MARCIA DALASTRA LAURENTI
Projetos de Pesquisa
Unidades Organizacionais
Departamento de Patologia, Faculdade de Medicina - Docente
LIM/50 - Laboratório de Patologia das Moléstias Infecciosas, Hospital das Clínicas, Faculdade de Medicina - Líder
LIM/50 - Laboratório de Patologia das Moléstias Infecciosas, Hospital das Clínicas, Faculdade de Medicina - Líder
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- Involvement of the Inflammasome and Th17 Cells in Skin Lesions of Human Cutaneous Leishmaniasis Caused by Leishmania (Viannia) panamensis(2020) GONZALEZ, K.; CALZADA, J. E.; CORBETT, C. E. P.; SALDANA, A.; LAURENTI, M. D.Localized cutaneous leishmaniasis (LCL) caused by Leishmania (Viannia) panamensis is an endemic disease in Panama. This condition causes ulcerated skin lesions characterized by a mixed Th1/Th2 immune response that is responsible for disease pathology. However, the maintenance of the in situ inflammatory process involves other elements, such as Th17 and inflammasome responses. Although these processes are associated with parasite elimination, their role in the increase in disease pathology cannot be discarded. Thus, the role in Leishmania infection is still unclear. In this sense, the present study aimed at characterizing the Th17 and inflammasome responses in the skin lesions of patients with LCL caused by L. (V.) panamensis to help elucidate the pathogenesis of this disease in Panama. Th17 and inflammasome responses were evaluated by immunohistochemistry (IHQ) in 46 skin biopsies from patients with LCL caused by L. (V.) panamensis. The Th17 immune response was assessed using CD3, CD4, RoR gamma t, IL-17, IL-6, IL-23, and TGF-beta 1 antibodies, and the inflammasome response was assessed by IL-1 beta, IL-18, and caspase-1 antibodies. The presence of the Th17 and inflammasome responses was evidenced by a positive reaction for all immunological markers in the skin lesions. An inverse correlation between the density of amastigotes and the density of RoR gamma t(+), IL-17(+), IL-1 beta(+), and caspase-1(+) cells was observed, but no correlation between Th17 and the inflammasome response with evolutionary disease pathology was reported. These data showed the participation of Th17 cells and the inflammasome in the inflammatory response of the skin lesions of LCL caused by L. (V.) panamensis infection. These results suggest a role in the control of tissue parasitism of IL-17 and the activation of the NLRP3 inflammasome dependent on IL-1 beta but cannot exclude their role in the development of disease pathology.
- Th17 lymphocytes in atypical cutaneous leishmaniasis caused byLeishmania (L.) infantum chagasiin Central America(2020) FLORES, Gabriela Venicia Araujo; PACHECO, Carmen Maria Sandoval; OCHOA, Wilfredo Humberto Sosa; GOMES, Claudia Maria Castro; ZUNIGA, Concepcion; CORBETT, Carlos P.; LAURENTI, Marcia DalastraSkin lesions in nonulcerated cutaneous leishmaniasis (NUCL) caused byLeishmania (L.) infantum chagasiare characterized by a mononuclear inflammatory infiltrate in the dermis, which is composed mainly of lymphocytes, followed by macrophages, few plasma cells and epithelioid granulomas with mild tissue parasitism. Previous studies have shown that the main population of lymphocytes present in the dermal infiltrate is CD8(+)T cells, followed by CD4(+)T cells, which are correlated with IFN-gamma(+)cells. To improve the knowledge of cellular immune responses in NUCL, skin biopsies were submitted to immunohistochemistry using anti-ROR-gamma t, anti-IL-17, anti-IL-6, anti-TGF-beta, and anti-IL-23 antibodies to characterize the involvement of Th17 cells in the skin lesions of patients affected by NUCL. ROR-gamma t(+), IL-17(+), IL-6(+), TGF-beta(+)and IL-23(+)cells were observed in the dermal inflammatory infiltrate of NUCL skin lesions. A positive correlation between CD4(+)T-lymphocytes and ROR-gamma t(+)and IL-17(+)cells suggests that some of the CD4(+)T-lymphocytes in NUCL could be Th17 lymphocytes. Moreover, a positive correlation between ROR-gamma t(+)cells and TGF-beta(+), IL-6(+), IL-17(+)and IL-23(+)cells could indicate the role of these cytokines in the differentiation and maintenance of Th17 lymphocytes. Our findings improve knowledge of the pathogenesis of this rare and atypical clinical form of leishmaniasis.
- Chromosomal segments may explain the antibody response cooperation for canine leishmaniasis pathogenesis(2020) BATISTA, Luis F. S.; TORRECILHA, Rafaela B. P.; SILVA, Rafaela B.; UTSUNOMIYA, Yuri T.; SILVA, Thais B. F.; TOMOKANE, Thaise Y.; PACHECO, Acacio D.; BOSCO, Anelise M.; PAULAN, Silvana C.; ROSSI, Claudio N.; COSTA, Gustavo N. O.; MARCONDES, Mary; CIARLINI, Paulo C.; NUNES, Caris M.; MATTA, Vania L. R.; LAURENTI, Marcia D.Visceral leishmaniasis (VL) is marked by hyperactivation of a humoral response secreting high quantity of immunoglobulins (Igs) that are inaccessible to intracellular parasites. Here we investigated the contributions of the antibody response to the canine leishmaniasis pathogenesis. Using correlation and genome-wide association analysis, we investigated the relationship of anti-Leishmania infantum immunoglobulin classes levels with parasite burden, clinical response, renal/hepatic biochemical, and oxidative stress markers in dogs from endemic areas of VL. Immunoglobulin G (IgG) and IgA were positively correlated with parasite burden on lymph node and blood. Increased IgG, IgA and IgE levels were associated with severe canine leishmaniasis (CanL) whereas IgM was elevated in uninfected exposed dogs. Correlations of IgM, IgG and IgA with creatinine, urea, AST and ALT levels in the serum were suggested an involvement of those Igs with renal and hepatic changes. The correlogram of oxidative radicals and antioxidants revealed a likely relationship of IgM, IgG and IgA with oxidative stress and lipid pemxidation in the blood, suggested as mechanisms mediating tissue damage and CanL worsening. The gene mapping on chromosomal segments associated with the quantitative variation of immunoglobulin classes identified genetic signatures involved with reactive oxygen species generation, phagolysosome maturation and rupture, free iron availability, Thl/Th2 differenciation and, immunoglobulin clearance. The findings demonstrated the roles of the antibody response as resistance or susceptibility markers and mediators of CanL pathogenesis. In addition we pinpointed candidate genes as potential targets for the therapy against the damage caused by exacerbated antibody response and parasitism in VL.
- Reactivity of purified and axenic amastigotes as a source of antigens to be used in serodiagnosis of canine visceral leishmaniasis(2020) SILVA, Thais Bruna Ferreira da; SILVEIRA, Fernando Tobias; TOMOKANE, Thaise Yumie; BATISTA, Luis Fabio da Silva; NUNES, Juliana Barbosa; MATTA, Vania Lucia Ribeiro da; PASSERO, Luiz Felipe Domingues; LAURENTI, Marcia DalastraAlthough there is a great diversity of techniques and antigens used in the serodiagnosis of canine visceral leishmaniasis (CVL), total sensitivity and specificity have not yet been found. Since the use of amastigote forms in the indirect immunofluorescence assay has shown an improvement in the specificity of the test for the diagnosis of CVL, the performance of amastigotes forms of L. (L.) infantum chagasi as antigen source were evaluated in automatized ELISA WA using crude antigen of axenic amastigote and purified amastigote from spleen of hamster chronically infected comparing with ELISA using total antigen produced with promastigote forms of L. (L.) infantum chagasi. One hundred and fifteen sera from dogs with positive parasitological diagnosis by PCR were used. The animals were classified into 2 groups: symptomatic (n = 67) and asymptomatic (n = 48) animals, in accordance with the clinical signs and laboratory tests were. As control, ninety-four sera from dogs with negative parasitological diagnosis were included. No significant difference was found in sensitivity, specificity, predictive values and accuracy between ELISA using whole antigens produced with both axenic and purified amastigotes in comparison with promastigotes forms. Correlation and concordance between the three total antigens tested in ELISA was observed. According to the similar performance among antigens, data pointed out to use antigen from promastigote forms for diagnosing canine leishmaniasis, especially due the easily in the production, lower cost and the abundance of correlative literature.
- Topical buparvaquone nano-enabled hydrogels for cutaneous leishmaniasis(2020) LALATSA, Aikaterini; STATTS, Larry; JESUS, Jessica Adriana de; ADEWUSI, Olivia; DEA-AYUELA, Maria Auxiliadora; BOLAS-FERNANDEZ, Francisco; LAURENTI, Marcia Dalastra; PASSERO, Luiz Felipe Domingues; SERRANO, Dolores R.Leishmaniasis is a neglected disease presenting cutaneous, mucosal and visceral forms and affecting an estimated 12 million mostly low-income people. Treatment of cutaneous leishmaniasis (CL) is recommended to expedite healing, reduce risk of scarring, prevent parasite dissemination to other mucocutaneous (common with New World species) or visceral forms and reduce the chance of relapse, but remains an unmet need. Available treatments are painful, prolonged (> 20 days) and require hospitalisation, which increases the cost of therapy. Here we present the development of optimised topical self-nanoemulsifying drug delivery systems (SNEDDS) loaded with buparvaquone (BPQ, a hydroxynapthoquinone from the open Malaria Box) for the treatment of CL from New World species. The administration of topical BPQ-SNEDDS gels for 7 days resulted in a reduction of parasite load of 99.989 +/- 0.019% similar to the decrease achieved with intralesionally administered Glucantime (R) (99.873 +/- 0.204%) in a L. amazonensis BALB/c model. In vivo efficacy was supported by ex vivo permeability and in vivo tape stripping studies. BPQ-SNEDDS and their hydrogels demonstrated linear flux across non-infected CD-1 mouse skin ex vivo of 182.4 +/- 63.0 mu g cm(-2) h(-1) and 57.6 +/- 10.8 mu g cm(-2 )h(-1) respectively localising BPQ within the skin in clinically effective concentrations (227.0 +/- 45.9 mu g and 103.8 +/- 33.8 mu g) respectively. These levels are therapeutic as BPQ-SNEDDS and their gels showed nanomolar in vitro efficacy against L. amazonensis and L. braziliensis amastigotes with excellent selectivity index toward parasites versus murine macrophages. In vivo tape stripping experiments indicated localisation of BPQ within the stratum corneum and dermis. Histology studies confirmed the reduction of parasitism and indicated healing in animals treated with BPQ-SNEDDS hydrogels. These results highlight the potential clinical capability of nano-enabled BPQ hydrogels towards a non-invasive treatment for CL.
- LEISHMANICIDAL ACTIVITY in vivo OF A MILTEFOSINE DERIVATIVE IN Mesocricetus auratus(2020) SILVA, Joana C. da; NUNES, Juliana B.; GONTIJO, Vanessa S.; MALAQUIAS, Luiz Cosme C.; FREITAS, Rossimiriam P. de; ALVES, Rosemeire B.; COLOMBO, Fabio A.; LAURENTI, Marcia D.; MARQUES, Marcos J.Visceral leishmaniasis (VL) is a chronic and systemic disease; if untreated, it can cause death in a large number of cases. The therapy is based on the use of antimonials, which have been used for over 50 years. However, cases of resistance have been reported in some countries. In this context, miltefosine (MIL) was introduced to treat antimonial unresponsive cases. Nonetheless, in recent years MIL unresponsive and relapse cases of VL have increasingly been reported. In the current study, the therapeutic potential of compound 5-(4-(3-methanesulfonatepropyl)-1H-1,2,3-triazol-1-yl)dodecyl methanesulfonate (C11), an MIL derivative, was assessed in an experimental VL hamster model. For this purpose, golden hamsters (Mesocricetus auratus) were infected with Leishmania (L.) infantum chagasi and treated daily for 10 days with C11 and MIL administered orally; in addition, Glucantime (GLU), peritoneal route, were administered at 15, 10, 50 mg/kg body weight/day, respectively. Twenty four hours after the end of treatment the animals were euthanatized; and the specimens were collected to evaluate the relative mRNA expression of cytokines IFN-gamma, TNF-alpha, IL-17, TGF-beta, IL-4 and IL-10 in fragments of the spleen and liver; moreover, the parasitism in these organs was evaluated as well as the main histopathological alterations. The C11-treated animals showed greater expression of IL-17 and TNF-alpha cytokines and reduced expression of IL-10 in the spleen in comparison to the infected untreated group (UTG) (p <0.05). The C11 and GLU groups showed a significant reduction in the IgG levels in comparison to the UTG group (p <0.05). Moreover, the C11-treated animals had fewer parasites in the spleen than the UTG animals (reduction of 95.9%), as well as a greater preservation of white pulp architecture in the spleen than the UTG, GLU and MIL groups (p <0.05). For the liver, the animals from the C11 and MIL groups showed a significant increase in TNF-alpha relative expression in comparison to the UTG animals, which would explain the increase in the number of granulomas and the reduction in the parasitic load (p <0.05). Combined, these findings indicate that C11 is an interesting compound that should be considered for the development of new drugs against VL, mainly due to its leishmanicidal effect and immunostimulating action.
- Tolnaftate inhibits ergosterol production and impacts cell viability of Leishmania sp.(2020) YAMAMOTO, Eduardo Seiji; JESUS, Jessica Adriana de; BEZERRA-SOUZA, Adriana; BRITO, Juliana R.; LAGO, Joao Henrique G.; LAURENTI, Marcia Dalastra; PASSERO, Luiz Felipe DominguesLeishmaniasis is an infectious disease caused by protozoan parasites of the genus Leishmania. The treatment of all forms of leishmaniasis relies on first-line drug, pentavalent antimonial, and in cases of drug failure, the second-line drug amphotericin B has been used. Besides the high toxicity of drugs, parasites can be resistant to antimonial in some areas of the World, making it necessary to perform further studies for the characterization of new antileishmanial agents. Thus, the aim of the present work was to evaluate the leishmanicidal activity of tolnaftate, a selective reversible and non-competitive inhibitor of the fungal enzyme squalene epoxidase, which is involved in the biosynthesis of ergosterol, essential to maintain membrane physiology in fungi as well as trypanosomatids. Tolnaftate eliminated promastigote forms of L. (L.) amazonensis, L. (V.) braziliensis and L. (L.) infantum (EC50 similar to 10 mu g/mL and SI similar to 20 for all leishmanial species), and intracellular amastigote forms of all studied species (EC50 similar to 23 mu g/mL in infections caused by dermatotropic species; and 11.7 mu g/mL in infection caused by viscerotropic species) with high selectivity toward parasites [SI similar to 8 in infections caused by dermatotropic species and 17.4 for viscerotropic specie]. Promastigote forms of L. (L.) amazonensis treated with the EC50 of tolnaftate displayed morphological and physiological changes in the mitochondria and cell membrane. Additionally, promastigote forms treated with tolnaftate EC50 reduced the level of ergosterol by 5.6 times in comparison to the control parasites. Altogether, these results suggest that tolnaftate has leishmanicidal activity towards Leishmania sp., is selective, affects the cell membrane and mitochondria of parasites and, moreover, inhibits ergosterol production in L. (L.) amazonensis.
- Ursolic Acid Potentializes Conventional Therapy in Experimental Leishmaniasis(2020) JESUS, Jessica Adriana; SILVA, Thays Nicolli Fragoso da; YAMAMOTO, Eduardo Seiji; LAGO, Joao Henrique G.; LAURENTI, Marcia Dalastra; PASSERO, Luiz Felipe DominguesUrsolic acid (UA) is a triterpene with a broad array of pharmacological activities. In leishmaniasis, UA killed different species of parasites, and it was active in the experimental model of cutaneous and visceral leishmaniasis. Thus, the objective of this work was to study the therapeutic efficacy of the conventional drugs amphotericin B (AmB) or glucantime (Glu) combined with UA in experimental visceral and cutaneous leishmaniasis, respectively. L. (L.) infantum-infected hamsters were treated with AmB alone or combined with UA. L. (L.) amazonensis-infected BALB/c mice were treated with Glu alone or combined with UA. Animals were treated for 15 consecutive days by intraperitoneal or intralesional routes. Following one week after the last dose, the tissue parasitism and cellular immune responses were analyzed. Hamsters treated with 0.2 and 1.0 mg/kg of AmB plus 1.0 mg/kg of UA showed low hepatic and splenic parasitisms; however, AmB given as monotherapy did not reduce the number of viable parasites in the spleen of treated animals. In cutaneous leishmaniasis, Glu given as monotherapy was inactive at 2.0 mg/kg, showed mild activity at 10.0 mg/kg, and at 50.0 mg/kg was highly active at eliminating parasites in the skin. When animals were treated with Glu plus UA, higher leishmanicidal activity was observed in comparison to all groups treated with monotherapy schemes, and such activity was related to lesion improvement and upregulation of IFN-gamma production. Altogether, data suggest that the association of drugs for the treatment of leishmaniasis can increase the efficiency of the treatment and decrease the toxicity associated to the conventional drugs.
- Lipophosphoglycans from dermotropic Leishmania infantum are more pro-inflammatory than those from viscerotropic strains(2020) CARDOSO, Camila A.; V, Gabriela Araujo; SANDOVAL, Carmen M.; NOGUEIRA, Paula M.; ZUNIGA, Concepcion; SOSA-OCHOA, Wilfredo H.; LAURENTI, Marcia D.; SOARES, Rodrigo P.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(beta 1,4)Man(alpha 1)-PO4 common to all LPGs. Peritoneal macrophages from BALB/c and C57BL/6 were exposed to LPG for nitric oxide (NO) and cytokine (TNF-alpha 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.
- New record of preclinical diagnosis of American visceral leishmaniasis in Amazonian Brazil encourages optimizing disease control(2020) LIMA, L.; VASCONCELOS-DOS-SANTOS, T.; CAMPOS, M.; RAMOS, P.K.; GOMES, C.; LAURENTI, M.; MATTA, V. da; CORBETT, C.; SILVEIRA, F.The clinical-immunological spectrum of human Leishmania (L.) infantum chagasi-infection in Amazonian Brazil has recently been reviewed based on the combined use of the delayed-type hypersensitivity (DTH) and indirect fluorescence antibody test (IFAT-IgG/IgM), both with homologous L. (L.) infantum chagasi-antigens, and associated with the clinical evaluation of infected individuals. This diagnostic approach has allowed to identify the broadest clinical-immunological spectrum of human L. (L.) infantum chagasi-infection composed by five clinical-immunological profiles of infection: three asymptomatic, 1) Asymptomatic Infection (AI) [DTH+/++++, IFAT−], 2) Subclinical Resistant Infection (SRI) [DTH+/++++, IFAT+/++], and 3) Indeterminate Initial Infection (III) [DTH−, IFAT+/++], and two symptomatic ones, 4) Symptomatic Infection (SI) [=American visceral leishmaniasis - AVL] and, 5) Subclinical Oligosymptomatic Infection (SOI), both with the same immune profile [DTH−, IFAT+++/++++]. Herein, we confirm for the third time the preclinical diagnosis of AVL through IgM-antibody response in an early asymptomatic case of infection (profile III), a 17-year-old boy who evolved to AVL (=profile SI) six weeks after the initial infection diagnosis, confirming that the combined use of DTH and IFAT-(IgG/IgM) assays associated with the clinical evaluation of infected individuals is potentially useful for monitoring human L. (L.) infantum chagasi-infection in endemic areas as well as optimizing AVL control. © 2020