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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- Histological and neuronal changes in the duodenum of hamsters infected with Leishmania (Leishmania) infantum(2022) CAVALLONE, Italo N.; LIMA, Sarah K. S. de; OLIVEIRA, Karine S.; PASSERO, Luiz Felipe D.; LAURENTI, Marcia D.; JESUS, Jessica Adriana; MARINSEK, Gabriela P.; CHUCRI, Thais M.; MARI, Renata B.Visceral leishmaniasis is a neglected tropical disease caused by parasites belonging to the Leishmania genus that infect macrophages in different tissues such as the spleen, liver, lymph nodes, bone marrow, and intestine. Therefore, this study aimed to investigate the integrity of the intestinal tract and the nitrergic (NADPH-dp) and metabolically active (NADH-dp) myenteric neurons of the duodenum of golden hamsters infected with L. (L.) infantum. Therefore, thirty golden hamsters were divided into six groups (n = 5); three of them were infected with 2 x 107 promastigote forms of L. (L.) infantum by intraperitoneal route (Infected Group - IG) and three were inoculated with saline solution (control group - CG). After 30, 60 and 90 days post-infection (DPI) infected animals were euthanized and the liver, spleen and duodenum were collected to analyze tissue parasitism. The duodenum was processed using usual histological techniques to analyze the main changes that occurred during infection and histochemical techniques to phenotype myenteric neurons. Amastigote forms were observed in the spleen, liver, and duodenum during all experimental periods, and tissue parasitism in these organs increased significantly over time. At 30 DPI, reduction in muscle tunic, increase in the total intestinal wall and the number of goblet cells PAS+ was observed. At 60 DPI, an increase in intestinal crypts and intraepithelial lymphocytes was observed, and a reduction in intestinal villi was observed at 90 DPI, along with an increase in crypt size. Regarding neurons, an increase in the density of the NADPH-dp population was observed at 30 DPI, but at 60 and 90 DPI a significant reduction of this population was observed. In general, infection progression was observed to cause significant morphofunctional changes in the duodenum of infected hamsters.
- The antifungal compound butenafine eliminates promastigote and amastigote forms of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis(2016) BEZERRA-SOUZA, Adriana; YAMAMOTO, Eduardo S.; LAURENTI, Marcia D.; RIBEIRO, Susan P.; PASSERO, Luiz Felipe D.The production of ergosterol lipid, important for the Leishmania membrane homeostasis, involves different enzymes. This pathway can be blocked to azoles and allylamines drugs, such as Butenafine. The aim of the present work was to evaluate the anti-leishmanicidal activity of this drug in 2 major species of Leishmania responsible for causing the American tegumentar leishmaniasis (L (L.) amazonensis and L (V.) braziliensis). Butenafine eliminated promastigote forms of L amazonensis and L braziliensis with efficacy similar to miltefosine, a standard anti-leishmania drug. In addition, butenafine induced alterations in promastigote forms of L amazonensis that resemble programmed cell death. Butenafine as well as miltefosine presented mild toxicity in peritoneal macrophages, however, butenafine was more effective to eliminate intracellular amastigotes of both L amazonensis and L braziliensis, and this effect was not associated with elevated levels of nitric oxide or hydrogen peroxide. Taken together, data presented herein suggests that butenafine can be considered as a prototype drug able to eliminate L amazonensis and L braziliensis, etiological agents of anergic diffuse and mucocutaneous leishmaniasis, respectively.
- Thymic alterations resulting from experimental visceral leishmaniasis in a Syrian hamster (Mesocricetus auratus)(2023) MARCO, Karen Santos; BOREGIO, Jaqueline da Silva; JUSSIANI, Giulia Goncalves; FERREIRA, Laura Flavia Esperanca de Souza; FLORES, Gabriela Venicia Araujo; PACHECO, Carmen Maria Sandoval; LAURENTI, Marcia Dalastra; MACHADO, Gisele FabrinoBackground: The thymus is a lymphoid organ responsible for the development and maturation of T cells, which are part of the Th1, Th2, Th17, and Treg immune responses triggered by visceral leishmaniasis. The maturation and immunological development of T lymphocytes require a bidirectional interaction between the thymic microenvironment of epithelial cells, dendritic cells, and macrophages and the extracellular matrix with differentiating lymphocytes.Objectives: We evaluated the morphological characteristics and tissue distribution of hematopoietic and stromal cells in the thymuses of hamsters experimentally infected with Leishmania infantum, aiming to gain an insight into the pathophysiology of the disease.Methods: Fifteen hamsters were subjected to intraperitoneal experimental infection with 107 L. infantum pro-mastigotes (MHOM/BR/1972/BH46). The animals were divided into three groups, each comprising five infected hamsters, and were then euthanized 15, 60, and 120 days postinfection. The control groups consisted of three groups of five healthy hamsters euthanized simultaneously with the infected ones. Thymic morphology was evaluated through histopathology and the cell composition through immunohistochemistry. We used antibodies to mark mesenchymal cells (anti-vimentin), epithelial cells (anti-cytokeratin), macrophages (anti-MAC387), B lymphocytes (anti-CD79a), and T lymphocytes (anti-CD3). Immunohistochemistry was also used to mark the parasite in the thymus.Results: Infected and control hamsters showed no difference in thymic morphology and degree of atrophy. After 15 days of infection, CD3 + T lymphocytes in the thymus showed an increase that stabilized over time. At 120 days of infection, we detected a significant decrease in CD79a+ B lymphocytes. The parasite was present in the medullary and corticomedullary regions of 9 out of 15 hamsters. These findings confirm that the presence of a parasite can cause changes in a thymus cell population. However, further studies are needed to evaluate these changes' effects on the immune response of infected animals.
- Evaluation of Leishmania (Leishmania) infantum excreted-secreted antigens for detection of canine leishmaniasis(2016) PINEDO-CANCINO, Viviana; LAURENTI, Marcia Dalastra; KESPER, Norival; UMEZAWA, Eufrosina SetsuThe efficacy of tests with L (L.) infantum excreted-secreted antigens (ESA) to detect canine leishmaniasis (CanL) was evaluated using immunoblotting (ESA-blot), ELISA (ESA-ELISA) and ELISA with alkaline extract from promastigotes (PAE). Of one hundred fifty-five domestic dogs tested, 100 were suspected of CanL, 23 had other diseases and 32 were healthy. Sera from the dogs suspected of CanL were tested by immunohistochemistry (IHC), and 54% were confirmed to be infected by L (L.) infantum (38 symptomatic and 16 asymptomatic). Of these, 100% were positive by ESA-blot, ESA-ELISA and PAE-ELISA. In the ESA blot their sera recognized polypeptides in the 26.5-31.5 kDa region. Of the 46% of dogs with negative IHC, 44-53% tested positive in all three tests irrespective of clinical status. The twenty-three dogs with other diseases were negative by ESA-blot, but sera from 9% and 26% of them reacted with ESA-ELISA and PAE-ELISA, respectively. The 32 healthy dogs were negative in all the tests. ESA-blot showed good correlation with IHC in the detection of CanL and a high specificity index.
- Infectious Diseases in Free-Ranging Blonde Capuchins, Sapajus flavius, in Brazil(2017) BUENO, Marina Galvao; CATO-DIAS, Jose Luiz; LAROQUE, Plautino de Oliveira; VASCONCELLOS, Silvio Arruda; FERREIRA NETO, Jose Soares; GENNARI, Solange Maria; FERREIRA, Fernando; LAURENTI, Marcia Dalastra; UMEZAWA, Eufrosina Setsu; KESPER, Norival; KIRCHGATTER, Karin; GUIMARES, Lilian Oliveira; PAVANATO, Heloise Juliao; VALENCA-MONTENEGRO, Monica MafraThe main threats to primates worldwide are the degradation, fragmentation, and loss of their habitats; hunting (especially for bushmeat); and illegal trade. For many species, the most important threat is forest fragmentation, resulting in small populations that are restricted to isolated forest patches. In this situation, primates are particularly vulnerable to disease. The Endangered blonde capuchin (Sapajus flavius) is now restricted to a few forest patches in Northeast Brazil. We investigated the occurrence of parasites and bacterial diseases in one of three free-ranging groups of S. flavius in a small forest patch in Paraiba state, Northeast Brazil. We tested for antibodies against Leishmania spp., Trypanosoma cruzi, Toxoplasma gondii, Leptospira spp. (24 strains), and Brucella spp.. We used molecular analysis to detect Plasmodium spp., and evaluated blood smears for the presence of hemoparasites. All individuals tested negative for Leptospira spp. and B. abortus, but 8 of 48 (16%) presented antibodies for both Leishmania spp. and T. cruzi. We identified antibodies to T. gondii in 12% of the individuals tested. Plasmodium brasilianum infection was present in 4% of the individuals tested, and blood smears showed microfilariae parasites in 46% of the individuals tested. The occurrence of these infectious diseases in S. flavius may pose a significant threat in terms of reduced recruitment and poor survival rates, and an understanding of the influence of pathogens is crucial for the management of small populations of primates.
- In situ study of cellular immune response in human cutaneous lesions caused by Leishmania (Viannia) panamensis in Panama(2021) GONZALEZ, Kadir; CALZADA, Jose Eduardo; TOMOKANE, Thaise Yumie; PACHECO, Carmen Maria Sandoval; FLORES, Gabriela Venicia Araujo; GOMES, Claudia Maria Castro; CORBETT, Carlos Eduardo Pereira; SALDANA, Azael; LAURENTI, Marcia DalastraAims: Leishmaniasis is considered a disease with multiple clinical/immunopathological characteristics, depending on the immunity of the host and the species of the parasite. In Panama, the most prevalent species that causes localized cutaneous leishmaniasis (LCL) is Leishmania (Viannia) panamensis, and its immune response is poorly studied. Therefore, we evaluated by immunohistochemistry, the in situ immune response during this infection. Methods and Results: Biopsies from Panamanian patients with LCL were collected and processed by histological techniques. Infection by L. (V.) panamensis was demonstrated by isolation in culture and molecular characterization by Hsp70-RFLP. The in situ immune response was assessed by immunohistochemistry. The immune response was characterized by predominance of T cells, mainly CD8 cells that showed positive correlation with IFN-gamma and Granzyme B. CD4 cells presented positive correlation with both IFN-gamma and IL-13, pointed by mixed cellular immune response. Regulatory response was characterized by FoxP3 cells, which showed positive correlation to IL-10 but not with TGF-beta. Conclusions: L. (V.) panamensis infection triggers a mixed cellular immune response, characterized by the presence of pro-inflammatory, anti-inflammatory and regulatory elements in the skin lesion of Panamanian patients. These data contribute to a better understanding of the immunopathogenesis of Leishmania Viannia infection in Panama.
- Value of the oral swab for the molecular diagnosis of dogs in different stages of infection with Leishmania infantum(2016) ASCHAR, Mariana; OLIVEIRA, Eveline Tozzi Braga de; LAURENTI, Marcia Dalastra; MARCONDES, Mary; TOLEZANO, Jose Eduardo; HIRAMOTO, Roberto Mitsuyoshi; CORBETT, Carlos Eduardo P.; MATTA, Vania Lucia Ribeiro daThis study was based on the need to employ a sensitive and specific method with samples that could be easily collected for diagnosing dogs infected with Leishmania infantum. To this end, we used real time-PCR (qPCR) to assess the value of the oral swab (OS) in detecting infected sick dogs (SD; n = 62), including, for the first time, the analysis of apparently healthy infected dogs (AD; n =30), both from endemic areas for visceral leishmaniasis (VL). For comparison, we also evaluated the performance of the conjunctival swab (CS), blood (BL), lymph node (LN) and serology. We detected the presence of Leishmania DNA in the oral cavity in 62 out of the 92 dogs studied. The OS positivity (67.4%) was equivalent to the CS (68.5%) (p > 0.05), higher than BL (52.2%) (p <= 0.05), and lower than LN (84.8%) (p <= 0.05). OS and CS performed well in SD dogs (82.3% and 83.9%, respectively) but not in AD dogs (36.7% for both samples). BL showed the lowest positivity (52.2%) and provided equivalent results between AD (60.0%) and SD (48.4%) dogs (p > 0.05). LN yielded the highest positivity (84.8%), and it was also higher in the SD population (93.5%) compared to the AD population (66.7%) (p <= 0.05). Parasite load was high in LN, moderate in OS and CS, and low in BL, showing the relationship between the levels of parasitism and the positivity rates found in these samples. Serology was positive in 82.2% of the SD group and in 70% of the AD dogs (p > 0.05). Among the 20 seronegative dogs, seven (35%) were positive in either OS or CS, and 12 (60%) were positive when both noninvasive samples were jointly considered. The OS/CS combination resulted in a significant increase of positivity (p <= 0.05) for the AD dogs (from 36.7% to 63.4%), as well as OS/serology (80%) and OS/CS/serology (83.4%). For the SD population, positivity reached up to 95.2% with the same combinations, showing that combination of samples and/or tests is required for the identification of dogs infected with L. infantum and that the OS and CS combination based on qPCR notably improves the detection of both AD and SD dogs. In conclusion, OS proved to be a suitable sample for the molecular diagnosis of infected dogs with clinical signs of VL, but not for dogs with inapparent infection. For these, we recommend the combination of OS results with CS and/or serology in order to reach relevant positivity for L infantum. Finally, another advantage of using OS or both noninvasive samples is the increased likelihood of diagnosing seronegative dogs.
- Salivary gland homogenates from wild-caught sand flies Lutzomyia flaviscutellata and Lutzomyia (Psychodopygus) complexus showed inhibitory effects on Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis infection in BALB/c mice(2014) FRANCESQUINI, Fernanda C.; SILVEIRA, Fernando T.; PASSERO, Luiz Felipe D.; TOMOKANE, Thaise Y.; CARVALHO, Ana Kely; CORBETT, Carlos Eduardo P.; LAURENTI, Marcia D.During the natural transmission of Leishmania parasites, the infected sand fly female regurgitates promastigotes into the host's skin together with its saliva. It has been reported that vector saliva contains immunomodulatory molecules that facilitate the establishment of infection. Thus, the main objective of this study was to evaluate the specificity of Lutzomyia (Lu.) flaviscutellata and Lu.(Psychodopygus) complexus salivas on the infectivity of Leishmania (L.) (Leishmania) amazonensis and L.(Viannia) braziliensis, respectively. BALB/c mice were inoculated into the skin of hind footpad with L.(L.) amazonensis and L.(V.) braziliensis promastigotes in the absence or presence of Lu.flaviscutellata and Lu.(P.) complexus salivary gland homogenates (SGHs). The evolution of the infection was evaluated by lesion size, histopathological analysis and determination of the parasite load in the skin biopsies collected from the site of infection at 4 and 8weeks PI. The lesion size and the parasite load of both groups of mice infected in the presence of SGHs were smaller than the control groups. The histopathological features showed that the inflammatory reaction was less prominent in the groups of mice infected in the presence of both SGHs when compared to the control group. The results showed that the presence of SGHs of Lu.flaviscutellata and Lu.(P.) complexus led to induction of processes that were disadvantageous to parasite establishment during infection by L.(L.) amazonensis and L.(V.) braziliensis. An inhibitory effect on Leishmania infection could be observed in both groups inoculated with SGHs, especially when the SGH from Lu.(P.) complexus was used.
- Antileishmanial activity and ultrastructural changes of related tetrahydrofuran dineolignans isolated from Saururus cernuus L. (Saururaceae)(2019) BRITO, Juliana R.; PASSERO, Luiz Felipe D.; BEZERRA-SOUZA, Adriana; LAURENTI, Marcia D.; ROMOFF, Paulete; BARBOSA, Henrique; FERREIRA, Edgard A.; LAGO, Joao Henrique G.Objective This work describes the isolation of anti-Leishmania amazonensis metabolites from Saururus cernuus (Saururaceae). Additionally, ultrastructural changes in promastigotes were evidenced by electron microscopy. Methods The MeOH extract from the leaves of S. cernuus was subjected to bioactivity-guided fractionation. Anti-L. amazonensis activity of purified compounds was performed in vitro against promastigote and amastigote forms. Key findings Bioactivity-guided fractionation of the MeOH extract from the leaves of S. cernuus afforded two related tetrahydrofuran dineolignans: threo,threo-manassantin A (1) and threo,erythro-manassantin A (2). Compounds 1 and 2 displayed activity against promastigotes (EC50 of 35.4 +/- 7.7 and 17.6 +/- 4.2 mu m, respectively) and amastigotes (EC50 of 20.4 +/- 1.9 and 16.0 +/- 1.1 mu m, respectively), superior to that determined for the positive control miltefosine (EC50 of 28.7 +/- 3.5 mu m). Reduced cytotoxicity for host cells was observed for both compounds. Additionally, ultrastructural changes in promastigotes leading to an alteration of structural morphology were observed, as evidenced by electron microscopy. Furthermore, these compounds altered the morphology and physiology of the plasmatic membrane of L. amazonensis. Conclusions The obtained results indicated that dineolignans 1 and 2 could be considered as a scaffold for the design of novel and selective drug candidates for the treatment of leishmaniasis.
- Human cutaneous leishmaniasis: interferon-dependent expression of double-stranded RNA-dependent protein kinase (PKR) via TLR2(2011) VIVARINI, Aislan de Carvalho; PEREIRA, Renata de Meirelles Santos; TEIXEIRA, Karina Luiza Dias; CALEGARI-SILVA, Teresa Cristina; BELLIO, Maria; LAURENTI, Marcia Dalastra; CORBETT, Carlos Eduardo Pereira; GOMES, Claudia Maria de Castro; SOARES, Rodrigo Pedro; SILVA, Aristobolo Mendes; SILVEIRA, Fernando Tobias; LOPES, Ulisses GazosWe investigated the type I interferon (IFN-1)/PKR axis in the outcome of the Leishmania (Leishmania) amazonensis infection, along with the underlying mechanisms that trigger and sustain this signaling pathway. Reporter assays of cell extracts from RAW-264.7 macrophages infected with L. (L.) amazonensis or HEK-293T cells cotransfected with TLR2 and PKR promoter constructions were employed. Primary macrophages of TLR2-knockout (KO) or IFNR-KO mice were infected, and the levels of PKR, IFN-1, and superoxide dismutase 1 (SOD1) transcript levels were investigated and compared. Immunohistochemical analysis of human biopsy lesions was evaluated for IFN-1 and PKR-positive cells. Leishmania infection increased the expression of PKR and IFN-beta on induction of PKR-promoter activity. The observed effects required the engagement of TLR2. TLR2-KO macrophages expressed low IFN-beta and PKR levels postinfection with a reduced parasite load. We also revealed the requirement of PKR signaling for Leishmania-induced IFN-1 expression, responsible for sustaining PKR expression and enhancing infection. Moreover, during infection, SOD1 transcripts increased and were also enhanced when IFN-1 was added to the cultures. Remarkably, SOD1 expression was abrogated in infected, dominant-negative PKR-expressing cells. Finally, lesions of patients with anergic diffuse cutaneous leishmaniasis exhibited higher levels of PKR/IFN1-expressing cells compared to those with single cutaneous leishmaniasis. In summary, we demonstrated the mechanisms and relevance of the IFN-1/PKR axis in the Leishmania infection.-De Carvalho Vivarini, A., Pereira, R. M. S., Dias Teixeira, K. L., Calegari-Silva, T. C., Bellio, M., Laurenti, M. D., Corbett, C. E. P., de Castro Gomes, C. M., Soares, R. P., Mendes Silva, A., Silveira, F. T., Lopes, U. G. Human cutaneous leish-maniasis: interferon-dependent expression of double-stranded RNA-kinase (PKR) via TLR2. FASEB J. 25, 4162-4173 (2011). www.fasebj.org