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Leishmaniases are diseases caused by several Leishmania species, and many factors contribute to the development of the infection. Because the adaptive immune response does not fully explain the outcome of Leishmania infection and considering that the initial events are crucial in the establishment of the infection, we investigated one of the growth factors, the insulin-like growth factor-I (IGF-I), found in circulation and produced by different cells including macrophages and present in the skin where the parasite is inoculated. Here, we review the role of IGF-I in leishmaniasis experimental models and human patients. IGF-I induces the growth of different Leishmania species in vitro and alters the disease outcome increasing the parasite load and lesion size, especially in L. major- and L. amazonensis-infected mouse leishmaniasis. IGF-I affects the parasite interacting with the IGF-I receptor present on Leishmania. During Leishmania-macrophage interaction, IGF-I acts on the arginine metabolic pathway, resulting in polyamine production both in macrophages and Leishmania. IGF-I and cytokines interact with reciprocal influences on their expression. IL-4 is a hallmark of susceptibility to L. major in murine leishmaniasis, but we observed that IGF-I operates astoundingly as an effector element of the IL-4. Approaching human leishmaniasis, patients with mucosal, disseminated, and visceral diseases presented surprisingly low IGF-I serum levels, suggesting diverse effects than parasite growth. We observed that low IGF-I levels might contribute to the inflammatory response persistence and delayed lesion healing in human cutaneous leishmaniasis and the anemia development in visceral leishmaniasis. We must highlight the complexity of infection revealed depending on the Leishmania species and the parasite’s developmental stages. Because IGF-I exerts pleiotropic effects on the biology of interaction and disease pathogenesis, IGF-I turns up as an attractive tool to explore biological and pathogenic processes underlying infection development. IGF-I pleiotropic effects open further the possibility of approaching IGF-I as a therapeutical target.

A 31-year-old male patient developed an ulcer on the glans penis that evolved for three months without healing. We diagnosed it as leishmaniasis using polymerase chain reaction. No immunosuppression or associated diseases were observed. The patient was treated with meglumine antimoniate that cured the lesion in a month post-treatment. Here, we report this case of cutaneous leishmaniasis lesion at the unusual location of glans penis in an immunocompetent individual. The lesion likely developed due to the bite of a vector, highlighting the need for considering cutaneous leishmaniasis among differential diagnosis of sexually transmitted diseases in areas endemic for leishmaniasis.

Cutaneous leishmaniasis (CL) is a disfiguring disease caused by infection with Leishmania parasites and is characterised by parasitism of the dermis and chronic inflammation. Whilst T cell responses to Leishmania are essential for both parasite clearance and disease resolution they also drive inflammation, and clinical presentation reflects the balance of these opposing activities 1 . Pentavalent antimonials (e.g. sodium stibogluconate; SSG) remain the first line drugs for CL, even though treatment may be protracted and painful. Although evidence from animal models indicates that an effective clinical response to antimonials requires immune-drug synergy 2 , little is known about how this operates in human disease. Here, we studied formalin fixed paraffin embedded (FFPE) skin biopsies from patients in Sri Lanka with CL, at presentation and during intra-lesional SSG treatment. Immune-targeted transcriptomics in a test patient cohort indicated heightened immune checkpoint pathway expression at presentation. We confirmed reduced PD-L1 and IDO1 protein expression on treatment in a second validation cohort, using digital spatial profiling and quantitative immunohistochemistry. PD-L1 and IDO1 expression on CD68 + monocytes / macrophages was positively correlated with the degree of intracellular parasitism, as determined by parasite-specific RNA FISH. Our data support a model whereby the initial anti-leishmanial activity of antimonial drugs alleviates checkpoint inhibition of T cell immunity, thus facilitating immune-drug synergism and clinical cure. We suggest a need to evaluate shorter course SSG treatment and/or the use of checkpoint inhibition as an adjunct host directed therapy (HDT) in CL.

Human visceral leishmaniasis, a parasitic disease of major public health importance in developing countries, is characterized by variable degrees of severity of anemia, but the mechanisms underlying this change in peripheral blood have not been thoroughly explored. Here, we used an experimental model of visceral leishmaniasis in C57BL/6 mice to explore the basis of anemia following infection with Leishmania donovani. 28 days post infection,mice showed bone marrow dyserythropoiesis by myelogram, with a reduction of TER119+ CD71-/+ erythroblasts. Reduction of medullary erythropoiesis coincided with loss of CD169high bone marrow stromal macrophages and a reduction of CXCL12-expressing stromal cells. Although the spleen is a site of extramedullary erythropoiesis and erythrophagocytosis, splenectomy did not impact the extent of anemia or affect the repression of medullary hematopoiesis that was observed in infected mice. In contrast, these changes in bone marrow erythropoiesis were not evident in B6.Rag2 -/- mice, but could be fully reconstituted by adoptive transfer of IFNg-producing but not IFNg-deficient CD4+ T cells, mimicking the expansion of IFNg-producing CD4+ T cells that occurs during infection in wild type mice. Collectively, these data indicate that anemia during experimental murine visceral leishmaniasis can be driven by defects associated with the bone marrow erythropoietic niche, and that this represents a further example of CD4+ T cell-mediated immunopathology affecting hematopoietic competence.

This work was supported by Funda??o de Amparo ? Pesquisa do Estado de S?o Paulo (grant 2015/22075-8 and fellowship 2016/02622-7 to C.M.S.), by CAPES (fellowship to C.M.S.), by Conselho Nacional de Pesquisa (research fellowship to H.G.), and by Laborat?rios de Investiga??o M?dica (LIM-38-HC, FMUSP). This research was also supported by funding from the Bill and Melinda Gates Foundation under grants OPP49932 and OPP1084251 and by grant R01AI025038 from NIAID, National Institutes of Health. Universidade de S?o Paulo. Instituto de Medicina Tropical de S?o Paulo. S?o Paulo, SP, Brazil. Universidade de S?o Paulo. Instituto de Medicina Tropical de S?o Paulo. S?o Paulo, SP, Brazil. Universidade de S?o Paulo. Instituto de Medicina Tropical de S?o Paulo. S?o Paulo, SP, Brazil. Infectious Disease Research Institute. Seattle, WA, USA. Infectious Disease Research Institute. Seattle, WA, USA. Infectious Disease Research Institute. Seattle, WA, USA. Oswaldo Cruz Foundation. Aggeu Magalh?es Research Center. Department of Immunology. Recife, PE, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Funda??o de Medicina Tropical Dr. Heitor Vieira Dourado. Manaus, AM, Brazil. Funda??o de Medicina Tropical Dr. Heitor Vieira Dourado. Manaus, AM, Brazil / Universidade do Estado do Amazonas. Manaus, AM, Brazil. Funda??o de Medicina Tropical Dr. Heitor Vieira Dourado. Manaus, AM, Brazil Funda??o de Medicina Tropical Dr. Heitor Vieira Dourado. Manaus, AM, Brazil Funda??o de Medicina Tropical Dr. Heitor Vieira Dourado. Manaus, AM, Brazil / Universidade Nilton Lins. Manaus, AM, Brazil; Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil / Universidade Federal do Par?. N?cleo de Medicina Tropical. Bel?m, PA, Brazil. Universidade de S?o Paulo. Instituto de Medicina Tropical de S?o Paulo. S?o Paulo, SP, Brazil / Oswaldo Cruz Foundation. Aggeu Magalh?es Research Center. Department of Immunology. Recife, PE, Brazil. Universidade de S?o Paulo. Instituto de Medicina Tropical de S?o Paulo. S?o Paulo, SP, Brazil / Universidade de S?o Paulo. Faculdade de Medicina. S?o Paulo, SP, Brazil. American tegumentary leishmaniasis (ATL) (also known as cutaneous leishmaniasis [CL]) is caused by various species of protozoa of the genus Leishmania. The diagnosis is achieved on a clinical, epidemiological, and pathological basis, supported by positive parasitological exams and demonstration of leishmanin delayedtype hypersensitivity. Serological assays are not routinely used in the diagnosis because many are considered to have low sensitivity and the particular Leishmania species causing the disease can lead to variable performance. In the present study, we generated recombinant versions of two highly conserved Leishmania proteins, Leishmania (Viannia) braziliensis-derived Lb8E and Lb6H, and evaluated both in enzyme-linked immunosorbent assays (ELISA). Recombinant Lb6H (rLb6H) had better performance and reacted with 100.0% of the ATL and 89.4% of the VL samples. These reactions with rLb6H were highly specific (98.5%) when compared against those for samples from healthy control individuals. We then assessed rLb6H against sera from ATL patients infected with different species of Leishmania prevalent in Brazil [Leishmania (Leishmania) amazonensis, L. (Viannia) braziliensis, and L. (V.) guyanensis] and samples from patients with other infectious diseases. In analyses of 500 sera, ELISA using rLb6H detected all 219 ATL samples (sensitivity of 100.0%) with an overall specificity of 93.9% (considering healthy individuals and other infectious diseases patients). Only a minority of samples from Chagas disease patients possessed antibodies against rLb6H, and all of these responses were low (with a highest reactivity index of 2.2). Taken together, our data support further evaluation of rLb6H and the potential for its routine use in the serological diagnosis of ATL.

José Angelo Lauletta Lindoso,1 Jackson Maurício Lopes Costa,2 Igor Thiago Queiroz,3 Hiro Goto41Instituto de Infectologia Emilio Ribas-SES- SP, São Paulo, São Paulo, Brazil; 2Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia, Brazil; 3Departament of Infectious Diseases, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Sao Paulo, Brazil; 4Instituto de Medicina Tropical de São Paulo, and Faculdade de Medicina, Universidade de São Paulo, Sao Paulo, Sao Paulo, BrazilAbstract: Leishmaniases are vector-borne zoonotic diseases that are prevalent in tropical and subtropical areas in the world, with two million new cases occurring yearly. Visceral and tegumentary forms of leishmaniasis are known. The latter form may present as localized cutaneous or mucosal forms, disseminated, diffuse forms, or leishmaniasis recidiva cutis. Visceral leishmaniasis is caused by parasites of the species Leishmania (Leishmania) donovani and L. (L.) infantum, and tegumentary leishmaniasis is caused by 15 other species, with distinct distributions in the Old and New World. The varied clinical manifestations, the multitude of Leishmania species, and the increasing incidence of HIV coinfection make the diagnosis and treatment of leishmaniases complex. Since there are no solid data relating clinical manifestations, treatment outcomes and Leishmania species the decision regarding the best therapeutic option is almost entirely based on clinical manifestations. Because most of the literature is focused on leishmaniasis in the Old World, in this review we present data on the treatment of New World leishmaniasis in more detail. Ranked therapeutic options, clinical trials, and also observations, even with a restricted number of subjects, on treatment outcome of visceral and different forms of tegumentary leishmaniasis, are presented. Treatment for leishmaniasis in HIV-coinfected patients is addressed as well. Some of these data strongly suggest that the differences in the outcome of the treatment are related to the Leishmania species. Therefore, although it is not possible at most points of care to identify the species causing the infection – a process that requires a well equipped laboratory – the infecting species should be identified whenever possible. More recent approaches, such as the use of immunomodulators and immunotherapy, and the lines for development of new candidate drugs are mentioned.Keywords: tegumentary, visceral, therapy, HIV

As Leptospiroses são zoonoses graves de distribuição mundial que afetam o homem e outros animais. A infecção em animais, geralmente, é inaparente, ou os sintomas quando presentes são similares a outras infecções. Neste estudo foram colhidos soros de 119 ovinos e seus respectivos rins durante abate em feiras livres no município de Teresina-Piauí. Pela técnica de soroaglutinação microscópica (SAM) obtiveram-se 34 amostras sorológicas positivas para um ou mais sorovares de Leptospira spp., com taxa de ocorrência de 28,6% de anticorpos anti-leptospiras, sendo 23 casos de infecção para um único sorovar e 11 com coaglutinações para dois ou mais sorovares. Dentre os sorovares patogênicos, o de maior ocorrência foi o Autumnalis (29,4%). A análise histopatológica de 36 fragmentos de rins revelou alterações túbulo intersticiais em 33 (91,7%) animais soro-reagentes. Lesões tubulares foram observadas em 20 (55,5%) animais soro-reagentes. A presença de leptospiras, pela técnica de Warthin Starry, foi observada em 8 (22,20%) amostras positivas. Pela técnica de imunoperixidase, de 20 casos analisados, foi verificada a presença de leptospira em 12 (60%) de 20 amostras positivas. Nos animais soro-reagentes, o infiltrado inflamatório foi significantemente mais evidente na região córtico-medular e cortical do que na região medular (p=0,000), mas não houve diferença entre animais soro-reagentes e soro não-reagentes. Cilindros hialinos nos túbulos proximais estavam presentes em quantidade significantemente maior nos animais soro-reagentes comparados aos não-reagentes (p=0,0001). Em glomérulos, foi observada lesão discreta. Os resultados deste estudo mostram que ovinos soro-reagentes para leptospiras apresentam lesões renais túbulo intersticiais, com presença de leptospiras nos túbulos, o que confere a esses animais a condição de disseminadores da infecção. Lepitospirosis is a serious worldwide distribution disease which affects man and other animals. The infection is generally asymptomatic in animals. In cases whose symptoms are present, symptoms are similar to other infections. In the present study serum samples from 119 sheep and their kidneys were collected during their slaughter in outdoor markets in the city of Teresina, Piauí, Brazil. The Microscopic Agglutination Test (MAT) obtained 34 positive serological samples for one or more Leptospira spp. serovar with occurrence rate of 28.6% of leptospiral antibodies. There were 23 cases of infection for a single serovar, and 11 cases with coagglutination for two or more serovars. Autumnalis had the highest occurrence (29.4%) among the pathogenic serovars. The histopathological analysis of 36 kidney fragments revealed tubulo-interstitial alterations in 33 (91.7%) positive animals. Tubular lesions were observed in 20 (55.5%) positive animals. The Warthin Starry staning revealed the presence of Leptospira in 8 (22.20%) of the 36 positive samples. The immunoperoxidase staining revealed the presence of Leptospira in 12 (60%) of 20 positive samples. The inflammatory infiltrate in the positive animals was significantly more evident in the cortical-medullar and cortical regions than in the medullar region (p=0.000), however, there was no difference between positive and negative animals. The presence of hyaline casts in the proximal tubules was significantly higher in positive animals compared to the negative ones (p=0.0001). Discrete lesion was observed in glomeruli. In conclusion, the results from this study showed that sheep which are positive for Leptospira present tubulo-intersticial renal lesions with the presence of Leptospira in the tubules, conferring to these animals the condition of asymptomatic carriers.

Reactive oxygen species (ROS) production by immunological cells is known to cause damage to pathogens. Increasing evidence accumulated in the last decade has shown, however, that ROS (and redox signals) functionally regulate different cellular pathways in the host-pathogen interaction. These especially affect (i) pathogen entry through protein redox switches and redox modification (i.e., intra- and interdisulfide and cysteine oxidation) and (ii) phagocytic ROS production via Nox family NADPH oxidase enzyme and the control of phagolysosome function with key implications for antigen processing. The protein disulfide isomerase (PDI) family of redox chaperones is closely involved in both processes and is also implicated in protein unfolding and trafficking across the endoplasmic reticulum (ER) and towards the cytosol, a thiol-based redox locus for antigen processing. Here, we summarise examples of the cellular association of host PDI with different pathogens and explore the possible roles of pathogen PDIs in infection. A better understanding of these complex regulatory steps will provide insightful information on the redox role and coevolutional biological process, and assist the development of more specific therapeutic strategies in pathogen-mediated infections.

Acesso restrito: Texto completo. p. 566-568. Submitted by JURANDI DE SOUZA SILVA (jssufba@hotmail.com) on 2012-04-20T12:41:51Z No. of bitstreams: 1 __ac.els-cdn.com_S003592...5da97e17f0cfad0829dec221b8ae9.pdf: 105057 bytes, checksum: 5d1a0868d78012b6c2bb88fc2bc14e99 (MD5) Made available in DSpace on 2012-04-20T12:41:51Z (GMT). No. of bitstreams: 1 __ac.els-cdn.com_S003592...5da97e17f0cfad0829dec221b8ae9.pdf: 105057 bytes, checksum: 5d1a0868d78012b6c2bb88fc2bc14e99 (MD5) Previous issue date: 2010-08 Arginase activity has been related to leishmaniasis development, thus we studied the constitutive and insulin-like growth factor (IGF) I-induced arginase activity of Leishmania (Viannia) braziliensis isolates from patients with different clinical forms of American tegumentary leishmaniasis (ATL). Isolates from mucosal leishmaniasis presented higher basal levels of arginase activity than isolates from other clinical forms of ATL. Isolates from disseminated leishmaniasis that present mucosal lesion in some cases reached the arginase activity similar to that of isolates from mucosal leishmaniasis upon IGF-I stimulation. Differences in arginase activity may influence disease outcomes such as evolution to mucosal lesion in patients with L. (V.) braziliensis infection.

American tegumentary leishmaniasis presents as two major clinical forms: localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL). The immune response in leishmaniasis is efficiently evaluated by the response to Leishmania antigen through the Montenegro skin test (MST). Both LCL and MCL present positive response to MST, indicating that the patients present cell-mediated immunity against the parasite - Leishmania. In spite of the presence of immunity in MCL, this is not sufficient to stop disease progression and prevent resistance to treatment. In this study we demonstrated interleukin (IL) 2, 4, 5 and interferon (IFN) gamma expression in biopsies of MST of ten patients with American tegumentary leishmaniasis. The obtained results were compared between LCL (n = 5) and MCL (n = 5) patients. The MST of MCL patients displayed a higher expression of IL-2, IL-4 and IL-5, in comparison to LCL. There was no significant difference in IFN-gamma expression between groups. The obtained results suggest the role of IL-4 and IL-5 in the maintenance of the immunopathogenic mechanism of the destructive lesions that characterize MCL. A leishmaniose tegumentar americana apresenta duas formas clínicas mais comuns: a leishmaniose cutânea localizada e a leishmaniose cutâneo-mucosa. A imunidade da leishmaniose é avaliada pela resposta ao antígeno Leishmania através da Intradermorreação de Montenegro. Estas duas formas apresentam resposta positiva, indicando que o paciente apresenta imunidade celular contra o parasita Leishmania. Apesar da presença da imunidade celular na leishmaniose cutâneo-mucosa, esta não é suficiente para barrar a progressão da doença e a resistência ao tratamento. Neste estudo, detectamos quatro citocinas por imunohistoquímica, IL-2, IL-4, IL-5 e IFN-gama nas biópsias da intradermorreação de Montenegro de pacientes com leishmaniose tegumentar americana (n = 10), cinco com leishmaniose cutânea e cinco com cutâneo-mucosa. Os resultados mostraram uma alta expressão significativa de IL-2, IL-4, IL-5 na leishmaniose cutâneo-mucosa comparada com a leishmaniose cutânea localizada, mas sem diferença significante na expressão do IFN-γ entre os grupos. Estes resultados sugerem a importância da participação da citocina IL-4 e IL-5 na manutenção do mecanismo imunopatogênico das lesões destrutivas da forma cutâneo-mucosa.

While the control or progression of leishmaniasis depends on host immune responses, the initial inflammatory process represents a key event. This process involves the participation of several cytokines and growth factors induced during inflammation as well as factors already present at the site of infection such as insulin-like growth factor (IGF)-I. We have previously demonstrated a potential role for IGF-I in experimental cutaneous leishmaniasis based on the significant increase in lesion size seen in mice injected with Leishmania promastigotes preactivated with IGF-I. In the present study we show that preactivation of Leishmania (Leishmania) amazonensis promastigotes with IGF-I induces an increase in the actual number of parasites at the lesion site from seven days postinfection, in addition to a more intense inflammatory infiltrate. There was a higher numerical density of polymorphonuclear neutrophils from 3 to 24 h, and of mononuclear cells from 48 h of infection onward. A higher density of polymorphonuclear neutrophils and mononuclear cells harboring parasites was also observed. The most important observation, however, was that more parasites per cell were present, revealing that IGF-I appears to favour parasite growth within the macrophages. These results strongly suggest an important role for IGF-I in the development of cutaneous leishmaniasis, where it influences both the inflammatory process and parasite growth.

Minist?rio da Sa?de. Funda??o Nacional de Sa?de. Instituto Evandro Chagas. Bel?m, PA, Brasil. Universidade de S?o Paulo. Faculdade de Medicina. Departamento de Medicina Preventiva. S?o Paulo, SP, Brasil. Minist?rio da Sa?de. Funda??o Nacional de Sa?de. Instituto Evandro Chagas. Bel?m, PA, Brasil / Universidade de S?o Paulo. Instituto de Ci?ncias Biom?dicas. Departamento de Parasitologia. S?o Paulo, SP, Brasil.

Universidade de S?o Paulo. Faculdade de Medicina. Departament of Pathology. Laboratory of Pathology of Infectious Diseases. S?o Paulo, SP, Brasil. Universidade de S?o Paulo. Faculdade de Medicina. Departament of Pathology. Laboratory of Pathology of Infectious Diseases. S?o Paulo, SP, Brasil. Universidade de S?o Paulo. Faculdade de Medicina. Departament of Pathology. Laboratory of Pathology of Infectious Diseases. S?o Paulo, SP, Brasil. Funda??o Universit?ria do ABC. Faculdade de Medicina. Disciplina of Dermatology. S?o Paulo, SP, Brasil. Funda??o Universit?ria do ABC. Faculdade de Medicina. Disciplina of Dermatology. S?o Paulo, SP, Brasil. Minist?rio da Sa?de. Funda??o Nacional de Sa?de. Instituto Evandro Chagas. Bel?m, PA, Brasil. A case of leishmaniasis with multiple lesions due to Leishmania (Viannia) braziliensis infections was diagnosed in a male patient from north-east Brazil. The skin lesions were nodular and pustular and progressed to ulcers. Skin and the oral mucosae were also affected. The behaviour in culture and hamsters of the parasite recovered from a skin lesion was similar to other strains of L. (V.) braziliensis and the strain was characterized by monoclonal antibodies as belonging to serodeme I of this species. The histopathology of the skin lesions of the patient and hamster were of an inflammatory nature which extended through the dermis; leishmania in the tissues were more easily detected by the immunoenzymatic PAP method.

Orientadores: Selma Giorgio, Nahiara Esteves Zorgi Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Na leishmaniose visceral, os agentes etiológicos são a Leishmania infantum e a Leishmania donovani. Apesar da variedade dos fármacos disponíveis para o tratamento das leishmanioses, a maioria ocasiona efeitos colaterais, além de relatos de resistência. Atualmente não há vacinas disponíveis com uso permitido em humanos, somente vacinas veterinárias. Com o objetivo de desenvolver um sistema vacinal, produzimos um plasmídeo pFL-AMA, e transfectamos em promastigotas de L. infantum. Este plasmídeo tem a sua expressão somente na fase amastigota do parasita. Ao inserir sequências que codificam proteínas tóxicas (forma ativa da tripsina bovina e avidina de ovo) no plasmídeo, os parasitas transfectados morrem após diferenciarem-se em amastigota. Neste trabalho, foi avaliada e comparada a carga parasitária em baço, fígado e medula óssea de animais imunizados com a vacina experimental (L. infantum transfectadas com os plasmídeos com sequências que codificam proteínas tóxicas) e infectados com a linhagem selvagem (WT) de L. infantum, além de análise da resposta imunológica humoral e histopatologia. Os dados mostraram que quanto à carga parasitátia, camundongos imunizados apresentaram carga parasitária em baço, fígado e medula óssea após 3 semanas similares aos animais do grupo WT acompanhados da presença de células gigantes multinucleadas em baço e infiltrados linfocitários no fígado, além de aumento dos níveis de IgG, IgG1 e IgG2a. Após 10 semanas, os animais imunizados tiveram redução da carga parasitária acompanhado da diminuição dos níveis de anticorpos, além da presença de células gigantes multinucleadas em baço e infiltrados linfocitários no fígado. Com relação à proteção frente a infecção com L. infantum WT, os camundongos imunizados com parasitas transfectados foram satisfatoriamente protegidos nos dois protocolos de imunização utilizados, pois apresentaram cargas parasitárias nos órgãos afetados menores do que as de animais do grupo WT, acompanhado de altos níveis de IgG, IgG1 e IgG2a, sugerindo um mix de resposta Th1/Th2. Portanto, L. infantum transfectada com plasmídeo contendo sequências de proteínas tóxicas, que levam à morte do parasita quando da diferenciação em amastigotas, é um ótimo candidato vacinal contra a leishmaniose visceral Abstract: In the visceral leishmaniasis, the ethiologic agents are Leishmania infantum and Leishmania donovani. Despite the variety of available drugs to treat leishmaniasis, most of them lead to serious adverse effects and resistance has been reported. Nowadays, there are no legalized vacines available for humans, there are only veterinary vaccines. In order to develop a vaccinal system, our group developed the pFL-AMA plasmid, and we transfected it to L. infantum promastigotes. This plasmid is only expressed in amastigote form. Sequences which encode toxic proteins (bovine active trypsin and egg avidin) were inserted in this plasmid and the transfected parasites die after the differentiation process. In this work, the evaluation and comparison of parasite burden in the spleen, liver and the bone marrow of immunized animals with the experimental vaccine (transfected L. infantum with plasmid containing the toxic protein sequences) and infected animals with wild type line of L. infantum were performed together with the histopathological and humoral response analysis. The data showed that immunized mice presented parasite burden in spleen, liver and bone marrow similar to infected mice at 3 weeks post-immunization/infection together with the presence of mononuclear giant cells in the spleen, linfocitary infiltrates in the liver and high levels of IgG, IgG1 and IgG2a. At 10 weeks post-immunization/infection, the immunized mice presented a reduced parasite burden in all the organs together with reduced levels of the antibodies. In addition, giant multinucleated cells were observed in the spleen and linfocitary infiltrates in the liver. The protection in the face of re-infection with wild L. infantum ocurred in both imunization protocols, because the immunized animals presented reduced parasite burden in all affected organs when compared with WT group together with high levels of IgG, IgG1 e IgG2a suggesting a mixed Th1/Th2 response. So, the transfected L. infantum with plasmid expressing toxic proteins, wchich leads the parasite to death after the differeciation process, is a great vaccine candidate against visceral leishmaniasis Mestrado Relações Antrópicas, Meio Ambiente e Parasitologia Mestre em Biologia Animal CAPES 001 FAPESP 2018/23302-6 CNPQ 405581/2018-1

Orientador: Selma Giorgio Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: As células dendríticas (DCs) são potentes células apresentadoras de antígeno envolvidas principalmente na iniciação da resposta imune primária. As DCs podem ser isoladas a partir do cultivo de monócitos do sangue periférico humano realizado na presença de GM-CSF e IL-4. Protozoários do gênero Leishmania são parasitas intracelulares obrigatórios de células do Sistema Fagocítico Mononuclear como os macrófagos e as DCs. A L. amazonensis, espécie utilizada em nossos estudos, causa principalmente lesões cutâneas que podem se cronificar resultando num processo infeccioso com conseqüente hipóxia tecidual. Neste trabalho avaliamos o papel deletério/terapêutico da hipóxia em modelo in vitro de uma infecção intracelular, a leishmaniose, através da análise dos parâmetros comentados na seqüência. Culturas de DCs humanas infectadas com amastigotas de L. amazonensis foram expostas à hipóxia de 6% de oxigênio. A avaliação fenotípica das culturas indicou que a hipóxia, apesar de não afetar a viabilidade das células, reduziu a expressão dos marcadores de superfície celular CD80, CD86 e CD1a. A hipóxia não afetou a entrada do parasita nas DCs, mas modulou a atividade funcional das DCs, com diminuição da infecção em cerca de 30%. Na presença de ativadores como LPS e INF-g, as DCs foram capazes de reduzir a porcentagem de infecção em normóxia, sendo que a hipóxia não potencializou este efeito estimulador. No entanto, essas mesmas culturas ativadas e infectadas em hipóxia produziram menos IL-12 que as culturas controle em normóxia. O efeito da hipóxia sobre drogas leishmanicidas Anfotericina B, Glucantime e Miltefosine foram também avaliados. As curvas dose-resposta e análise das IC50s de Anfotericina B e Glucantime sugeriram que a hipóxia anulou ou diminuiu a ação das drogas nas culturas de DCs infectadas durante 48 horas de tratamento, quando comparados ao controle em normóxia. Já o Miltefosine agiu de forma semelhante tanto em normóxia como em hipóxia. Por fim, analisamos o processo de interação Leishmania-DC utilizando amastigotas isolados de lesões de camundongos C3H/nude (os quais não apresentam anticorpos na superfície). A porcentagem de infecção das DCs com amastigotas ¿nude¿ foi de 36%. A opsonização destes parasitas com soro humano elevou a porcentagem de infecção em torno de 40%. A inativação e a depleção de anticorpos destes soros diminuiu marcadamente a porcentagem de infecção proporcionada pela opsonização, indicando o envolvimento de receptores para Fc (FcR) e para Complemento (CRs) na fagocitose dos parasitas. A infecção das DCs também foi inibida em 50% após tratamento dos amastigotas com heparina. Nós não relacionamos os receptores de manose fucose com a entrada do amastigotas nas DCs. Nossos dados sugerem que o processo de infecção das DCs envolve a participação de pelo menos três interações receptor/ligante (FcR/anticorpos, CRs/complemento e proteoglicanas/proteína ¿ligadora¿ de heparina) Abstract: Dendritic cells (DCs) are antigen-presenting cells involved in the initiation of primary immune response. Human DC were obtained from peripheral blood monocytes after culturing with GM-CSF and IL-4. Leishmania parasites are intracellular protozoan that replicate in Mononuclear Phagocytic System cells, mainly macrophages and DCs. Leishmania amazonensis, a species used in our studies is associated with cutaneous infection that can become chronical lesions with a inflammatory process and small regions of hypoxia. In this work we evaluated the role of hypoxia in a in vitro model of intracellular infection, leishmaniasis. Human DCs infection with infected by L. amazonensis amastigotes were performed under normoxic (21% O2) and hypoxic (6% O2) conditions. The phenotypic analysis of DC cultured under hypoxic condition showed a reduced expression of CD80, CD86 and CD1a compared with normoxic control. In these cell cultures, viability was not affected. Hypoxia did not affect the amastigote entrance into DCs although it modulated their functional activity reducing 30% the percentage of infected DCs. Furthermore, hypoxia did not act synergistically with IFN-g plus LPS in DCs to induce killing of parasites. DCs activated and infected with amastigotes under hypoxic condition produced low levels of IL-12 compared with normoxic control. Moreover, in the abscence of stimulation, infected DCs did not produce IL-12. We also evaluated the leishmanicidal activity of Amphotericin-B, Glucantime and Miltefosine under normoxic and hypoxic conditions during 48 h. Under hypoxic condition, Amphotericin-B and Glucantime were less efective in L. amazonensis infected DCs. None effect of hypoxia was observed in Miltefosine was observed against L. amazonensis infected DC. Finally, we investigated in vitro mechanisms of entry into human DCs of L. amazonensis amastigotes isolated from lesions in nude mice (Am nude). The DC infection rate with Am nude was approximately 36%, while opsonization of Am nude with normal human serum and infected human serum increased the DC infection rates to 60% and 62%, respectively. Heat inactivation and depletion of antibodies in sera brought the DC infection rate down to 40%. The DC infection rate was inhibited after pre-treatment of Am nude with heparin. We were unable to implicate mannose-fucose receptors in the uptake of Am nude by DCs. Our data suggest that the ability of L. amazonensis amastigotes to infect human DCs involves the participation of at least three multiple receptor-ligand interactions, antibodies/FcR, complement components/CR and proteoglycans/heparin-binding protein Doutorado Imunologia Doutor em Genética e Biologia Molecular

Submitted by Hiolanda Rêgo (hiolandarego@gmail.com) on 2016-08-05T14:56:00Z No. of bitstreams: 1 Tese_ICS_Sara Timóteo Passos.pdf: 542336 bytes, checksum: ad11eb9ab0a2f6a49dec1696235594ce (MD5) Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2016-08-23T14:59:18Z (GMT) No. of bitstreams: 1 Tese_ICS_Sara Timóteo Passos.pdf: 542336 bytes, checksum: ad11eb9ab0a2f6a49dec1696235594ce (MD5) Made available in DSpace on 2016-08-23T14:59:19Z (GMT). No. of bitstreams: 1 Tese_ICS_Sara Timóteo Passos.pdf: 542336 bytes, checksum: ad11eb9ab0a2f6a49dec1696235594ce (MD5) CAPES; NIH – grant AI-30639 A leishmaniose de modo geral vem sendo amplamente estudada e diversas questões relacionadas aos aspectos imunológicos têm sido abordadas como caracterização e modulação da resposta imune. A linha de pesquisa abordada nesta tese visa caracterizar a resposta imune a antígenos recombinantes de Leishmania em pacientes com leishmaniose visceral e leishmaniose tegumentar e, avaliar aspectos relacionados à modulação da resposta imune na leishmaniose tegumentar. No primeiro artigo, tendo em vista a dificuldade do diagnóstico precoce e correto da leishmaniose visceral, avaliamos a capacidade de três antígenos recombinantes (rH2A, KMP11 e Proteína “Q”) de Leishmania serem utilizados em teste sorológico e realizar o diagnóstico diferencial entre infecção subclínica por L. chagasi e doença. A sensibilidade e a especificidade do teste sorológico para diagnóstico da leishmaniose visceral foi de 100% para os três antígenos recombinantes estudados e em relação ao diagnóstico diferencial o KMP11 foi o antígeno que apresentou melhor capacidade de ser utilizado para distinguir entre a doença ativa e a infecção por L. chagasi. Prosseguindo o estudo com antígenos recombinantes de Leishmania, no segundo artigo, mas visando avaliar aspectos relacionados à modulação da resposta imune em pacientes com leishmaniose cutânea e leishmaniose mucosa, células mononucleares do sangue periférico destes pacientes foram estimuladas com antígeno solúvel de Leishmania e adicionados os antígenos recombinantes LACK e KMP11 – antígenos indutores de IL-10. Os resultados encontrados mostraram que na leishmaniose cutânea, ambos os antígenos foram capazes de suprimir a resposta de IFN- através da produção de IL-10, mas na leishmaniose mucosa essa supressão não foi observada, mesmo havendo produção de IL-10 pelos antígenos recombinantes. Visando aprofundar o conhecimento da resposta imune em pacientes com leishmaniose cutânea e com leishmaniose mucosa, no terceiro artigo, avaliamos o papel de moléculas co-estimulatórias, marcadores de ativação de células T na modulação da resposta imune, além de determinar a capacidade de citocinas (IL-2, IL-12, IL-15) e de CTLA-4 em modular a produção de IFN-. Observamos que a intensidade da resposta Th1 na leishmaniose mucosa está associada com o aumento do número de células T efetoras ativadas. E que, a modulação não apropriada dessas células e a manutenção de uma resposta inflamatória persistente leva ao dano tecidual observado na leishmaniose mucosa. Portanto, os resultados aqui apresentados contribuíram nas questões relacionadas aos aspectos imunológicos em grupos de pacientes com leishmaniose visceral, leishmaniose cutânea e leishmaniose mucosa no que se refere ao diagnóstico sorológico e modulação da resposta imune.

Submitted by Hiolanda Rêgo (hiolandarego@gmail.com) on 2016-08-05T14:56:00Z No. of bitstreams: 1 Tese_ICS_Sara Timóteo Passos.pdf: 542336 bytes, checksum: ad11eb9ab0a2f6a49dec1696235594ce (MD5) Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2016-08-23T14:59:18Z (GMT) No. of bitstreams: 1 Tese_ICS_Sara Timóteo Passos.pdf: 542336 bytes, checksum: ad11eb9ab0a2f6a49dec1696235594ce (MD5) Made available in DSpace on 2016-08-23T14:59:19Z (GMT). No. of bitstreams: 1 Tese_ICS_Sara Timóteo Passos.pdf: 542336 bytes, checksum: ad11eb9ab0a2f6a49dec1696235594ce (MD5) CAPES; NIH – grant AI-30639 A leishmaniose de modo geral vem sendo amplamente estudada e diversas questões relacionadas aos aspectos imunológicos têm sido abordadas como caracterização e modulação da resposta imune. A linha de pesquisa abordada nesta tese visa caracterizar a resposta imune a antígenos recombinantes de Leishmania em pacientes com leishmaniose visceral e leishmaniose tegumentar e, avaliar aspectos relacionados à modulação da resposta imune na leishmaniose tegumentar. No primeiro artigo, tendo em vista a dificuldade do diagnóstico precoce e correto da leishmaniose visceral, avaliamos a capacidade de três antígenos recombinantes (rH2A, KMP11 e Proteína “Q”) de Leishmania serem utilizados em teste sorológico e realizar o diagnóstico diferencial entre infecção subclínica por L. chagasi e doença. A sensibilidade e a especificidade do teste sorológico para diagnóstico da leishmaniose visceral foi de 100% para os três antígenos recombinantes estudados e em relação ao diagnóstico diferencial o KMP11 foi o antígeno que apresentou melhor capacidade de ser utilizado para distinguir entre a doença ativa e a infecção por L. chagasi. Prosseguindo o estudo com antígenos recombinantes de Leishmania, no segundo artigo, mas visando avaliar aspectos relacionados à modulação da resposta imune em pacientes com leishmaniose cutânea e leishmaniose mucosa, células mononucleares do sangue periférico destes pacientes foram estimuladas com antígeno solúvel de Leishmania e adicionados os antígenos recombinantes LACK e KMP11 – antígenos indutores de IL-10. Os resultados encontrados mostraram que na leishmaniose cutânea, ambos os antígenos foram capazes de suprimir a resposta de IFN- através da produção de IL-10, mas na leishmaniose mucosa essa supressão não foi observada, mesmo havendo produção de IL-10 pelos antígenos recombinantes. Visando aprofundar o conhecimento da resposta imune em pacientes com leishmaniose cutânea e com leishmaniose mucosa, no terceiro artigo, avaliamos o papel de moléculas co-estimulatórias, marcadores de ativação de células T na modulação da resposta imune, além de determinar a capacidade de citocinas (IL-2, IL-12, IL-15) e de CTLA-4 em modular a produção de IFN-. Observamos que a intensidade da resposta Th1 na leishmaniose mucosa está associada com o aumento do número de células T efetoras ativadas. E que, a modulação não apropriada dessas células e a manutenção de uma resposta inflamatória persistente leva ao dano tecidual observado na leishmaniose mucosa. Portanto, os resultados aqui apresentados contribuíram nas questões relacionadas aos aspectos imunológicos em grupos de pacientes com leishmaniose visceral, leishmaniose cutânea e leishmaniose mucosa no que se refere ao diagnóstico sorológico e modulação da resposta imune.