishmaniose Cutânea/metabolismo , Adolescente , Adulto , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Feminino , Humanos , Índia , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/fisiologia , Leishmaniose Cutânea/parasitologia , Lipocalina-2/genética , Lipocalina-2/metabolismo , Macrófagos/metabolismo , Masculino , Monócitos/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Adulto Jovem
6.
Nanomedicine ; 24: 102121, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31672601

RESUMO

Cutaneous leishmaniasis (CL) is a neglected parasitic disease conventionally treated by multiple injections with systemically toxic drugs. Aiming at a more acceptable therapy, we developed lipid-core nanocapsules (LNCs) entrapping the potent antileishmanial chalcone (CH8) for topical application. Rhodamine-labeled LNC (Rho-LNC-CH8) was produced for imaging studies. LNC-CH8 and Rho-LNC-CH8 had narrow size distributions (polydispersity index <0.10), with similar mean sizes (~180 nm) by dynamic light scattering. In vitro, Rho-LNC-CH8 was rapidly internalized by extracellular Leishmania amazonensis parasites macrophages in less than 15 min. LNC-CH8 activated macrophage oxidative mechanisms more efficiently than CH8, and was more selectively toxic against the intracellular parasites. In vivo, topically applied Rho-LNC-CH8 efficiently permeated mouse skin. In L. amazonensis-infected mice, LNC-CH8 reduced the parasite load by 86% after three weeks of daily topical treatment, while free CH8 was ineffective. In conclusion, LNC-CH8 has strong potential as a novel topical formulation for CL treatment.


Assuntos
Antiprotozoários , Leishmaniose Cutânea/tratamento farmacológico , Lipídeos , Nanoestruturas , Administração Tópica , Animais , Antiprotozoários/química , Antiprotozoários/farmacologia , Cápsulas , Feminino , Leishmania/metabolismo , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/patologia , Lipídeos/química , Lipídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Nanoestruturas/química , Nanoestruturas/uso terapêutico
7.
BMC Res Notes ; 12(1): 803, 2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31831065

RESUMO

OBJECTIVE: The resistance to antimony-containing glucantime is a major obstacle to successful treatment, especially in endemic areas. Looking the molecular mechanisms involved in this drug resistance will help in choosing the best treatment. The aim of this study was to evaluate the expression of multidrug-resistance 1 (MDR1) and multidrug-resistance protein A (MRPA) genes in acute, chronic non-lupoid, and chronic lupoid forms of dry type cutaneous leishmaniasis (DTCL). RESULTS: MDR1 gene was over-expressed as 14.4- and 1.56-folds in the chronic lupoid and acute forms compared with the chronic non-lupoid form, respectively. Results comparison showed P < 0.05 between the chronic non-lupoid and acute groups, P < 0.01 between acute and chronic lupoid groups, and P < 0.001 between the chronic non-lupoid and chronic lupoid groups. MRPA gene was over-expressed as 266 and 17.7-fold in the chronic lupoid and chronic non-lupoid forms compared with the acute form, respectively. Statistical analysis showed P < 0.01 between the chronic non-lupoid and chronic lupoid groups, P < 0.05 between acute and chronic non-lupoid groups, and P < 0.001 between the acute and chronic lupoid groups.


Assuntos
Leishmaniose Cutânea/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Doença Aguda , Doença Crônica , Expressão Gênica , Humanos , Leishmaniose Cutânea/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Pele/metabolismo , Regulação para Cima/genética
8.
Rev Soc Bras Med Trop ; 52: e20190361, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31800923

RESUMO

INTRODUCTION: Cutaneous leishmaniasis is caused by protozoa of the genus Leishmania and transmission occurs through the bite of sandflies. It is an infectious disease, which affects skin and mucosa. The aim was to quantify the macrophages M1 and M2 and the annexin A1 expression in the skin lesions of patients with cutaneous leishmaniasis. METHODS: Skin biopsies from patients (n = 50) were analyzed and classified according to the lesion type as: exudative cellular reaction, exudative granulomatous reaction, exudative necrotic reaction, exudative necrotic-granulomatous reaction. Using the immunofluorescence technique, macrophages were identified by CD163 marker, differentiated by anti-MHCII and anti-CD206 antibodies, and annexin A1 expression was determined by arbitrary unit (a.u.) densitometry. RESULTS: In M1 macrophages, a greater expression of this protein was observed in the exudative cellular reaction type lesions (136.3 ± 2.6 a.u., assuming mean and standard derivation) when compared to the expression in the lesions of exudative granulomatous reaction, exudative necrotic reaction and exudative necrotic-granulomatous reaction patients (108.0 ± 2.3, 121.6 ± 3.2 and 124.7 ± 2.4 a.u., respectively). Regarding M2 macrophages, it was observed that patients with exudative cellular reaction lesion also had a higher expression of this protein (128.8 ± 2.6 a.u.), when compared to the expression in the lesions of exudative granulomatous reaction, exudative necrotic reaction and exudative necrotic-granulomatous reaction patients (105.6 ± 2, 113.9 ± 2.8, 114.3 ± 2.1 a.u., respectively). CONCLUSIONS: These data suggest that annexin A1 is assisting macrophages in the phagocytosis process of patients with exudative cellular reaction lesion type.


Assuntos
Anexina A1/metabolismo , Leishmaniose Cutânea/metabolismo , Macrófagos/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anexina A1/análise , Biópsia , Feminino , Imunofluorescência , Humanos , Leishmaniose Cutânea/patologia , Macrófagos/parasitologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Adulto Jovem
9.
Front Immunol ; 10: 2442, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31749794

RESUMO

The early inflammatory skin micromilieu affects resistance in experimental infection with Leishmania major. We pursue the concept that macrophages, which take up parasites during early infection, exert decisive influence on the inflammatory micromilieu after infection. In order to analyze their distinctive potential, we identified differentially regulated genes of murine granuloma macrophages (GMΦ) from resistant and susceptible mice after their infection with metacyclic Leishmania major. We found induction of several cytokines in GMΦ from both strains and a stronger upregulation of the transcription factor aryl hydrocarbon receptor (AhR) in GMΦ from resistant mice. Using both an AhR agonist and antagonist we demonstrated that AhR is involved in Leishmania-induced production of TNF in macrophages. In vivo, single local injection of an AhR agonist in early lesions of susceptible mice caused an increased induction of Tnf and other cytokines in the skin. Importantly, local agonist treatment led to a reduction of disease severity, reduced parasite loads and a weaker Th2 response. Our results demonstrate that local activation of AhR has a beneficial effect in experimental leishmaniasis.


Assuntos
Citocinas/metabolismo , Leishmania major/fisiologia , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/parasitologia , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais , Animais , Citocinas/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Humanos , Leishmaniose Cutânea/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Monócitos
10.
Artigo em Inglês | MEDLINE | ID: mdl-31637219

RESUMO

Disease manifestation after infection with cutaneous Leishmania species is the result of a complex interplay of diverse factors, including the immune status of the host, the infecting parasite species, or the parasite load at the lesion site. Understanding how these factors impact on the pathology of cutaneous leishmaniasis (CL) may provide new targets to manage the infection and improve clinical outcome. We quantified the relative expression of 170 genes involved in a diverse range of biological processes, in the skin biopsies from patients afflicted with CL caused by infection with either L. major or L. tropica. As compared to healthy skin, CL lesions bear elevated levels of transcripts involved in the immune response, and conversely, present a significant downregulation in the expression of genes involved in epidermal integrity and arginine or fatty acid metabolism. The expression of transcripts encoding for cytotoxic mediators and chemokines in lesions was inversely correlated with the expression of genes involved in epidermal integrity, suggesting that cytotoxicity is a major mediator of CL pathology. When comparing the transcriptional profiles of lesions caused by either L. major or L. tropica, we found them to be very similar, the later presenting an aggravated inflammatory/cytotoxic profile. Finally, we identified genes positively correlated with the parasite load in lesions. Among others, these included Th2 or regulatory cytokines, such as IL4 or IL10. Remarkably, a single gene among our dataset, encoding for tryptophan-2,3-deoxygenase (TDO), presented a negative correlation with the parasite load, suggesting that its expression may restrict parasite numbers in lesions. In agreement, treatment of macrophages infected with L. major in vitro with a TDO inhibitor led to an increase in parasite transcripts. Our work provides new insights into the factors that impact CL pathology and identifies TDO as a restriction factor for cutaneous Leishmania.


Assuntos
Perfilação da Expressão Gênica , Leishmaniose Cutânea/genética , Transcriptoma , Triptofano Oxigenase/genética , Animais , Arginina/metabolismo , Biópsia , Linhagem Celular , Biologia Computacional/métodos , Citocinas/genética , Citocinas/metabolismo , Epiderme/metabolismo , Epiderme/parasitologia , Epiderme/patologia , Ácidos Graxos/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Leishmania , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos , Triptofano Oxigenase/metabolismo
11.
Sensors (Basel) ; 19(21)2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31661834

RESUMO

Cutaneous leishmaniasis (CL) is a neglected tropical disease that requires novel tools for its understanding, diagnosis, and treatment follow-up. In the cases of other cutaneous pathologies, such as cancer or cutaneous ulcers due to diabetes, optical diffuse reflectance-based tools and methods are widely used for the investigation of those illnesses. These types of tools and methods offer the possibility to develop portable diagnosis and treatment follow-up systems. In this article, we propose the use of a three-layer diffuse reflectance model for the study of the formation of cutaneous ulcers caused by CL. The proposed model together with an inverse-modeling procedure were used in the evaluation of diffuse-reflectance spectral signatures acquired from cutaneous ulcers formed in the dorsal area of 21 golden hamsters inoculated with Leishmanisis braziliensis. As result, the quantification of the model's variables related to the main biological parameters of skin were obtained, such as: diameter and volumetric fraction of keratinocytes, collagen; volumetric fraction of hemoglobin, and oxygen saturation. Those parameters show statistically significant differences among the different stages of the CL ulcer formation. We found that these differences are coherent with histopathological manifestations reported in the literature for the main phases of CL formation.


Assuntos
Leishmaniose Cutânea/patologia , Úlcera Cutânea/patologia , Pele/química , Espectrofotometria/métodos , Animais , Colágeno/fisiologia , Cricetinae , Modelos Animais de Doenças , Processamento Eletrônico de Dados , Feminino , Hemoglobinas/química , Leishmaniose Cutânea/metabolismo , Masculino , Mesocricetus , Oxigênio/química , Pele/patologia , Úlcera Cutânea/metabolismo , Úlcera Cutânea/parasitologia
12.
Microb Pathog ; 137: 103738, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31513893

RESUMO

INTRODUCTION: Cutaneous leishmaniasis is a dermal disease caused by several species of the genus Leishmania. It is an endemic disease with 1.2 million new cases occurring annually and mostly in developing countries. Oxidative stress is a condition of an imbalance in oxidant/antioxidant which may play a role in many different pathologic conditions. For the first time in this study, we introduced isoprostane as a reliable index for oxidative stress in patients suffering from leishmaniasis. We also investigated the possible relation between quantitative CRP and this disease. METHOD AND MATERIAL: We collected 5 ml blood of 30 patients in addition to the same sample of the control healthy group. After applying appropriate methods, the plasma and serum specimens were extracted in order to conduct oxidant-antioxidant balance and CRP tests in serum as well as measuring isoprostane factor in plasma. STATISTICAL ANALYSIS: We used T-student, ANOVA as well as linear regression to analyze the gathered data with a 0.05 confidence interval in SPSS environment. RESULTS: The results showed a significant difference between the two groups in terms of the oxidant-antioxidant balance. Also, isoprostane and quantitative CRP levels were substantially higher in patients. There was no significant relationship between the mentioned factors and wound size and number. CONCLUSION: Leishmania Amastigotes plays an important role in disturbing the oxidant-antioxidant balance resulting in inflammation and stress in patients. Furthermore, isoprostane was confirmed as a reliable index for evaluating oxidative stress in patients.


Assuntos
Antioxidantes/análise , Proteína C-Reativa/análise , Isoprostanos/sangue , Leishmaniose Cutânea/sangue , Leishmaniose Cutânea/metabolismo , Oxidantes/sangue , Estudos de Casos e Controles , Feminino , Humanos , Irã (Geográfico) , Masculino , Estresse Oxidativo
13.
FASEB J ; 33(12): 13367-13385, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31553893

RESUMO

Heme is an essential molecule synthetized through a broadly conserved 8-step route that has been lost in trypanosomatid parasites. Interestingly, Leishmania reacquired by horizontal gene transfer from γ-proteobacteria the genes coding for the last 3 enzymes of the pathway. Here we show that intracellular amastigotes of Leishmania major can scavenge heme precursors from the host cell to fulfill their heme requirements, demonstrating the functionality of this partial pathway. To dissect its role throughout the L. major life cycle, the significance of L. major ferrochelatase (LmFeCH), the terminal enzyme of the route, was evaluated. LmFeCH expression in a heterologous system demonstrated its activity. Knockout promastigotes lacking lmfech were not able to use the ferrochelatase substrate protoporphyrin IX as a source of heme. In vivo infection of Phlebotomus perniciosus with knockout promastigotes shows that LmFeCH is not required for their development in the sandfly. In contrast, the replication of intracellular amastigotes was hampered in vitro by the deletion of lmfech. However, LmFeCH-/- parasites produced disease in a cutaneous leishmaniasis murine model in a similar way as control parasites. Therefore, although L. major can synthesize de novo heme from macrophage precursors, this activity is dispensable being an unsuited target for leishmaniasis treatment.-Orrego, L. M., Cabello-Donayre, M., Vargas, P., Martínez-García, M., Sánchez, C., Pineda-Molina, E., Jiménez, M., Molina, R., Pérez-Victoria, J. M. Heme synthesis through the life cycle of the heme auxotrophic parasite Leishmania major.


Assuntos
Ferroquelatase/metabolismo , Heme/biossíntese , Leishmania major/crescimento & desenvolvimento , Leishmaniose Cutânea/metabolismo , Proteínas de Protozoários/metabolismo , Psychodidae/metabolismo , Virulência , Sequência de Aminoácidos , Animais , Coproporfirinogênio Oxidase/metabolismo , Feminino , Ferroquelatase/química , Ferroquelatase/genética , Leishmaniose Cutânea/parasitologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Conformação Proteica , Protoporfirinogênio Oxidase/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Psychodidae/parasitologia , Homologia de Sequência
14.
Infect Immun ; 87(11)2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31451620

RESUMO

Cutaneous leishmaniasis is characterized by vascular remodeling. Following infection with Leishmania parasites, the vascular endothelial growth factor A (VEGF-A)/VEGF receptor 2 (VEGFR-2) signaling pathway mediates lymphangiogenesis, which is critical for lesion resolution. Therefore, we investigated the cellular and molecular mediators involved in VEGF-A/VEGFR-2 signaling using a murine model of infection. We found that macrophages are the predominant cell type expressing VEGF-A during Leishmania major infection. Given that Leishmania parasites activate hypoxia-inducible factor 1α (HIF-1α) and this transcription factor can drive VEGF-A expression, we analyzed the expression of HIF-1α during infection. We showed that macrophages were also the major cell type expressing HIF-1α during infection and that infection-induced VEGF-A production is mediated by ARNT/HIF activation. Furthermore, mice deficient in myeloid ARNT/HIF signaling exhibited larger lesions without differences in parasite numbers. These data show that L. major infection induces macrophage VEGF-A production in an ARNT/HIF-dependent manner and suggest that ARNT/HIF signaling may limit inflammation by promoting VEGF-A production and, thus, lymphangiogenesis during infection.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Leishmaniose Cutânea/metabolismo , Macrófagos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Células Cultivadas , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Leishmania major , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Pele/metabolismo , Pele/parasitologia , Fator A de Crescimento do Endotélio Vascular/genética
15.
Photobiomodul Photomed Laser Surg ; 37(5): 298-304, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31084559

RESUMO

Objective: In this study, we evaluated the effectiveness of photodynamic therapy (PDT) for the treatment of experimental cutaneous leishmaniasis (CL) and the profile of macrophages activation markers. Background: Leishmaniasis is an infectious disease caused by parasites of the genus Leishmania. CL is caused by Leishmania major in the old world and by Leishmania braziliensis in the Americas. Considering the targeted organs, PDT may constitute a valuable therapeutic intervention. Macrophages are the host cells of Leishmania in mammals and may be classified into type M1 or M2 depending on the pattern of activation. Methods: BALB/c mice were infected in the foot pad with 1 × 106 amastigotes of L. braziliensis and treated with 5-aminolevulinic acid (5-ALA), visible light, or 5-ALA-PDT. The ex vivo mRNA expression levels of interleukin-10, tumor necrosis factor-α (TNF-α), arginase-1, heme oxygenase ( Hmox), and induced nitric oxide synthase (iNOS) were quantities as markers of macrophage activation with distinct ability to kill intracellular parasite. Results: The parasite load decreased significantly in the group treated with PDT compared with the other groups. The iNOS relative mRNA was higher in the group treated with PDT and light only compared with the group without treatment, whereas iNOS/arginase ratio was significantly higher only in the PDT group. The expression of TNF-α was significantly higher in 5-ALA and light compared with PDT and control group. No significant difference was observed in the expression of the other markers evaluated. Conclusions: Both, light and 5-ALA-PDT were able to upregulate iNOS expression only; 5-ALA-PDT was able to reduce parasite burden. The increase in the iNOS levels suggests it might participate in the antimicrobial mechanisms triggered by 5-ALA-PDT; although parasite death mechanism was not completely clarified, the results presented in this study suggest that macrophage activation may contribute to parasite control.


Assuntos
Ácido Aminolevulínico/uso terapêutico , Leishmaniose Cutânea/terapia , Ativação de Macrófagos/efeitos da radiação , Fotoquimioterapia , Fármacos Fotossensibilizantes/uso terapêutico , Animais , Modelos Animais de Doenças , Interleucina-10/metabolismo , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Carga Parasitária , Fator de Necrose Tumoral alfa/metabolismo
16.
ACS Infect Dis ; 5(8): 1295-1305, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31094195

RESUMO

Cutaneous leishmaniasisis is the most common clinical form of leishmaniasis and one of the most relevant neglected diseases. It is known that the progress of the disease is species specific and the host's immune response plays an important role in its outcome. However, the pathways that lead to parasite clearance or survival remain unknown. In this work, skin tissue from mice experimentally infected with L. amazonensis, one of the causative agents of cutaneous leishmaniasis in the Amazon region, L. major, another causative agent of cutaneous leishmaniasis in Africa, the Middle East, China, and India, or lipopolysaccharides from Escherichia coli as an inflammation model were investigated using label-free proteomics to unveil Leishmania-specific protein alterations. Proteomics is a powerful tool to investigate host-pathogen relationships to address biological questions. In this work, proteins from mice skin biopsies were identified and quantified using nano-LC coupled with tandem mass spectrometry analyses. Integrated Proteomics Pipeline was used for peptide/protein identification and quantification. Western blot was used for validation of protein quantification by mass spectrometry, and protein pathways were predicted using Ingenuity Pathway Analysis. In this proteomics study, several proteins were pointed out as hypothetical targets to guide future studies on Leishmania-specific modulation of proteins in the host. We identified hundreds of exclusively modulated proteins after Leishmania spp. infection and 17 proteins that were differentially modulated in the host after L. amazonensis or L. major infection.


Assuntos
Interações Hospedeiro-Patógeno , Leishmania braziliensis/patogenicidade , Leishmania major/patogenicidade , Leishmaniose Cutânea/metabolismo , Proteômica , Pele/metabolismo , Animais , Biópsia , Feminino , Inflamação , Leishmaniose Cutânea/patologia , Camundongos , Camundongos Endogâmicos BALB C , Mapas de Interação de Proteínas , Proteínas/análise , Pele/parasitologia , Pele/patologia , Espectrometria de Massas em Tandem
17.
Molecules ; 24(7)2019 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-30934998

RESUMO

Leishmaniasis is a neglected tropical disease caused by members of the Leishmania genus of parasitic protozoa that cause different clinical manifestations of the disease. Current treatment options for the cutaneous disease are limited due to severe side effects, poor efficacy, limited availability or accessibility, and developing resistance. Essential oils may provide low cost and readily available treatment options for leishmaniasis. In-vitro screening of a collection of 52 commercially available essential oils has been carried out against promastigotes of Leishmania amazonensis. In addition, cytotoxicity has been determined for the essential oils against mouse peritoneal macrophages in order to determine selectivity. Promising essential oils were further screened against intracellular L. amazonensis amastigotes. Three essential oils showed notable antileishmanial activities: frankincense (Boswellia spp.), coriander (Coriandrum sativum L.), and wintergreen (Gualtheria fragrantissima Wall.) with IC50 values against the amastigotes of 22.1 ± 4.2, 19.1 ± 0.7, and 22.2 ± 3.5 µg/mL and a selectivity of 2, 7, and 6, respectively. These essential oils could be explored as topical treatment options for cutaneous leishmaniasis.


Assuntos
Antiprotozoários/química , Leishmania/química , Óleos Voláteis/química , Animais , Antiprotozoários/farmacologia , Boswellia/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Coriandrum/química , Avaliação Pré-Clínica de Medicamentos/métodos , Concentração Inibidora 50 , Leishmaniose Cutânea/metabolismo , Macrófagos Peritoneais/química , Camundongos Endogâmicos BALB C , Óleos Voláteis/farmacologia
18.
Methods Mol Biol ; 1971: 315-349, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980313

RESUMO

Experimental cutaneous leishmaniasis of mice is a valuable model to study the immune response to the protozoan pathogen Leishmania and to define mechanisms of parasite control and resolution of inflammation as well as of parasite evasion and chronicity of disease. In addition, over many years Leishmania-infected mice have been successfully used to analyze the function of newly discovered immune cell types, transcription factors, cytokines, and effector mechanisms in vivo. In this chapter we present detailed protocols for the culture, propagation, and inoculation of Leishmania promastigotes, the monitoring of the course of cutaneous infection, the determination of the tissue parasite burden and for the phenotyping of the ensuing immune response. The focus lies on the L. major mouse model, but an overview on other established models of murine cutaneous leishmaniasis is also provided.


Assuntos
Modelos Animais de Doenças , Leishmania/crescimento & desenvolvimento , Leishmaniose Cutânea/parasitologia , Estágios do Ciclo de Vida , Carga Parasitária , Animais , Doença Crônica , Inflamação/metabolismo , Inflamação/parasitologia , Inflamação/patologia , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/patologia , Camundongos , Camundongos Endogâmicos BALB C
19.
Immun Inflamm Dis ; 7(3): 95-104, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30997749

RESUMO

INTRODUCTION: Zoonotic cutaneous leishmaniasis (ZCL), due to infection by Leishmania (L). major, is characterized by polymorphic clinical manifestations which could be attributed to the host's immune response. In this study we investigated the involvement of cytotoxic cells on the outcome of the disease. METHODS: Expression of granzyme B (GrB), granulysine (Grly), and interferon (IFN)-γ was evaluated within ZCL lesion specimens using the technique of real-time quantitative polymerase chain reaction (RT-qPCR). Immunohistochemical staining was performed using anti-CD3, CD4, CD8, CD56, GrB, and IFN-γ antibodies to identify the phenotype of GrB and IFN-γ-producing cells. RESULTS: GrB and Grly mRNA was detected within 75% and 80% of ZCL lesions, respectively. Statistical analysis demonstrated a significant correlation between levels of GrB and Grly. Interestingly, expression of these molecules correlates negatively with the lesion's age. The highest levels were measured in early lesions (E-ZCL) (lesion age ≤1 month) comparing to late lesions (L-ZCL) (lesion age >1 month). Otherwise, IFN-γ mRNA was detected only within 56% and a positive correlation was found between levels of this cytokine and those of GrB. Immunohistochemical analysis showed that GrB is produced essentially by CD8+ T cells whereas IFN-γ is produced by both CD4+ and CD8+ T cells. CONCLUSION: Together our results demonstrate the presence of cytotoxic cells producing GrB and Grly within leishmaniasis cutaneous lesions.


Assuntos
Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Linfócitos T Citotóxicos/imunologia , Zoonoses/imunologia , Adulto , Animais , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Criança , Pré-Escolar , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Expressão Gênica/imunologia , Granzimas/genética , Granzimas/imunologia , Granzimas/metabolismo , Humanos , Lactente , Recém-Nascido , Interferon gama/genética , Interferon gama/metabolismo , Leishmania major/parasitologia , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/parasitologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Pele/imunologia , Pele/parasitologia , Linfócitos T Citotóxicos/metabolismo , Zoonoses/parasitologia
20.
Biomed Microdevices ; 21(1): 8, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30617619

RESUMO

Current therapeutic options against cutaneous leishmaniasis are plagued by several weaknesses. The effective topical delivery of an antileishmanial drug would be useful in treating some forms of cutaneous leishmaniasis. Toward this end, a microneedle based delivery approach for the antileishmanial drug amphotericin B was investigated in murine models of both New World (Leishmania mexicana) and Old World (Leishmania major) infection. In the L. mexicana model, ten days of treatment began on day 35 post infection, when the area of nodules averaged 9-15 mm2. By the end of the experiment, a significant difference in nodule area was observed for all groups receiving topical amphotericin B at 25 mg/kg/day after application of microneedle arrays of 500, 750, and 1000 µM in nominal length compared to the group that received this dose of topical amphotericin B alone. In the L. major model, ten days of treatment began on day 21 post infection when nodule area averaged 51-65 mm2 in the groups. By the end of the experiment, there was no difference in nodule area between the group receiving 25 mg/kg of topical amphotericin B after microneedle application and any of the non-AmBisome groups. These results show the promise of topical delivery of amphotericin B via microneedles in treating relatively small nodules caused by L. mexicana. These data also show the limitations of the approach against a disseminated L. major infection. Further optimization of microneedle delivery is needed to fully exploit this strategy for cutaneous leishmaniasis treatment.


Assuntos
Anfotericina B/farmacologia , Sistemas de Liberação de Medicamentos , Leishmania mexicana/metabolismo , Leishmaniose Cutânea/tratamento farmacológico , Agulhas , Animais , Sistemas de Liberação de Medicamentos/instrumentação , Sistemas de Liberação de Medicamentos/métodos , Feminino , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/patologia , Camundongos , Camundongos Endogâmicos BALB C
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