Browsing by Author "Lemos, Denise da Silveira"

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    Analysis using canine peripheral blood for establishing in vitro conditions for monocyte differentiation into macrophages for Leishmania chagasi infection and T-cell subset purification.
    (2013) Viana, Kelvinson Fernandes; Soares, Rodrigo Dian de Oliveira Aguiar; Roatt, Bruno Mendes; Resende, Lucilene Aparecida; Lemos, Denise da Silveira; Oliveira, Rodrigo Corrêa de; Martins Filho, Olindo Assis; Moura, Sandra Aparecida Lima de; Zanini, Marcos Santos; Araújo, Márcio Sobreira Silva; Reis, Alexandre Barbosa; Giunchetti, Rodolfo Cordeiro
    Canine visceral leishmaniasis (CVL) is a parasitic disease endemic in many countries, anddogs present as the major natural reservoir of the parasite, Leishmania chagasi (syn. L.infantum). Biomarkers in the canine immune system is an important technique in thecourse of developing vaccines and treatment strategies against CVL. New methodologiesfor studying the immune response of dogs during Leishmania infection and after receivingvaccines and treatments against CVL would be useful. In this context, we used peripheralblood mononuclear cells (PBMCs) from healthy dogs to evaluate procedures related to (i)establishment of in vitro conditions of monocytes differentiated into macrophages infectedwith L. chagasi and (ii) purification procedures of T-cell subsets (CD4+and CD8+) usingmicrobeads. Our data demonstrated that after 5 days of differentiation, macrophages wereable to induce significant phagocytic and microbicidal activity after L. chagasi infectionand also showed increased frequency of parasitism and a higher parasite load. Although N-acetyl- _-d-glucosaminidase (NAG) levels presented similar levels of macrophage cultureand L. chagasi infection, a progressive decrease in myeloperoxidase (MPO) levels was ahallmark over 5 days of culture. High purity levels (>90%) of CD4 and CD8 T cells wereobtained on a magnetic separation column. We concluded that monocytes differentiatedinto macrophages at 5 days and displayed an intermediate frequency of parasitism andparasite load 72 h after L. chagasi infection. Furthermore, the purification system usingcanine T-lymphocyte subsets obtained after 5 days of monocyte differentiation provedefficient for CD4 or CD8 T-cell purification (≥90%). The in vitro analysis using L. chagasi-infected macrophages and purified T cells presented a prospective methodology that couldbe incorporated in CVL vaccine and treatment studies that aim to analyze the microbicidalpotential induced by specific CD4+and/or CD8+T cells.
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    Antigenicity of a whole parasite vaccine as promising candidate against canine leishmaniasis.
    (2008) Giunchetti, Rodolfo Cordeiro; Reis, Alexandre Barbosa; Lemos, Denise da Silveira; Martins Filho, Olindo Assis; Oliveira, Rodrigo Corrêa de; Bethony, Jeffrey Michael; Vale, André Macedo; Quetz, Josiane da Silva; Bueno, Lilian Lacerda; Silva, João Carlos França da; Nascimento, Evaldo do; Mayrink, Wilson; Fujiwara, Ricardo Toshio
    Human visceral leishmaniasis, one of the most important zoonoses, is caused by the protozoaLeishmania chagasi(syn. L. infantum ) and is present as a fatal disease common in South America and Europe where dogs and wild canids are the main reservoirs. A vaccine against visceral leishmaniasis would be an important tool in the control of this disease in dogs. Although the current strategies for vac-cination against leishmaniasis are based on the use of recombinant antigens, killed vaccines are still attractive in terms of stability of their biochemical composition and antigenicity, cost, and safety. Here we evaluate the immunogenicity of a whole parasite vaccine as a prom-ising candidate against canine leishmaniasis, demonstrated by cellular reactivity, changes in the cellular profile of the peripheral blood and by the differential production of immunoglobulins. Our results showed that immunization elicited mainly a strong cellular reactivity and increase in T-lymphocytes, particularly the subpopulation CD8 + that would be related to the control of tissue parasitism. In addi-tion, a higher production of anti- Leishmania total IgG, characterized by mixed isotypes profile (IgG1 and IgG2), was demonstrated.
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    Association of liposome-encapsulated trivalent antimonial with ascorbic acid: an effective and safe strategy in the treatment of experimental visceral. leishmaniasis.
    (2014) Castro, Renata Alves de Oliveira e; Barcellos, Neila Marcia Silva; Licio, Carolina Souza Andrade; Souza, Janine Braga de; Testasicca, Miriam Conceição de Souza; Ferreira, Flávia Monteiro; Batista, Maurício Azevedo; Lemos, Denise da Silveira; Moura, Sandra Aparecida Lima de; Frezard, Frederic Jean Georges; Rezende, Simone Aparecida
    Visceral leishmaniasis (VL) is a chronic debilitating disease endemic in tropical and subtropical areas, caused by protozoan parasites of the genus Leishmania. Annually, it is estimated the occurrence of 0.2 to 0.4 million new cases of the disease worldwide. Considering the lack of an effective vaccine the afflicted population must rely on both, an accurate diagnosis and successful treatment to combat the disease. Here we propose to evaluate the efficacy of trivalent antimonial encapsulated in conventional liposomes, in association with ascorbic acid, by monitoring its toxicity and efficacy in BALB/c mice infected with Leishmania infantum. Methodology/Principal Findings:: Infected mice were subjected to single-dose treatments consisting in the administration of either free or liposome-encapsulated trivalent antimony (SbIII), in association or not with ascorbic acid. Parasite burden was assessed in the liver, spleen and bone marrow using the serial limiting dilution technique. After treatment, tissue alterations were examined by histopathology of liver, heart and kidney and confirmed by serum levels of classic biomarkers. The phenotypic profile of splenocytes was also investigated by flow cytometry. Treatment with liposome-encapsulated SbIII significantly reduced the parasite burden in the liver, spleen and bone marrow. Co-administration of ascorbic acid, with either free SbIII or its liposomal form, did not interfere with its leishmanicidal activity and promoted reduced toxicity particularly to the kidney and liver tissues. Conclusions/Significance:: Among the evaluated posological regimens treatment of L. infantum-infected mice with liposomal SbIII, in association with ascorbic acid, represented the best alternative as judged by its high leishmanicidal activity and absence of detectable toxic effects. Of particular importance, reduction of parasite burden in the bone marrow attested to the ability of SbIII-carrying liposomes to efficiently reach this body compartment.
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    Association of water extract of green propolis and liposomal meglumine antimoniate in the treatment of experimental visceral leishmaniasis.
    (2014) Ferreira, Flávia Monteiro; Castro, Renata Alves de Oliveira e; Batista, Maurício Azevedo; Rossi, Fernanda Mendes de Oliveira; Lemos, Denise da Silveira; Frezard, Frederic Jean Georges; Moura, Sandra Aparecida Lima de; Rezende, Simone Aparecida
    This work investigated the use of water extract of green propolis (WEP) and its association with free or liposomal meglumine antimoniate (MA) for the treatment of murine visceral leishmaniasis. Mice infected with Leishmania infantum were treated with oral doses of WEP associated or not with a single dose of liposomal MA by intraperitoneal route. Parasite burden was assessed in the liver and spleen by limiting dilution assay, and alterations in the spleen cellular phenotype were evaluated by flow cytometry. Tissue damage was assessed by determination of biochemical markers of the liver, heart, and kidney function and histopathological analysis of the liver and spleen. Our data showed that treatmentwith WEP was able to reduce parasite load in the liver but not in the spleen. On the other hand, liposomal MA reduced parasite load in both organs. Unexpectedly, there was no synergism with the combination of WEP and liposomal MA in reducing the parasite load. The histopathological analysis showed that administration of WEP, liposomal MA, or their association was able to protect the liver and spleen fromlesions caused by infection. No alteration in the profile of spleen cells by flow cytometry or in the liver, heart, and kidney functions by biochemical markers due to any of the treatments was observed. These results demonstrate that althoughWEP was able to significantly reduce the liver parasite load, its association with liposomalMA did not lead to significant improvement in reducing parasite load. On the other hand, treatment with WEP and/or liposomal MA protected the liver and spleen from lesions caused by the infection.
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    Baccharis trimera inhibits reactive oxygen species production through PKC and down-regulation p47phox phosphorylation of NADPH oxidase in SK Hep-1 cells.
    (2017) Araújo, Glaucy Rodrigues de; Rabelo, Ana Carolina Silveira; Meira, Janaína Serenato; Rossoni Júnior, Joamyr Victor; Borges, William de Castro; Cota, Renata Guerra de Sá; Batista, Maurício Azevedo; Lemos, Denise da Silveira; Souza, Gustavo Henrique Bianco de; Brandão, Geraldo Célio; Chaves, Míriam Martins; Costa, Daniela Caldeira
    Baccharis trimera, popularly known as ‘‘carqueja’’, is a native South-American plant possessing a high concentration of polyphenolic compounds and therefore high antioxidant potential. Despite the antioxidant potential described for B. trimera, there are no reports concerning the signaling pathways involved in this process. So, the aim of the present study was to assess the influence of B. trimera on the modulation of PKC signaling pathway and to characterize the effect of the nicotinamide adenine dinucleotide phosphate oxidase enzyme (NOX) on the generation of reactive oxygen species in SK Hep-1 cells. SK-Hep 1 cells were treated with B. trimera, quercetin, or rutin and then stimulated or notwith PMA/ionomycin and labeled with carboxy H2DCFDA for detection of reactive oxygen species by flow cytometer. The PKC expression by Western blot and enzyme activity was performed to evaluate the influence of B. trimera and quercetin on PKC signaling pathway. p47phox and p47phox phosphorylated expression was performed byWestern blot to evaluate the influence of B. trimera on p47phox phosphorylation. The results showed that cells stimulated with PMA/ionomycin (activators of PKC) showed significantly increased reactive oxygen species production, and this production returned to baseline levels after treatment with DPI (NOX inhibitor). Both B. trimera and quercetin modulated reactive oxygen species production through the inhibition of PKC protein expression and enzymatic activity, also with inhibition of p47phox phosphorylation. Taken together, these results suggest that B. trimera has a potentialmechanism for inhibiting reactive oxygen species production through the PKC signaling pathway and inhibition subunit p47phox phosphorylation of nicotinamide adenine dinucleotide phosphate oxidase.
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    Canine visceral leishmaniasis biomarkers and their employment in vaccines.
    (2019) Giunchetti, Rodolfo Cordeiro; Silveira, Patricia; Resende, Lucilene Aparecida; Leite, Jaqueline Costa; Melo Júnior, Otoni Alves de Oliveira; Alves, Marina Luiza Rodrigues; Costa, Laís Moreira; Lair, Daniel Ferreira; Chaves, Vinícius Rossi; Soares, Ingrid dos Santos; Mendonça, Ludmila Zanandreis de; Lanna, Mariana Ferreira; Ribeiro, Helen Silva; Gonçalves, Ana Alice Maia; Santos, Thaiza Aline Pereira; Roatt, Bruno Mendes; Soares, Rodrigo Dian de Oliveira Aguiar; Souza, Juliana Vitoriano de; Moreira, Nádia das Dores; Siqueira, Fernando Augusto Mathias; Cardoso, Jamille Mirelle de Oliveira; Vital, Wendel Coura; Galdino, Alexsandro Sobreira; Viana, Kelvinson Fernandes; Martins Filho, Olindo Assis; Lemos, Denise da Silveira; Dutra, Walderez Ornelas; Reis, Alexandre Barbosa
    The natural history of canine visceral leishmaniasis (CVL) has been well described, particularly with respect to the parasite load in different tissues and immunopathological changes according to the progression of clinical forms. The biomarkers evaluated in these studies provide support for the improvement of the tools used in developing vaccines against CVL. Thus, we describe the major studies using the dog model that supplies the rationale for including different biomarkers (tissue parasitism, histopathology, hematological changes, leucocytes immunophenotyping, cytokines patterns, and in vitro co-culture systems using purified T-cells subsets and macrophages infected with L. infantum) for immunogenicity and protection evaluations in phases I and II applied to pre-clinical and clinical vaccine trials against CVL. The search for biomarkers related to resistance or susceptibility has revealed a mixed cytokine profile with a prominent proinflammatory immune response as relevant for Leishmania replication at low levels as observed in asymptomatic dogs (highlighted by high levels of IFN-γ and TNF-α and decreased levels in IL-4, TGF-β and IL-10). Furthermore, increased levels in CD4+ and CD8+ T-cell subsets, presenting intracytoplasmic proinflammatory cytokine balance, have been associated with a resistance profile against CVL. In contrast, a polyclonal B-cell expansion towards plasma cell differentiation contributes to high antibody production, which is the hallmark of symptomatic dogs associated with high susceptibility in CVL. Finally, the different studies used to analyze biomarkers have been incorporated into vaccine immunogenicity and protection evaluations. Those biomarkers identified as resistance or susceptibility markers in CVL have been used to evaluate the vaccine performance against L. infantum in a kennel trial conducted before the field trial in an area known to be endemic for visceral leishmaniasis. This rationale has been a guiding force in the testing and selection of the best vaccine candidates against CVL and provides a way for the veterinary industry to register commercial immunobiological products.
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    Cellular immunophenotypic profile in the splenic compartment during canine visceral leishmaniasis.
    (2014) Reis, Alexandre Barbosa; Carvalho, Andréa Teixeira de; Giunchetti, Rodolfo Cordeiro; Roatt, Bruno Mendes; Vital, Wendel Coura; Nicolato, Roney Luiz de Carvalho; Lemos, Denise da Silveira; Oliveira, Rodrigo Corrêa de; Martins Filho, Olindo Assis
    To determine the role of the spleen in the pathogenesis of canine visceral leishmaniasis (CVL), we analyzed cellular immunophenotypic profiles of 52 dogs naturally infected with Leishmania infantum, clinically classified as follows: asymptomatic dogs-I (AD-I), seroneg-ative/PCR+; asymptomatic dogs-II (AD-II), seropositive/PCR+; oligosymptomatic dogs (OD) and symptomatic dogs (SD). Seven non-infected dogs (CD) were included as a control group. AD-II presented higher levels of CD8+ T splenocytes and lower TCD4+/TCD8+ ratio in com-parison with CD. OD and SD showed lower percentages of CD21+ as compared with AD-II. All seropositive dogs presented lower levels of CD45RA+ than CD. Regardless of the stimuli used, the proliferation index from splenocytes in vitro was inversely correlated with clini-cal status. After LSA stimulation, there was a higher percentage of specific CD8+ T in AD-II than CD and non-stimulated culture. In contrast, splenocytes from SD under in vitro LSA stimulation induced decreased MHC-II+ expression in comparison with all groups, and non-stimulated culture. In conclusion, the role of CD8+ T splenocytes seems to be important for an effective immunological response, a hallmark of asymptomatic CVL, whereas the pro-nounced loss of MHC-II expression upon LSA stimulation is a biomarker of symptomatic CVL.
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    Clinical forms of canine visceral Leishmaniasis in naturally Leishmania infantum – infected dogs and related myelogram and hemogram changes.
    (2013) Nicolato, Roney Luiz de Carvalho; Abreu, Raquel Trópia de; Roatt, Bruno Mendes; Soares, Rodrigo Dian de Oliveira Aguiar; Reis, Levi Eduardo Soares; Carvalho, Maria das Graças; Carneiro, Cláudia Martins; Giunchetti, Rodolfo Cordeiro; Bouillet, Leoneide Érica Maduro; Lemos, Denise da Silveira; Vital, Wendel Coura; Reis, Alexandre Barbosa
    Hematological analysis has limited applications for disease diagnosis in Leishmania infantum–infected dogs, but it can be very important in evaluating the clinical forms of the disease and in understanding the evolution of canine visceral leishmaniasis (CVL) pathogenesis. Recently, we demonstrated that alterations in leucopoiesis and erythropoiesis are related to clinical status and bone marrow parasite density in dogs naturally infected by L. infantum. To further characterize these alterations, we evaluated the association between the hematological parameters in bone marrow and peripheral blood alterations in groups of L. infantum–infected dogs: asymptomatic I (AD-I: serum negative/PCR+), asymptomatic II (AD-II: serum positive), oligosymptomatic (OD), and symptomatic (SD). Results were compared with those from noninfected dogs (NID). The SD group was found to present a decrease in erythropoietic lineage with concomitant reductions in erythrocytes, hemoglobin, and hematocrit parameters, resulting in anemia. The SD group also had increased neutrophils and precursors and decreased band eosinophils and eosinophils, leading to peripheral blood leucopenia. In the AD-II group, lymphocytosis occurred in both the peripheral blood and the bone marrow compartments. The SD group exhibited lymphocytosis in the bone marrow, with lymphopenia in the peripheral blood. In contrast, the AD-I group, showed no significant changes suggestive of CVL, presenting normal counts in bone marrow and peripheral blood. Our results showed for the first time that important changes in hematopoiesis and hematological parameters occur during ongoing CVL in naturally infected dogs, mainly in symptomatic disease. Taken together, our results based on myelogram and hemogram parameters enable better understanding of the pathogenesis of the anemia, lymphocytosis, and lymphopenia, as well as the leucopenia (eosinopenia and monocytopenia), that contribute to CVL prognosis.
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    Cytokine and nitric oxide patterns in dogs immunized with LBSap vaccine, before and after experimental challenge with Leishmania chagasi plus saliva of Lutzomyia longipalpis.
    (2013) Resende, Lucilene Aparecida; Roatt, Bruno Mendes; Soares, Rodrigo Dian de Oliveira Aguiar; Viana, Kelvinson Fernandes; Mendonça, Ludmila Zanandreis de; Lanna, Mariana Ferreira; Lemos, Denise da Silveira; Oliveira, Rodrigo Corrêa de; Martins Filho, Olindo Assis; Fujiwara, Ricardo Toshio; Carneiro, Cláudia Martins; Reis, Alexandre Barbosa; Giunchetti, Rodolfo Cordeiro
    In the studies presented here, dogs were vaccinated against Leishmania (Leishmania) cha-gasi challenge infection using a preparation of Leishmania braziliensis promastigote proteinsand saponin as adjuvant (LBSap). Vaccination with LBSap induced a prominent type 1immune response that was characterized by increased levels of interleukin (IL-) 12 andinterferon gamma (IFN- _) production by peripheral blood mononuclear cells (PBMC) uponstimulation with soluble vaccine antigen. Importantly, results showed that this type ofresponsiveness was sustained after challenge infection; at day 90 and 885 after L. chagasichallenge infection, PBMCs from LBSap vaccinated dogs produced more IL-12, IFN- _ andconcomitant nitric oxide (NO) when stimulated with Leishmania antigens as comparedto PBMCs from respective control groups (saponin, LB- treated, or non-treated controldogs). Moreover, transforming growth factor (TGF)- _ decreased in the supernatant of SLcA-stimulated PBMCs in the LBSap group at 90 days. Bone marrow parasitological analysisrevealed decreased frequency of parasitism in the presence of vaccine antigen. It is con-cluded that vaccination of dogs with LBSap vaccine induced a long-lasting type 1 immuneresponse against L. chagasi challenge infection.
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    Empreendedorismo universitário.
    (2019) Freitas, Alan Ferreira de; Galdino, Alexsandro Sobreira; Cunha, Bernardo Gonçalves da; Toledo, Cristiane Bittencourt Barroso; Gonçalves, Daniel Bonoto; Carvalho, Dárlinton Barbosa Feres; Lemos, Denise da Silveira; Pereira, Eduardo Bento; Soares, Eduardo Emrich; Colares, Heloísa Carneiro; Magalhães, Juliana Teixeira de; Nogueira, Lais Moreira; Lunardi, Laura Martuscelli; Felicori, Liza; Oliveira, Lucilene Aparecida Resende; Speziali, Marcelo Gomes; Paz, Mariana Campos da; Mohallem, Nelcy Della Santina; Silveria, Patricia; Granjeiro, Paulo Afonso; Mariano, Reysla; Oliveira, Rita de Cássia; Giunchetti, Rodolfo Cordeiro; Bianchi, Rodrigo Fernando; Lomeo, Rosangela da Silva; Oliveira, Sabrina Feliciano; Paiva, Sofia Larissa da Costa; Monteiro, Soraia A.; Silva, Tuânia Natacha Lopes
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    Evaluation of a prototype flow cytometry test for serodiagnosis of canine visceral leishmaniasis.
    (2013) Ker, Henrique Gama; Vital, Wendel Coura; Soares, Rodrigo Dian de Oliveira Aguiar; Roatt, Bruno Mendes; Moreira, Nádia das Dores; Carneiro, Cláudia Martins; Machado, Evandro Marques de Menezes; Carvalho, Andréa Teixeira de; Martins Filho, Olindo Assis; Giunchetti, Rodolfo Cordeiro; Araújo, Márcio Sobreira Silva; Coelho, Eduardo Antônio Ferraz; Lemos, Denise da Silveira; Reis, Alexandre Barbosa
    Canine visceral leishmaniasis (CVL) is considered one of the most important canine protozoan diseases of zoonotic concern (1). Various species of Phlebotomus and Lutzomyia sandflies are the potential vectors for the pathogenic agent Leishmania infantum (2). In some European, Asian, and African countries and in America, infection in dogs is associated with a risk of human disease (3–5). In Brazil, the Ministry of Health, through the Visceral Leishmaniasis Control and Surveillance Program (VLCSP), has instituted specific measures to reduce morbidity and case fatality rates, including treating human cases, instituting vector control, and, an action that is unique in the world, sacrificing all seropositive/infected dogs and prohibiting the treatment of CVL (6). During the last decade, the criteria for eliminating infected animals were based on enzyme-linked immunosorbent assays (ELISAs) for screening and indirect immunofluorescence antibody tests (IFATs) for the confirmatory diagnosis of CVL (6, 7). That these tests may lead to false-positive results due to crossreactivity with other parasitic diseases is well known (8, 9). Recently, this approach was modified, and testing is now based on a dual-path platform (DPP) for screening and an ELISA for confirmation (10). However, Grimaldi et al. (11) evaluated the DPP test for the serodiagnosis of CVL and showed that it does not perform well in detecting asymptomatic dogs from areas where canine disease is endemic. It has been shown that vaccination with Leishmune may lead to seroconversion in healthy dogs (10). The vaccination of dogs has increasingly become a common practice in areas in Brazil where CVL is endemic; recently, in addition to the Leishmune vaccine, the Leish-Tec vaccine has become available commercially, and new candidates, such as the LBSap vaccine, are being studied (12– 15). In this sense, seroconversion has become an important problem for surveillance/control programs that employ conventional methodologies in their seroepidemiological surveys, because it can lead to the unnecessary euthanasia of healthy dogs. Nevertheless, the role of vaccination in the diagnosis of CVL still has not been studied sufficiently.
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    Higher expression of CCL2, CCL4, CCL5, CCL21, and CXCL8 chemokines in the skin associated with parasite density in canine Visceral Leishmaniasis.
    (2012) Souza, Daniel Menezes; Cota, Renata Guerra de Sá; Carneiro, Cláudia Martins; Souza, Juliana Vitoriano de; Giunchetti, Rodolfo Cordeiro; Carvalho, Andréa Teixeira de; Lemos, Denise da Silveira; Oliveira, Guilherme Corrêa de; Oliveira, Rodrigo Corrêa de; Reis, Alexandre Barbosa
    Background: The immune response in the skin of dogs infected with Leishmania infantum is poorly understood, and limited studies have described the immunopathological profile with regard to distinct levels of tissue parasitism and the clinical progression of canine visceral leishmaniasis (CVL). Methodology/Principal Findings: A detailed analysis of inflammatory cells (neutrophils, eosinophils, mast cells, lymphocytes, and macrophages) as well as the expression of chemokines (CCL2, CCL4, CCL5, CCL13, CCL17, CCL21, CCL24, and CXCL8) was carried out in dermis skin samples from 35 dogs that were naturally infected with L. infantum. The analysis was based on real-time polymerase chain reaction (PCR) in the context of skin parasitism and the clinical status of CVL. We demonstrated increased inflammatory infiltrate composed mainly of mononuclear cells in the skin of animals with severe forms of CVL and high parasite density. Analysis of the inflammatory cell profile of the skin revealed an increase in the number of macrophages and reductions in lymphocytes, eosinophils, and mast cells that correlated with clinical progression of the disease. Additionally, enhanced parasite density was correlated with an increase in macrophages and decreases in eosinophils and mast cells. The chemokine mRNA expression demonstrated that enhanced parasite density was positively correlated with the expression of CCL2, CCL4, CCL5, CCL21, and CXCL8. In contrast, there was a negative correlation between parasite density and CCL24 expression. Conclusions/Significance: These findings represent an advance in the knowledge about skin inflammatory infiltrates in CVL and the systemic consequences. Additionally, the findings may contribute to the design of new and more efficient prophylactic tools and immunological therapies against CVL.
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    In vitro Infectivity of strains isolated from dogs naturally infected with Leishmania infantum present a distinct pathogenic profile in hamsters.
    (2020) Resende, Lucilene Aparecida; Soares, Rodrigo Dian de Oliveira Aguiar; Moreira, Nádia das Dores; Ferreira, Sidney de Almeida; Lanna, Mariana Ferreira; Cardoso, Jamille Mirelle de Oliveira; Mathias, Fernando Augusto Siqueira; Vital, Wendel Coura; Mariano, Reysla Maria da Silveira; Leite, Jaqueline Costa; Silveira, Patricia; Carvalho, Tatiane Furtado de; Santos, Renato Lima; Lemos, Denise da Silveira; Martins Filho, Olindo Assis; Dutra, Walderez Ornelas; Reis, Alexandre Barbosa; Giunchetti, Rodolfo Cordeiro
    Visceral leishmaniasis (VL) is a severe disease caused by Leishmania infantum. Dogs are the parasite’s main reservoir, favoring its transmission in the urban environment. The analysis of L. infantum from infected dogs contributes to the identification of more virulent parasites, thereby supporting basic and applied studies such as vaccinal and therapeutic strategies. We proposed the in vitro and in vivo characterization of L. infantum strains from naturally infected dogs from a VL endemic area based on an infectivity and pathogenicity analysis. DH82 canine macrophages were infected in vitro with different strains for infectivity analysis, showing distinct infectivity profiles. The strains that showed greater and lesser infectivity using in vitro analyses (616 and 614, respectively) were used to infect hamsters for pathogenicity analysis. The group infected with strain 616 showed 100% survival while the group infected with strain 614 showed 50% after seven months of follow up. Furthermore, the 614 strain induced more noticeable clinicopathological changes and biochemical abnormalities in liver function, along with high inflammation and parasite load in the liver and spleen. We confirmed high variability of infectivity and pathogenicity in L. infantum strains from infected dogs. The results support the belief that screening for L. infantum infectivity using in vitro experiments is inadequate when it comes to selecting the most pathogenic strain.
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    Kinetics of phenotypic and functional changes in mouse models of sponge implants : rational selection to optimize protocols for specific biomolecules screening purposes.
    (2020) Lanna, Mariana Ferreira; Resende, Lucilene Aparecida; Soares, Rodrigo Dian de Oliveira Aguiar; Miranda, Marina Barcelos de; Mendonça, Ludmila Zanandreis de; Melo Júnior, Otoni Alves de Oliveira; Mariano, Reysla Maria da Silveira; Leite, Jaqueline Costa; Silveira, Patricia; Oliveira, Rodrigo Corrêa de; Dutra, Walderez Ornelas; Reis, Alexandre Barbosa; Martins Filho, Olindo Assis; Moura, Sandra Aparecida Lima de; Lemos, Denise da Silveira; Giunchetti, Rodolfo Cordeiro
    The sponge implant has been applied as an important in vivo model for the study of inflammatory processes as it induces the migration, proliferation, and accumulation of inflammatory cells, angiogenesis, and extracellular matrix deposition in its trabeculae. The characterization of immune events in sponge implants would be useful in identifying the immunological events that could support the selection of an appropriate experimental model (mouse strain) and time post-implant analysis in optimized protocols for novel applications of this model such as in biomolecules screening. Here, the changes in histological/morphometric, immunophenotypic and functional features of infiltrating leukocytes (LEU) were assessed in sponge implants for Swiss, BALB/c, and C57BL/6 mice. A gradual increase of fibrovascular stroma and a progressive decrease in LEU infiltration, mainly composed of polymorphonuclear cells with progressive shift toward mononuclear cells at late time-points were observed over time. Usually, Swiss mice presented a more prominent immune response with late mixed pattern (pro-inflammatory/anti-inflammatory: IL-2/IFN-γ/IL-4/IL-10/IL-17) of cytokine production. While BALB/c mice showed an early activation of the innate response with a controlled cytokine profile (low inflammatory potential), C57BL/6 mice presented a typical early pro-inflammatory (IL-6/TNF/IFN-γ) response with persistent neutrophilic involvement. A rational selection of the ideal time-point/mouse-lineage would avoid bias or tendentious results. Criteria such as low number of increased biomarkers, no recruitment of cytotoxic response, minor cytokine production, and lower biomarker connectivity (described as biomarker signature analysis and network analysis) guided the choice of the best time-point for each model (Day5/Swiss; Day7/BALB/c; Day6/C57BL/6) with wide application for screening purposes, such as identification of therapeutic biomolecules, selection of antigens/adjuvants, and follow-up of innate and adaptive immune response to vaccines candidates.
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    Multicomponent LBSap vaccine displays immunological and parasitological profiles similar to those of Leish-Tec® and Leishmune® vaccines against visceral leishmaniasis.
    (2016) Mendonça, Ludmila Zanandreis de; Resende, Lucilene Aparecida; Lanna, Mariana Ferreira; Soares, Rodrigo Dian de Oliveira Aguiar; Roatt, Bruno Mendes; Castro, Renata Alves de Oliveira e; Batista, Maurício Azevedo; Lemos, Denise da Silveira; Estanislau, Juliana de Assis Silva Gomes; Fujiwara, Ricardo Toshio; Rezende, Simone Aparecida; Martins Filho, Olindo Assis; Oliveira, Rodrigo Corrêa de; Dutra, Walderez Ornelas; Reis, Alexandre Barbosa; Giunchetti, Rodolfo Cordeiro
    Background: In past years, many researchers have sought canine visceral leishmaniasis (CVL) prevention through the characterization of Leishmania antigens as vaccine candidates. Despite these efforts, there is still no efficient vaccine for CVL control. Methods: In the present study, we performed a pre-clinical vaccine trial using BALB/c mice to compare the effects of the multicomponent LBSap vaccine with those of Leish-Tec® and Leishmune®. Blood was collected to determine the frequency of peripheral blood cells and to evaluate hematologic and immunophenotypic parameters. Liver and spleen samples were collected for parasitological quantification, and spleen samples were used to access the cytokine profile. Results: When measuring total IgG and IgG1 anti-Leishmania levels after the third vaccination and L. infantum challenge, it was evident that all vaccines were able to induce humoral immune response. Regarding the innate immune response, increased levels of NK CD3-CD49+ cells were the hallmark of all vaccinated groups, whereas only the Leish-Tec® group displayed a high frequency of CD14+ monocytes after L. infantum challenge. Moreover, CD3+CD4+ T cells were the main circulating lymphocytes induced after L. infantum challenge with all evaluated vaccines. Importantly, after L. infantum challenge, splenocytes from the Leishmune® vaccine produced high levels of IL-2, whereas a prominent type 1 immune response was the hallmark of the LBSap vaccine, which presented high levels of IL-2, IL-6, TNF-α, and IFN-γ. The efficacy analysis using real-time polymerase chain reaction demonstrated a reduction in the parasitism in the spleen (Leishmune®: 64 %; LBSap: 42 %; and Leish-Tec®: 36 %) and liver (Leishmune®: 71 %; LBSap: 62 %; and Leish-Tec®: 48 %). Conclusions: The dataset led to the conclusion that the LBSap vaccination was able to induce immune and efficacy profiles comparable with those of commercial vaccines, thus demonstrating its potential as a promising vaccine candidate for visceral leishmaniasis control.
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    Protective effect of ions against cell death induced by acid stress in Saccharomyces.
    (2009) Sant'Ana, Gilzeane dos Santos; Paes, Lisvane da Silva; Paiva, Argentino F. Vieira; Fietto, Luciano Gomes; Tótola, Antônio Helvécio; Trópia, Maria José Magalhães; Lemos, Denise da Silveira; Lucas, Cândida Manuel Ribeiro Simões; Fietto, Juliana Lopes Rangel; Brandão, Rogélio Lopes; Castro, Ieso de Miranda
    Saccharomyces boulardii is a probiotic used to prevent or treat antibiotic-induced gastrointestinal disorders and acute enteritis. For probiotics to be effective they must first be able to survive the harsh gastrointestinal environment. In this work, we show that S. boulardii displayed the greatest tolerance to simulated gastric environments compared with several Saccharomyces cerevisiae strains tested. Under these conditions, a pH 2.0 was the main factor responsible for decreased cell viability. Importantly, the addition of low concentrations of sodium chloride (NaCl) protected cells in acidic conditions more effectively than other salts. In the absence of S. boulardii mutants, the protective effects of Na1 in yeast viability in acidic conditions was tested using S. cerevisiae Na1-ATPases (ena1-4), Na1/H1 antiporter (nha1D) and Na1/H1 antiporter prevacuolar (nhx1D) null mutants, respectively. Moreover, we provide evidence suggesting that this protection is determined by the plasma membrane potential, once altered by low pH and low NaCl concentrations. Additionally, the absence or low expression/activity of Ena proteins seems to be closely related to the basal membrane potential of the cells.