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Item Vaccination with formulation of nanoparticles loaded with Leishmania amazonensis antigens confers protection against experimental visceral leishmaniasis in hamster.(2023) Cabrera González, Marco Antonio; Gonçalves, Ana Alice Maia; Ottino, Jennifer; Leite, Jaqueline Costa; Resende, Lucilene Aparecida; Melo Júnior, Otoni Alves de Oliveira; Silveira, Patricia; Cardoso, Mariana Santos; Fujiwara, Ricardo Toshio; Bueno, Lilian Lacerda; Santos, Renato Lima; Carvalho, Tatiane Furtado de; Garcia, Giani Martins; Paes, Paulo Ricardo de Oliveira; Galdino, Alexsandro Sobreira; Chávez Fumagalli, Miguel Angel; Melo, Marilia Martins; Lemos, Denise da Silveira; Martins Filho, Olindo Assis; Dutra, Walderez Ornelas; Mosqueira, Vanessa Carla Furtado; Giunchetti, Rodolfo CordeiroVisceral leishmaniasis (VL) is a fatal disease caused by the protozoa Leishmania infantum for which dogs are the main reservoirs. A vaccine against canine visceral leishmaniasis (CVL) could be an important tool in the control of human and CVL by reducing the infection pressure of L. infantum. Despite the CVL vaccine available on the market, the Brazilian Ministry of Health did not implement the use of it in their control programs. In this sense, there is an urgent need to develop more efficient vaccines. In this study, the association between two polymeric nanoformulations, (poly (D, L-lactic) acid (PLA) polymer) loading Leishmania amazonensis antigens, was evaluated as a potential immunobiological agent against VL using golden hamsters as an experimental model. The results indicated that no significant adverse reactions were observed in animals vaccinated with LAPSmP. LAPSmP presented similar levels of total anti-Leishmania IgG as compared to LAPSmG. The LAPSmP and LAPSmG groups showed an intense reduction in liver and spleen parasitic load by qPCR. The LAPSmP and LAPSmG vaccines showed exceptional results, indicating that they may be promising candidates as a VL vaccine.Item Nanoformulations with Leishmania braziliensis antigens triggered controlled parasite burden in vaccinated golden hamster (Mesocricetus auratus) against visceral leishmaniasis.(2022) Ottino, Jennifer; Leite, Jaqueline Costa; Melo Júnior, Otoni Alves de Oliveira; Cabrera González, Marco Antonio; Carvalho, Tatiane Furtado de; Garcia, Giani Martins; Batista, Maurício Azevedo; Silveira, Patrícia; Cardoso, Mariana Santos; Bueno, Lilian Lacerda; Fujiwara, Ricardo Toshio; Santos, Renato Lima; Paes, Paulo Ricardo de Oliveira; Lemos, Denise da Silveira; Martins Filho, Olindo Assis; Galdino, Alexsandro Sobreira; Chávez Fumagalli, Miguel Angel; Dutra, Walderez Ornelas; Mosqueira, Vanessa Carla Furtado; Giunchetti, Rodolfo CordeiroLeishmaniasis is a widespread vector-borne disease in Brazil, with Leishmania (Leishmania) infantum as the primary etiological agent of visceral leishmaniasis (VL). Dogs are considered the main reservoir of this parasite, whose treatment in Brazil is restricted to the use of veterinary medicines, which do not promote a parasitological cure. Therefore, efficient vaccine development is the best approach to Canine Visceral Leishmaniasis (CVL) control. With this in mind, this study used hamsters (Mesocricetus auratus) as an experimental model in an anti-Leishmania preclinical vaccine trial to evaluate the safety, antigenicity, humoral response, and effects on tissue parasite load. Two novel formulations of nanoparticles made from poly(D, L-lactic) acid (PLA) polymer loading Leishmania braziliensis crude antigen (LB) exhibiting two different particle sizes were utilized: LBPSmG (570 nm) and LBPSmP (388 nm). The results showed that the nanoparticles were safe and harmless to hamsters and were antigenic with the induction in LBSap, LBPSmG, and LBPSmG groups of total anti-Leishmania IgG antibodies 30 days after challenge, which persists 200 days in LBSap and LBPSmP. At the same time, a less pronounced hepatosplenomegaly in LBSap, LBPSmG, and LBPSmP was found when compared to control groups, as well as a less pronounced inflammatory infiltrate and granuloma formation in the spleen. Furthermore, significant reductions of 84%, 81%, and 90% were observed in spleen parasite burden accessed by qPCR in the LBSap, LBPSmG, and LBPSmP groups, respectively. In this way, LBSap, LBPSmG, and LBPSmP formulations showed better results in vaccinated and L. infantum-challenged animals in further reducing parasitic load in the spleen and attenuating lesions in liver and splenic tissues. This results in safe, harmless nanoformulation vaccines with significant immunogenic and infection control potential. In addition, animals vaccinated with LBPSmP had an overall reduction in parasite burden in the spleen, indicating that a smaller nanoparticle could be more efficient in targeting antigen-presenting cells.Item Phenotypic features of circulating leucocytes as immunological markers for clinical status and bone marrow parasite density in dogs naturally infected by Leishmania chagasi.(2006) Reis, Alexandre Barbosa; Carvalho, Andréa Teixeira de; Giunchetti, Rodolfo Cordeiro; Guerra, Luanda Liboreiro; Carvalho, Maria das Graças; Mayrink, Wilson; Genaro, Odair; Oliveira, Rodrigo Corrêa de; Martins Filho, Olindo AssisCanine visceral leishmaniasis (CVL) manifests itself as a broad clinical spectrum ranging from asymptomatic infection to patent severe disease. Despite relevant findings suggesting changes on lymphocytes subsets regarding the CVL clinical forms, it still remains to be elucidated whether a distinct phenotypic profile would be correlated with degree of tissue parasite density.Herein, we have assessed the correlation between the clinical status as well as the impact of bone marrow parasite density on the phenotypic profile of peripheral blood leucocytes in 40 Brazilian dogs naturally infected by Leishmania chagasi. Our major findings describe the lower frequency of B cells and monocytes as the most important markers of severe CVL. Our main statistically significant findings reveal that the CD8+ T cell subset reflects most accurately both the clinical status and the overall bone marrow parasite density, as increased levels of CD8+ lymphocytes appeared as the major phenotypic feature of asymptomatic disease and dogs bearing a low parasite load. Moreover, enhanced major histocompatibility complex (MHC)-II density as well as a higher CD45RB/CD45RA expression index seems to represent a key element to control disease morbidity. The association between clinical status, bone marrow parasitism and CD8+ T cells re-emphasizes the role of the T cellmediated immune response in the resistance mechanisms during ongoing CVL. Higher levels of circulating T lymphocytes (both CD4+ and CD8+ T cells) and lower MHC-II expression by peripheral blood lymphocytes seem to be the key for the effective immunological response, a hallmark of asymptomatic CVL.Item Serological screening confirms the re-emergence of canine leishmaniasis in urban and rural areas in Governador Valadares, Vale do Rio Doce, Minas Gerais, Brazil.(2007) Malaquias, Luiz Cosme Cotta; Romualdo, Rodrigo do Carmo; Anjos Junior, José Batista do; Giunchetti, Rodolfo Cordeiro; Oliveira, Rodrigo Corrêa de; Reis, Alexandre BarbosaThis study performed clinical, serological and parasitological assessments in dogs from Vale do Rio Doce, in Minas Gerais State, Brazil, a region considered as a ‘controlled endemic’ area for canine visceral leishmaniosis (CVL). Nevertheless, there are signs that CVL in dogs may be re-emerging as a programme to control the disease was interrupted in the 1990s. The majority of the animals examined presented various symptoms associated with CVL. The enzyme-linked immunosorbent assay test indicated 13.7 and 12.4% of positivity of dogs from the urban and rural areas, respectively. According to indirect immunofluorescence assay test and TRALd tests, 18.2 and 42.2% of dogs in the rural area were seropositive, respectively. Parasitism in seropositive dogs was confirmed by in vitro tissue culture. Sand flies of the genus Lutzomyia, which are able to transmit both cutaneous and visceral leishmaniosis, were found in the area. The results provide a strong evidence of the re-emergence of CVL in this region.Item Histopathological features, parasite density and cell phenotype of the popliteal lymph node in canine visceral leishmaniasis.(2008) Giunchetti, Rodolfo Cordeiro; Martins Filho, Olindo Assis; Carneiro, Cláudia Martins; Mayrink, Wilson; Marques, Marcos José; Tafuri, Washington Luiz; Oliveira, Rodrigo Corrêa de; Reis, Alexandre BarbosaWhile enlargement of popliteal lymph nodes (LN) is frequently described in canine visceral leishmaniasis (CVL), there are few histopathologic studies of lymph nodes during this chronic immunopathological condition.Besides a detailed histopathologic analysis, we have characterized the parasite load andmajor immunophenotypic features of theLNin Leishmania (Leishmania) chagasi-infected dogs. Our major histopathological findings highlight that hypertrophy/hyperplasia of LN cortical and medullary zones was the principal characteristic observed in asymptomatic dogs (AD), whereas atrophy of LN cortical zone was predominant in symptomatic animals (SD). The LN parasite density detected by anti-Leishmania immunohistochemical assay or expressed as Leishman Donovan Units was also highly correlated with the skin parasitism, the most reliable parameter to decode the clinical status of CVL. The major LN immunophenotypic changes during ongoing CVL were an increased frequency of T-lymphocytes, particularly CD8+ T-cells, upregulation of MHC-II expression by lymphocytes and decreased levels of CD21+ B-cells. Our findings further demonstrated that changes in the LNB-lymphocyte compartment exhibited a negative correlation with the skin parasite load. Conversely, we also showed evidence for a positive association between skin parasitismandLNT-cell-mediated immunity, suggesting thatT-cells, especiallyCD8+ lymphocytes, may have a Type-2 immunological profile in this lymphoid tissue in response to CVL.Item Evaluation of the influence of tissue parasite density on hematological and phenotypic cellular parameters of circulating leukocytes and splenocytes during ongoing canine visceral leishmaniasis.(2009) Guerra, Luanda Liboreiro; Carvalho, Andréa Teixeira de; Giunchetti, Rodolfo Cordeiro; Martins Filho, Olindo Assis; Reis, Alexandre Barbosa; Oliveira, Rodrigo Corrêa deDuring Leishmania infection, tissue parasitism at different sites may differ and imply distinct immunopathological patterns during canine visceral leishmaniasis (CVL). For this reason, we have assessed by flow cytometry the impact of spleen and skin parasite density on the phenotypic profile of splenocytes and circulating leukocytes of 40 Brazilian dogs naturally infected by Leishmania chagasi categorized according to splenic and cutaneous parasite load. Our major statistically significant findings demonstrated that dogs with splenic high parasitism presented a significant decrease in absolute counts of CD5+ T lymphocytes in comparison with dogs presenting splenic medium parasitism. Moreover, a decrease in the absolute number of circulating monocytes was observed as a hallmark of high parasitism. The increased frequency of CD8+ T cells is associated with low splenic parasitism during CVL. Although we did not found any significant differences between the immunophenotypic analysis performed in circulating lymphocytes according to cutaneous parasite load, there were negative correlations between CD5+ and CD8+ T cells and cutaneous parasite density reemphasizes the role of T cell-mediated immune response in resistance mechanisms during ongoing CVL. These results add new insights about the pathogenesis of CVL and may help in the establishment of additional tools for future studies on drugs and vaccine approaches.Item Qualitative and quantitative immunohistochemical evaluation of iNOS expression in the spleen of dogs naturally infected with leishmania chagasi.(2011) Santos, Fernando Rocha dos; Vieira, Paula Melo de Abreu; Oliveira, Rodrigo Corrêa de; Giunchetti, Rodolfo Cordeiro; Carneiro, Cláudia Martins; Reis, Alexandre Barbosa; Malaquias, Luiz Cosme CottaNitric oxide (NO), the product of the nitric oxide synthase enzymes has been detected in Leishmania-infected animals. Besides its role on the immunity to infection, the role of NO and the inducible nitric oxide synthase (iNOS) in the pathogenesis of canine visceral leishmaniasis (CVL) is not well understood. This study aimed at evaluating immunohistochemically the iNOS expression in the spleen of dogs naturally infected (ID) with Leishmania (L.) chagasi compared with non-infected dogs (NID). The ID was grouped according to the clinical form and the parasite load. Symptomatic dogs (SD) presented higher parasite load in relation to oligosymptomatic (OD) and asymptomatic (AD). The qualitative expression of iNOS was observed only in ID. SD presented strong and prominent labeling of iNOS, followed by OD and AD. Quantitatively, the results showed that the median expression of iNOS was higher in SD and OD compared to NID. Also, dog spleens with high parasitism load showed marked iNOS expression. Taken together, the results suggest that the expression of iNOS in the spleen of infected dogs with CVL was associated with clinical worsening of the disease and with high parasitism.Item Immunotherapy and immunochemotherapy in visceral leishmaniasis : promising treatments for this neglected disease.(2014) Roatt, Bruno Mendes; Soares, Rodrigo Dian de Oliveira Aguiar; Vital, Wendel Coura; Ker, Henrique Gama; Moreira, Nádia das Dores; Souza, Juliana Vitoriano de; Giunchetti, Rodolfo Cordeiro; Carneiro, Cláudia Martins; Reis, Alexandre BarbosaLeishmaniasis has several clinical forms: self-healing or chronic cutaneous leishmaniasis or post-kala-azar dermal leishmaniasis; mucosal leishmaniasis; visceral leishmaniasis (VL), which is fatal if left untreated.The epidemiology and clinical features of VL vary greatly due to the interaction of multiple factors including parasite strains, vectors, host genetics, and the environment. Human immunodeficiency virus infection augments the severity of VL increasing the risk of developing active disease by 100–2320 times. An effective vaccine for humans is not yet available. Resistance to chemotherapy is a growing problem in many regions, and the costs associated with drug identification and development, make commercial production for leishmaniasis, unattractive.The toxicity of currently drugs, their long treatment course, and limited efficacy are significant concerns. For cutaneous disease, many studies have shown promising results with immunotherapy/immunochemotherapy, aimed to modulate and activate the immune response to obtain a therapeutic cure. Nowadays, the focus of many groups centers on treating canine VL by using vaccines and immunomodulators with or without chemotherapy. In human disease, the use of cytokines like interferon-g associated with pentavalent antimonials demonstrated promising results in patients that did not respond to conventional treatment. In mice, immunomodulation based on monoclonal antibodies to remove endogenous immunosuppressive cytokines (interleukin-10) or block their receptors, antigen-pulsed syngeneic dendritic cells, or biological products like Pam3Cys (TLR ligand) has already been shown as a prospective treatment of the disease. This review addresses VL treatment, particularly immunotherapy and/or immunochemotherapy as an alternative to conventional drug treatment in experimental models, canine VL, and human disease.Item 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 BarbosaCanine 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.Item LBSapSal-vaccinated dogs exhibit increased circulating T-lymphocyte subsets (CD4+ and CD8+) as well as a reduction of parasitism after challenge with Leishmania infantum plus salivary gland of Lutzomyia longipalpis.(2014) Soares, Rodrigo Dian de Oliveira Aguiar; Roatt, Bruno Mendes; Ker, Henrique Gama; Moreira, Nádia das Dores; Mathias, Fernando Augusto Siqueira; Cardoso, Jamille Mirelle de Oliveira; Gontijo, Nelder de Figueiredo; Romero, Oscar Bruna; Carvalho, Andréa Teixeira de; Martins Filho, Olindo Assis; Oliveira, Rodrigo Corrêa de; Giunchetti, Rodolfo Cordeiro; Reis, Alexandre BarbosaBackground: The development of a protective vaccine against canine visceral leishmaniasis (CVL) is an alternative approach for interrupting the domestic cycle of Leishmania infantum. Given the importance of sand fly salivary proteins as potent immunogens obligatorily co-deposited during transmission of Leishmania parasites, their inclusion in an anti-Leishmania vaccine has been investigated in the last few decades. In this context, we previously immunized dogs with a vaccine composed of L. braziliensis antigens plus saponin as the adjuvant and sand fly salivary gland extract (LBSapSal vaccine). This vaccine elicited an increase in both anti-saliva and anti-Leishmania IgG isotypes, higher counts of specific circulating CD8+ T cells, and high NO production. Methods: We investigated the immunogenicity and protective effect of LBSapSal vaccination after intradermal challenge with 1 × 107 late-log-phase L. infantum promastigotes in the presence of sand fly saliva of Lutzomyia longipalpis. The dogs were followed for up to 885 days after challenge. Results: The LBSapSal vaccine presents extensive antigenic diversity with persistent humoral and cellular immune responses, indicating resistance against CVL is triggered by high levels of total IgG and its subtypes (IgG1 and IgG2); expansion of circulating CD5+, CD4+, and CD8+ T lymphocytes and is Leishmania-specific; and reduction of splenic parasite load. Conclusions: These results encourage further study of vaccine strategies addressing Leishmania antigens in combination with proteins present in the saliva of the vector.