RESUMO
In spite of several decades of research, an effective vaccine against schistosomiasis remains elusive. The radiation-attenuated (RA) cercarial vaccine is still the best model eliciting high protection levels, although the immune mechanisms have not yet been fully characterized. In order to identify genes and pathways underlying protection we investigated patterns of gene expression in PBMC and skin draining Lymph Nodes (LN) from mice using two exposure comparisons: vaccination with 500 attenuated cercariae versus infection with 500 normal cercariae; one versus three doses. Vaccinated mice were challenged with 120 normal parasites. Integration of PBMC and LN data from the infected group revealed early up-regulation of pathways associated with Th2 skewing and polarization of IgG antibody profiles. Additionally, hemostasis pathways were downregulated in infected mice, correlating with platelet reduction, potentially a mechanism to assist parasite migration through capillary beds. Conversely, up regulation of such mechanisms after vaccination may explain parasite blockade in the lungs. In contrast, a single exposure to attenuated parasites revealed early establishment of a Th1 bias (signaling of IL-1, IFN-γ; and Leishmania infection). Genes encoding chemokines and their receptors were more prominent in vaccinated mice, indicating an enhanced capacity for inflammation, potentially augmenting the inhibition of intravascular migration. Increasing the vaccinations from one to three did not dramatically elevate protection, but there was a clear shift towards antibody-mediated effectors. However, elements of the Th1 bias were still evident. Notable features after three vaccinations were markers of cytotoxicity (including IL-6 and NK cells) together with growth factors and their receptors (FGFR/VEGF/EGF) and the apoptosis pathway. Indeed, there is evidence for the development of anergy after three vaccinations, borne out by the limited responses detected in samples after challenge. We infer that persistence of a Th1 response puts a limit on expression of antibody-mediated mechanisms. This feature may explain the failure of multiple doses to drive protection towards sterile immunity. We suggest that the secretions of lung stage parasites would make a novel cohort of antigens for testing in protection experiments.
Assuntos
Hemostasia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Vacinas Protozoárias/administração & dosagem , Schistosoma mansoni/imunologia , Esquistossomose mansoni/prevenção & controle , Biologia de Sistemas , Animais , Cercárias/imunologia , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Hemostasia/genética , Interações Hospedeiro-Parasita , Peptídeos e Proteínas de Sinalização Intercelular/genética , Linfonodos/imunologia , Linfonodos/metabolismo , Linfonodos/parasitologia , Camundongos Endogâmicos C57BL , Análise em Microsséries , Vacinas Protozoárias/imunologia , Schistosoma mansoni/patogenicidade , Esquistossomose mansoni/imunologia , Esquistossomose mansoni/metabolismo , Esquistossomose mansoni/parasitologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th1/parasitologia , Equilíbrio Th1-Th2 , Células Th2/imunologia , Células Th2/metabolismo , Células Th2/parasitologia , Fatores de Tempo , Transcriptoma , Vacinação , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologiaRESUMO
Visceral leishmaniasis (VL) is an important zoonotic vector-borne disease and domestic dogs are considered the main domiciliary and peri-domiciliary reservoir of Leishmania (Leishmania) infantum in South America. Distinct eco-epidemiological scenarios associated to the prevalence of the disease, clusters of parasite genotypes and chemotypes of vectors population are described in Brazil, especially in the state of São Paulo (SP). In this context, the purpose of the present study is to evaluate the clinical signs, histopathological lesions, parasite load and cytokine profile by immunohistochemistry (IHC) in popliteal lymph nodes of canines naturally infected with L. infantum, from different municipalities of the state of SP. Eighty-three dogs with VL, 61 from northwest SP (NWSP) and 22 from southeast SP (SESP), were clinically classified in stage II, with no babesiosis and ehrlichiosis. Subcapsular inflammatory infiltration and histiocytosis were significantly higher in the SESP group (pâ¯=â¯0.0128; 0.0077, respectively). On the other hand, dogs from NWSP revealed 4.6-fold significantly higher parasite burden (pâ¯=â¯0.0004) and higher IHC scores of IL-1ß (pâ¯=â¯0.0275) and IL-4 (pâ¯=â¯0.0327) in the popliteal lymph node tissues, which may be associated with the susceptibility and progression of the disease in these dogs. Differences in immune response profile associated with higher parasite load in dogs can also contribute to explain the distinct eco-epidemiological patterns of VL in specific geographic regions.
Assuntos
Citocinas/imunologia , Doenças do Cão/imunologia , Leishmania infantum , Leishmaniose Visceral/veterinária , Linfonodos/parasitologia , Animais , Brasil/epidemiologia , Doenças do Cão/epidemiologia , Doenças do Cão/parasitologia , Doenças do Cão/patologia , Cães , Feminino , Leishmania infantum/imunologia , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/patologia , Linfonodos/patologia , Masculino , Carga ParasitáriaRESUMO
In most of the world Toxoplasma gondii is comprised of archetypal types (types I, II and III); however, South America displays several non-archetypal strains. This study used an experimental mouse model to characterize the immune response and parasite kinetics following infection with different parasite genotypes. An oral inoculation of 50 oocysts per mouse from T. gondii M4 type II (archetypal, avirulent), BrI or BrIII (non-archetypal, virulent and intermediate virulent, respectively) for groups (G)2, G3 and G4, respectively was used. The levels of mRNA expression of cytokines, immune compounds, cell surface markers and receptor adapters [interferon gamma (IFNγ), interleukin (IL)-12, CD8, CD4, CD25, CXCR3 and MyD88] were quantified by SYBR green reverse transcription-quantitative polymerase chain reaction. Lesions were characterized by histology and detection by immunohistochemistry established distribution of parasites. Infection in G2 mice was mild and characterized by an early MyD88-dependent pathway. In G3, there were high levels of expression of pro-inflammatory cytokines IFNγ and IL-12 in the mice showing severe clinical symptoms at 811 days post infection (dpi), combined with the upregulation of CD25, abundant tachyzoites and tissue lesions in livers, lungs and intestines. Significant longer expression of IFNγ and IL-12 genes, with other Th1-balanced immune responses, such as increased levels of CXCR3 and MyD88 in G4, resulted in survival of mice and chronic toxoplasmosis, with the occurrence of tissue cysts in brain and lungs, at 14 and 21 dpi. Different immune responses and kinetics of gene expression appear to be elicited by the different strains and non-archetypal parasites demonstrated higher virulence.
Assuntos
Toxoplasma/fisiologia , Toxoplasmose Animal/parasitologia , Animais , Antígenos CD/metabolismo , Gatos , Citocinas/metabolismo , DNA Complementar/biossíntese , DNA de Protozoário/isolamento & purificação , Feminino , Genótipo , Imuno-Histoquímica , Linfonodos/parasitologia , Linfonodos/patologia , Mesentério , Camundongos , Fator 88 de Diferenciação Mieloide/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real , Receptores CXCR3/metabolismo , Baço/parasitologia , Baço/patologia , Toxoplasma/classificação , Toxoplasma/genética , Toxoplasma/imunologia , Toxoplasmose Animal/imunologia , Toxoplasmose Animal/patologiaRESUMO
Trypanosoma rangeli is a non-pathogenic protozoan parasite that infects mammals, including humans, in Chagas disease-endemic areas of South and Central America. The parasite is transmitted to a mammalian host when an infected triatomine injects metacyclic trypomastigotes into the host's skin during a bloodmeal. Infected mammals behave as parasite reservoirs for several months and despite intensive research, some major aspects of T. rangeli-vertebrate interactions are still poorly understood. In particular, many questions still remain unanswered, e.g. parasite survival and development inside vertebrates, as no parasite multiplication sites have yet been identified. The present study used an insect bite transmission strategy to investigate whether the vector inoculation spot in the skin behave as a parasite-replication site. Histological data from the skin identified extracellular parasites in the dermis and hypodermis of infected mice in the first 24 hours post-infection, as well as the presence of inflammatory infiltrates in a period of up to 7 days. However, qPCR analyses demonstrated that T. rangeli is eliminated from the skin after 7 days of infection despite being still consistently found on circulating blood and secondary lymphoid tissues for up to 30 days post-infection. Interestingly, significant numbers of parasites were found in the spleen and mesenteric lymph nodes of infected mice during different periods of infection and steady basal numbers of flagellates are maintained in the host's bloodstream, which might behave as a transmission source to insect vectors. The presence of parasites in the spleen was confirmed by fluorescent photomicrography of free and cell-associated T. rangeli forms. Altogether our results suggest that this organ could possibly behave as a T. rangeli maintenance hotspot in vertebrates.
Assuntos
Doença de Chagas/transmissão , Linfonodos/parasitologia , Pele/parasitologia , Baço/parasitologia , Trypanosoma rangeli/isolamento & purificação , Animais , América Central/epidemiologia , Doença de Chagas/epidemiologia , Modelos Animais de Doenças , Interações Hospedeiro-Parasita , Humanos , Mordeduras e Picadas de Insetos/parasitologia , Insetos Vetores/parasitologia , Camundongos , Rhodnius/parasitologia , Sepse/parasitologia , América do Sul/epidemiologiaAssuntos
Doença de Chagas/diagnóstico , Linfonodos/patologia , Linfadenopatia/etiologia , Idoso , Linfócitos B/ultraestrutura , Cardiomiopatias/cirurgia , Doença de Chagas/patologia , Transplante de Coração , Humanos , Linfonodos/parasitologia , Linfadenopatia/patologia , Macrófagos/parasitologia , Macrófagos/ultraestrutura , Masculino , México/etnologia , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/patologia , Pseudolinfoma/etiologia , Pseudolinfoma/patologia , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
The inadequacy of available treatments for leishmaniasis has presented up to 40% therapeutic failure. This fact suggests an urgency in the discovery of new drugs or alternative approaches for treating this disease. The objective of this study was to evaluate the antileishmanial activity of combined therapy between crotamine (CTA) from Crotalus durissus terrificus and the pentavalent antimonial Glucantime® (GLU). The assays were in vitro performed measuring the inhibition of Leishmania amazonensis amastigotes, followed by the evaluation of cellular production of cytokines and nitrites. After that, analytical methods were performed in order to characterize the molecules involved in the study by Mass Spectrometry, molecular affinity through an in silico assay and Surface Plasmon Resonance. In vivo experiments with BALB/c mice were performed by analyzing parasitemia, lesion size and immunological mediators. In the in vitro experiments, the pharmacological association improved the inhibition of the amastigotes, modulated the production of cytokines and nitric oxide. The therapy improved the effectiveness of the GLU, demonstrating a decreased parasitemia in the infected tissues. Altogether, the results suggest that the combined approach with CTA and GLU may be a promising alternative for the treatment of cutaneous leishmaniasis.
Assuntos
Antiprotozoários/uso terapêutico , Venenos de Crotalídeos/uso terapêutico , Crotalus , Leishmania mexicana/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Antimoniato de Meglumina/uso terapêutico , Animais , Antiprotozoários/farmacologia , Venenos de Crotalídeos/farmacologia , Combinação de Medicamentos , Interleucina-12/sangue , Interleucina-12/metabolismo , Leishmania mexicana/isolamento & purificação , Linfonodos/parasitologia , Macrófagos Peritoneais , Espectrometria de Massas , Antimoniato de Meglumina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Óxido Nítrico/metabolismo , Nitritos/análise , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The diagnosis of canine visceral leishmaniasis (CVL) has been a problem for public health services due to the variety of clinical signs similar to other diseases and low sensitivity and specificity of available tests. In this sense, our main objective was to develop a simple, rapid, and accurate quantitative real-time PCR (qPCR) diagnosis for CVL. Thus, low-invasive samples from bone marrow (BM), popliteal lymph nodes (PLN), and conjunctival swabs (CS) were selected from negative and VL-positive dogs, using as gold standard, immunological and parasitological tests performed with different tissues. Oligonucleotides for Leishmania infantum kDNA were designed and the limit of quantification and amplification efficiency of the qPCR were determined using tissue-specific standards produced with DNA from those different tissues, mixed with DNA from a known amount of L. infantum promastigotes. Endogenous control was used to validate a comparative Ct method, and tissue parasite concentrations were estimated by comparison with tissue-specific reference standard samples. The overall analysis of the qPCR data suggests the following ranking for tissue choice: PLN > BM > CS. Finally, we have concluded that this molecular approach simplifies and accelerates the quantitative diagnostic process because it is easy to perform, requiring no DNA dosing or standard curve application, and it shows good diagnostic parameters, especially when using popliteal lymph node samples.
Assuntos
Doenças do Cão/diagnóstico , Leishmania infantum/genética , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/veterinária , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Medula Óssea/parasitologia , DNA de Cinetoplasto/genética , Doenças do Cão/parasitologia , Cães , Leishmaniose Visceral/parasitologia , Linfonodos/parasitologia , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Sensibilidade e Especificidade , Baço/parasitologiaRESUMO
INTRODUCTION: In canine visceral leishmaniasis (CVL), lymphopenia, and the disorganization of lymphoid organs such as spleen and lymph nodes have been demonstrated. However, the involvement of thymus in CVL has not been evaluated so far. Herein, we investigated whether the thymus can be colonized by Leishmania infantum in naturally infected dogs. METHODS: Thymus were obtained from 16 of 58 dogs and samples of this organ were submitted to immunohistochemistry for laminin and fibronectin detection, histopathology, in situ hybridization and polymerase chain reaction (PCR) targeting the gene ITS-1 for Leishmania and sequenced. Samples of spleen, skin and popliteal lymph nodes were collected and submitted to immunohistochemistry and parasitological culture followed by multilocus enzyme electrophoresis. RESULTS: L. infantum was identified in all dogs. DNA and amastigote forms of Leishmania were detected in the thymus from 16 dogs by PCR and in eight by immunohistochemistry. Besides thymus, parasites were detected in spleen, lymph nodes, and skin. A granulomatous or pyogranulomatous thymitis was observed in eight dogs associated to intact amastigotes forms of this parasite. Fibronectin deposition in thymus was higher in dogs with more clinical signs. CONCLUSIONS: These results demonstrate that the thymus of dogs can be parasitized by L. infantum, which may generate inflammatory reactions leading to alterations in thymic microarchitecture.
Assuntos
DNA de Protozoário/isolamento & purificação , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/veterinária , Timo/parasitologia , Animais , DNA de Protozoário/genética , Doenças do Cão/parasitologia , Cães , Feminino , Leishmania infantum/genética , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/patologia , Linfonodos/parasitologia , Masculino , Carga Parasitária , Pele/parasitologia , Baço/parasitologiaRESUMO
The control measures against visceral leishmaniasis (VL) include a precise diagnosis of disease, the treatment of human cases, and reservoir and vector controls. However, these are insufficient to avoid the spread of the disease in specific countries worldwide. As a consequence, prophylactic vaccination could be interesting, although no effective candidate against human disease is available. In the present study, the Leishmania infantum amastin protein was evaluated regarding its immunogenicity and protective efficacy against experimental VL. BALB/c mice immunized with subcutaneous injections of the recombinant protein with or without liposome/saponin (Lip/Sap) as an adjuvant. After immunization, half of the animals per group were euthanized and immunological evaluations were performed, while the others were challenged with L. infantum promastigotes. Forty-five days after infection, the animals were euthanized and parasitological and immunological evaluations were performed. Results showed the development of a Th1-type immune response in rAmastin-Lip and rAmastin-Sap/vaccinated mice, before and after infection, which was based on the production of protein and parasite-specific IFN-γ, IL-12, GM-CSF, and nitrite, as well as the IgG2a isotype antibody. CD4+ T cells were mainly responsible for IFN-γ production in vaccinated mice, which also presented significant reductions in parasitism in their liver, spleen, draining lymph nodes, and bone marrow. In addition, PBMC cultures of treated VL patients and healthy subjects stimulated with rAmastin showed lymphoproliferation and higher IFN-γ production. In conclusion, the present study shows the first case of an L. infantum amastin protein associated with distinct delivery systems inducing protection against L. infantum infection and demonstrates an immunogenic effect of this protein in human cells.
Assuntos
Leishmania infantum/imunologia , Leishmaniose Visceral/imunologia , Proteínas de Protozoários/imunologia , Adjuvantes Imunológicos/farmacologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/parasitologia , Células Cultivadas , Feminino , Humanos , Imunidade/imunologia , Interferon gama/imunologia , Leishmaniose Visceral/parasitologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/parasitologia , Linfonodos/imunologia , Linfonodos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/imunologia , Células Th1/imunologia , Células Th1/parasitologiaRESUMO
In immunocompromised patients, visceral leishmaniasis (VL) can present with atypical clinical symptoms that include poor response to treatment. No optimal therapeutic regimen is available for such cases. In a splenectomized male patient, we observed a disseminated form of the disease in the liver, bone marrow, lymph nodes, and gastrointestinal tract. There was an apparent clinical improvement when he was initially treated with liposomal amphotericin B (L-AmB), but this was followed by a relapse involving severe clinical symptoms. He was finally treated successfully with a combination of L-AmB, meglumine antimoniate, and pentamidine isethionate. It is important to include asplenia as an immunosuppressive condition that induces exotic VL pathologies. In such cases, combination anti-Leishmania drug therapy should be considered.