RESUMO
American Tegumentary Leishmaniasis (ATL) is a disease of high severity and incidence in Brazil, in addition to being a worldwide concern in public health. Leishmania amazonensis is one of the etiological agents of ATL, and the inefficiency of control measures, associated with the high toxicity of the treatment and the lack of effective immunoprophylactic strategies, makes the development of vaccines indispensable and imminent. In this light, the present study proposes to elaborate a chimeric protein (rChiP), based on the fusion of multiple epitopes of CD4+/CD8+ T cells, identified in the immunoproteome of the parasites L. amazonensis and L. braziliensis. The designed chimeric protein was tested in the L. amazonensis murine model of infection using the following formulations: 25 µg of the rChiP in saline (rChiP group) and 25 µg of the rChiP plus 25 µg of MPLA-PHAD® (rChiP+MPLA group). After completing immunization, CD4+ and CD8+ T cells, stimulated with SLa-Antigen or rChiP, showed an increased production of nitric oxide and intracytoplasmic pro-inflammatory cytokines, in addition to the generation of central and effector memory T cells. rChiP and rChiP+MPLA formulations were able to promote an effective protection against L. amazonensis infection determined by a reduction in the development of skin lesions and lower parasitic burden. Reduction in the development of skin lesions and lower parasitic burden in the vaccinated groups were associated with an increase of nitrite, CD4+/CD8+IFN-γ+TNF-α+ and CD4+/CD8+CD44highCD62Lhigh/low T cells, IgGTotal, IgG2a, and lower rates of IgG1 and CD4+/CD8+IL-10+. This data suggests that proposed formulations could be considered potential tools to prevent ATL.
RESUMO
In the context of neglected diseases, tegumentary leishmaniasis (TL) presents an emerging and re-emerging character in the national territory and in the world. The treatment of TL has limitations, such as intravenous administration route, high toxicity, and high treatment costs. Thus, several researchers work on new therapeutic strategies to improve the effectiveness of the treatment of leishmaniasis. In this light, the present study used a topical formulation, containing 8-hydroquinoline (8-HQN), for the treatment of Balb/c mice infected with L. amazonensis. After the treatment, the mean diameter of the lesion was measured, as well as the parasite load in organs and immunological parameters associated with the treatment. The results showed that the animals treated with 8-HQN 5%, when compared to controls, showed a reduction in the mean diameter of the lesion and in the parasite load. The animals treated with the ointment showed a type 1 cellular immune response profile associated with the production of cytokines such as INF-γ and TNF-α. In addition, the treatment did not demonstrate toxicity to mice. Therefore, the topical formulation containing 8-HQN 5% is a promising candidate in the topical treatment and could be considered, in the future, as an alternative for the treatment of TL.
Assuntos
Leishmaniose Cutânea , Camundongos Endogâmicos BALB C , Oxiquinolina , Carga Parasitária , Animais , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/parasitologia , Camundongos , Oxiquinolina/administração & dosagem , Oxiquinolina/química , Feminino , Administração Tópica , Antiprotozoários/administração & dosagem , Antiprotozoários/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Citocinas/metabolismo , Pomadas , Interferon gama , Modelos Animais de DoençasRESUMO
Tegumentary leishmaniasis (TL) is the main clinical manifestation of leishmaniasis, and it can cause the infected hosts to self-healing cutaneous lesions until mutilating scars in mucosal membranes, particularly in the nose and throat. The treatment against disease presents problems, and the diagnosis is hampered by variable sensitivity and/or specificity of the tests. In this context, the development of prophylactic vaccines could be considered as a strategy to control the disease. Previously, we showed that the recombinant LiHyp1 protein plus adjuvant protected mice from infection with Leishmania infantum, which causes visceral leishmaniasis. In the present study, we tested whether rLiHyp1 could induce protection against infection with L. amazonensis, a parasite species able to cause TL. We immunized BALB/c mice with rLiHyp1 plus saponin (rLiHyp1/S) or incorporated in micelles (rLiHyp1/M) as adjuvants and performed parasitological and immunological evaluations before and after infection. Results showed that after in vitro stimulation from spleen cell cultures using rLiHyp1 or a Leishmania antigenic extract (SLA), rLiHyp1/S and rLiHyp1/M groups developed a Th1-type immune response, which was characterized by high levels of IFN-γ, IL-2, TNF-α and IL-12 cytokines, nitrite, and IgG2a isotype antibodies when compared to values found in the control (saline, saponin, micelles alone) groups, which showed higher levels of anti-SLA IL-4, IL-10, and IgG1 antibodies before and after challenge. In addition, mice receiving rLiHyp1/S or rLiHyp1/M presented significant reductions in the lesion average diameter and parasite load in the infected tissue and internal organs. Blood samples were collected from healthy subjects and TL patients to obtain PBMC cultures, which were in vitro stimulated with rLiHyp1 or SLA, and results showed higher lymphoproliferation and IFN-γ production after stimulus using rLiHyp1, as compared to values found using SLA. These results suggest that rLiHyp1 plus adjuvant was protective against experimental TL and could also be considered for future studies as a vaccine candidate against human disease.
Assuntos
Leishmania infantum , Leishmaniose Visceral , Leishmaniose , Saponinas , Humanos , Animais , Camundongos , Micelas , Leucócitos Mononucleares/metabolismo , Proteínas Recombinantes , Leishmaniose Visceral/parasitologia , Adjuvantes Imunológicos , Citocinas/metabolismo , Vacinação , Camundongos Endogâmicos BALB C , Antígenos de Protozoários/genéticaRESUMO
Collagen deposition is a common event in chronic inflammation, and canine Leishmaniosis (CanL) is generally associated with a long and chronic evolution. Considering that the kidney shows fibrinogenic changes during CanL, and the balance of cytokines/chemokines regulates the profibrinogenic and antifibrinogenic immune responses differently, it can be hypothesized that the balance of cytokines/chemokines can be differentially expressed in the renal tissue in order to determine the expression of collagen depositions in the kidneys. This study aimed to measure collagen deposition and to evaluate cytokine/chemokine expressions in the kidney by means of qRT-PCR in sixteen Leishmania-infected dogs and six uninfected controls. Kidney fragments were stained with hematoxylin & eosin (H&E), Masson's Trichrome, Picrosirius Red, and Gomori's reticulin. Intertubular and adventitial collagen depositions were evaluated by the morphometric approach. Cytokine RNA expressions were measured by means of qRT-PCR to identify molecules involved in chronic collagen depositions in kidneys with CanL. Collagen depositions were related to the presence of clinical signs, and more intense intertubular collagen depositions occurred in infected dogs. Adventitial collagen deposition, as morphometrically measured by the average area of the collagen, was more intense in clinically affected dogs than in subclinically infected dogs. TNF-α/TGF-ß, MCP1/IL-12, CCL5/IL-12, IL-4/IFN-γ, and IL-12/TGF-ß expressions were associated with clinical manifestations in dogs with CanL. The IL-4/IFN-α ratio was more commonly expressed and upregulated in clinically affected dogs, and downregulated in subclinically infected dogs. Furthermore, MCP-1/IL-12 and CCL5/IL-12 were more commonly expressed in subclinically infected dogs. Strong positive correlations were detected between morphometric values of interstitial collagen depositions and MCP-1/IL-12, IL-12, and IL-4 mRNA expression levels in the renal tissues. Adventitial collagen deposition was correlated with TGF-ß, IL-4/IFN-γ, and TNF-α/TGF-ß. In conclusion, our results showed the association of MCP-1/IL-12 and CCL5/IL-12 ratios with an absence of clinical signs, as well as an IL-4/IFN-α ratio with adventitial and intertubular collagen depositions in dogs with visceral leishmaniosis.
Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Leishmaniose , Animais , Cães , Quimiocinas , Colágeno , Citocinas , Interferon gama , Interleucina-12/genética , Interleucina-4 , Rim/metabolismo , Leishmaniose/veterinária , Fator de Crescimento Transformador beta , Fator de Necrose Tumoral alfa , Quimiocina CCL2/metabolismoRESUMO
Leishmania amazonensis can cause cutaneous and visceral clinical manifestations of leishmaniasis in infected hosts. Once the treatment against disease is toxic, presents high cost, and/or there is the emergence of parasite-resistant strains, alternative means through which to control the disease must be developed. In this context, immunotherapeutics combining known drugs with immunogens could be applied to control infections and allow hosts to recover from the disease. In this study, immunotherapeutics protocols associating mimotopes selected by phage display and amphotericin B (AmpB) were evaluated in L. amazonensis-infected mice. Immunogens, A4 and A8 phages, were administered alone or associated with AmpB. Other animals received saline, AmpB, a wild-type phage (WTP), or WTP/AmpB as controls. Evaluations performed one and thirty days after the application of immunotherapeutics showed that the A4/AmpB and A8/AmpB combinations induced the most polarized Th1-type immune responses, which reflected in significant reductions in the lesion's average diameter and in the parasite load in the infected tissue and distinct organs of the animals. In addition, the combination also reduced the drug toxicity, as compared to values found using it alone. In this context, preliminary data presented here suggest the potential to associate A4 and A8 phages with AmpB to be applied in future studies for treatment against leishmaniasis.
RESUMO
In the present study, an immunoproteomic approach using Leishmania infantum parasites isolated from naturally infected dogs from an endemic region of the disease, was carried out to identify new antigens to be used in the diagnosis of canine visceral leishmaniasis (CVL). Protein extracts, obtained from parasites isolated from asymptomatic (CanLA) and symptomatic (CanLS) dogs, were used to perform the two-dimensional gels. Western Blotting assays were carried out by employing a pool of sera from dogs with visceral leishmaniasis (CanLA or CanLS), healthy dogs from an endemic area, or dogs with similar diseases associated with cross-reactions (babesiosis and ehrlichiosis). With these results, it was possible to exclude the spots that showed a cross-reactivity of the sera from groups of healthy dogs, and those with babesiosis or ehrlichiosis. Taken together, 20 proteins were identified, 15 of which have already been described in the literature and 5 of which are hypothetical. An immunogenomic screen strategy was applied to identify conserved linear B-cell epitopes in the identified hypothetical proteins. Two peptides were synthesized and tested in ELISA experiments as a proof of concept for the validation of our immunoproteomics findings. The results demonstrated that the antigens presented sensitivity and specificity values ranging from 81.93% to 97.59% and 78.14 to 85.12%, respectively. As a comparative antigen, a preparation of a Leishmania extract showed sensitivity and specificity values of 75.90% and 74.88%, respectively. The present study was able to identify proteins capable of being used for the serodiagnosis of canine visceral leishmaniasis.
Assuntos
Babesiose , Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Animais , Cães , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/veterinária , Antígenos de Protozoários , Doenças do Cão/parasitologia , Ensaio de Imunoadsorção Enzimática/métodos , Sensibilidade e Especificidade , Testes Sorológicos/métodosRESUMO
Vaccination against visceral leishmaniasis (VL) should be considered as a safe and effective measure to disease control; however, few vaccines are available against canine VL and there is no an approved human vaccine. In this context, in the present study, we evaluated the endonuclease III (ENDO) protein, which was recently showed to be antigenic for human disease, as a vaccine candidate against Leishmania infantum infection. The recombinant protein (rENDO) was administered in BALB/c mice alone or associated with saponin (rENDO/Sap) or micelles (rENDO/Mic) as adjuvants. Controls received saline, saponin or empty micelles. Results showed that both rENDO/Sap and rENDO/Mic compositions induced higher levels of IFN-γ, IL-12, TNF-α, and GM-CSF cytokines, besides nitrite and IgG2a isotype antibodies, before and after challenge infection, which were related to both CD4+ and CD8+ T cell subtypes. The immunological results contributed to significant reductions in the parasite load found in the spleens, livers, bone marrows and draining lymph nodes of the vaccinated animals. In general, mice immunized with rENDO/Mic presented a slightly higher Th1-type cellular and humoral immune response, as compared to those receiving rENDO/Sap. In addition, saponin caused a slight to moderate inflammatory edema in their vaccinated footpads, which was not observed when micelles were used with rENDO. In addition, a preliminary analysis showed that the recombinant protein was immunogenic to human cells cultures, since PBMCs from treated VL patients and healthy subjects showed higher lymphoproliferation and IFN-γ production in the culture supernatants. In conclusion, data suggest that rENDO could be considered as a candidate to be evaluated in future studies as vaccine to protect against VL.
Assuntos
Leishmania infantum , Vacinas contra Leishmaniose , Leishmaniose Visceral , Leishmaniose , Saponinas , Humanos , Animais , Cães , Camundongos , Micelas , Proteínas Recombinantes , Leishmaniose/prevenção & controle , Adjuvantes Imunológicos , Camundongos Endogâmicos BALB C , Antígenos de ProtozoáriosRESUMO
The pathogenesis of Chronic Chagas Cardiomyopathy (CCC) is still not fully understood, and the persistence of the parasite in tissues seems to be essential for the onset and progression of heart disease, tissue destruction, and chronic inflammation. It is clear that the polarity found between the asymptomatic (IND) and cardiac clinical forms refers mainly to the mechanisms involved in the regulation of the host's immune response. Thus, to elucidate aspects of the susceptibility of host phagocytes to T. cruzi infection, the present study explored novel aspects of innate immune response, integrating data on susceptibility to infection and intracellular replication, using monocyte-derived macrophages from CCC patients, together with memory CD4+ T-cells (CD45RO+). The isolation of PBMC was conducted by means of in vitro infection assay with T. cruzi trypomastigotes and flow cytometry analysis of the intracytoplasmic cytokine production by CD4+T-cells. Our findings indicated that monocytes derived from individuals with CCC are more susceptible to the infection and replication of intracellular amastigotes. Moreover, the stimulation of CD4+ T-cells from CCC patients, together with T. cruzi trypomastigotes, induces a predominance of a regulatory response over a type 1 response, demonstrated by an increase in IL-10 production and a reduction in the IFN-γ and IFN-γ/IL-10. Suppression of the function of monocyte-derived macrophages, from CCC patients, to control trypomastigote infection and intracellular replication sheds light on a potential susceptibility of these cells isolated from peripheral blood, which may reflect the ineffectiveness of parasite control by phagocytes in cardiac tissues, which can subsequently result in serious heart disease.
Assuntos
Cardiomiopatia Chagásica , Doença de Chagas , Trypanosoma cruzi , Humanos , Interleucina-10 , Leucócitos Mononucleares , Linfócitos T , Macrófagos , ImunidadeRESUMO
Tegumentary leishmaniasis (TL) is a disease of high severity and incidence in Brazil, and Leishmania braziliensis is its main etiological agent. The inefficiency of control measures, such as high toxicity and costs of current treatments and the lack of effective immunoprophylactic strategies, makes the development of vaccines indispensable and imminent. In this light, the present work developed a gene encoding multiple T-cell (CD4+/CD8+) epitope, derived from conserved proteins found in Leishmania species and associated with TL, to generate a chimeric protein (rMEP/TL) and compose a vaccine formulation. For this, six T-cell epitopes were selected by immunoinformatics approaches from proteins present in the amastigote stage and associated with host-parasite interactions. The following formulations were then tested in an L. braziliensis murine infection model: rMEP/TL in saline or associated with MPLA-PHAD®. Our data revealed that, after immunization (three doses; 14-day intervals) and subsequent challenging, rMEP/TL and rMEP/TL + MPLA-vaccinated mice showed an increased production of key immunological biomarkers of protection, such as IgG2a, IgG2a/IgG1, NO, CD4+, and CD8+ T-cells with IFN-γ and TNF-α production, associated with a reduction in CD4+IL-10+ and CD8+IL-10+ T-cells. Vaccines also induced the development of central (CD44highCD62Lhigh) and effector (CD44highCD62Llow) memory of CD4+ and CD8+ T-cells. These findings, associated with the observation of lower rates of parasite burdens in the vaccinated groups, when compared to the control groups, suggest that immunization with rMEP/TL and, preferably, associated with an adjuvant, may be considered an effective tool to prevent TL. KEY POINTS: ⢠Rational design approaches for vaccine development. ⢠Central and effector memory of CD4+ and CD8+ T-cells. ⢠Vaccine comprised of rMEP/TL plus MPLA as an effective tool to prevent TL.
Assuntos
Vacinas contra Leishmaniose , Leishmaniose , Animais , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Epitopos de Linfócito T/genética , Imunoglobulina G , Interleucina-10/metabolismo , Leishmaniose/prevenção & controle , Vacinas contra Leishmaniose/genética , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Leishmaniasis is a parasitic disease caused by Leishmania protozoa, which presents a large spectrum of clinical manifestations. In the present study, a quinoline derivative salt named N-(2-((7-chloroquinolin-4-yl)amino)ethyl)-N-(prop-2-yn-1-yl)prop-2-yn-1-aminium chloride or QDS3 was in vitro and in vivo tested against L. infantum by means of its incorporation in Poloxamer 407-based polymeric micelles (QDS3/M). The in vitro antileishmanial activity of QDS3 and QDS3/M was investigated in L. infantum promastigotes, axenic amastigotes and infected macrophages. BALB/c mice were infected with L. infantum, and parasitological parameters were evaluated 1 and 15 days post-treatment by determining the parasite load by a limiting dilution assay, besides a quantitative PCR (qPCR) method. Immunological response was assessed based on production of cellular cytokines, as well as by quantification of nitrite levels and specific antibodies. In vitro results showed that QDS3 free or in micelles presented effective antileishmanial action against both parasite stages, being more effective in amastigotes. In vivo data showed that treatment using QDS3 or QDS3/M reduced the parasite load in the livers, spleens, draining lymph nodes (dLN) and bone marrows of the treated animals, 1 and 15 days after treatment, when compared to values found in the control groups. Additionally, treated mice developed a polarized Th1-type immune response, with higher levels of IL-12, IFN-γ, GM-CSF and nitrite, besides high production of specific IgG2a antibodies, when compared to the controls. Parasitological and immunological data obtained using the micellar composition were better than the others. In conclusion, QDS3, mainly when applied in a delivery adjuvant system, could be considered for future studies as therapeutic candidate against VL.
Assuntos
Antiprotozoários , Leishmania infantum , Leishmaniose Visceral , Leishmaniose , Quinolinas , Animais , Antiprotozoários/uso terapêutico , Leishmaniose/parasitologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Micelas , Nitritos/uso terapêutico , Polímeros/uso terapêutico , Quinolinas/uso terapêuticoRESUMO
Diagnosis of tegumentary leishmaniasis (TL) is essential to avoid permanent damage and severe functional sequelae and there is an urgent need to discover new antigens. The present study aimed to comprehensively evaluate the potential use of the Tryparedoxin Peroxidase (TryP) as an antigen for serological tests. The proposal integrates data from immunoproteomics with immunoinformatics, in addition to a precise analysis of protein levels in the evolutionary stages of the parasite by flow cytometry. To evaluate the performance in the diagnosis of TL, Enzyme-Linked Immunosorbent Assay (ELISA) assays were performed using the recombinant protein and the respective B-cell epitope, followed by an analysis of the contribution of this peptide in the recognition of the protein by patients, evaluated by serum depletion assays. We showed that the TryP has a linear B-cell epitope with high divergence compared to orthologs from Trypanosoma cruzi and Homo sapiens. The results also show high expression and positive cells for TryP (TryP+) in the infective metacyclic promastigotes (MET) and intracellular (24 and 48 hours) stages. From the depletion assays, it was possible to confirm the contribution of the peptide in the specific recognition of the TryP protein by patients with TL (13.7-15.9%). ELISA using the peptide showed high performance in the diagnosis compared to the recombinant TryP (rTryP), Soluble Leishmania braziliensis Antigen (sLba) and Immunofluorescence Assay (IFA) with accuracy of 94.29, 89.29, 65.00 and 37.14%, respectively). We can conclude that the MNEPAPP peptide is a potential antigen for the diagnosis of TL.
Assuntos
Leishmaniose Cutânea , Leishmaniose , Anticorpos Antiprotozoários , Antígenos de Protozoários/genética , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos de Linfócito B , Humanos , Leishmaniose Cutânea/parasitologia , Peptídeos , Peroxidases , Proteínas de Protozoários/genéticaRESUMO
Treatment against visceral leishmaniasis (VL) presents problems by the toxicity of drugs, high cost and/or emergence of resistant strains. The diagnosis is hampered by variable sensitivity and/or specificity of tests. In this context, prophylactic vaccination could represent a control measure against disease. In this study, the protective efficacy of Leishmania LiHyC protein was evaluated in a murine model against Leishmania infantum infection. LiHyC was used as recombinant protein (rLiHyC) associated with saponin (rLiHyC/S) or Poloxamer 407-based polymeric micelles (rLiHyC/M) to immunize mice. Animals received also saline, saponin or empty micelles as controls. The immunogenicity was evaluated before and after the challenge, and results showed that vaccination with rLiHyC/S or rLiHyC/M induced the production of high levels of interferon-gamma (IFN-γ), interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor in cell culture supernatants, as well as higher IFN-γ expression evaluated by RT-qPCR and involvement from CD4+ and CD8+ T-cell subtypes producing IFN-γ, tumor necrosis factor-α and IL-2. A positive lymphoproliferative response was also found in cell cultures from vaccinated animals, besides high levels of rLiHyC- and parasite-specific nitrite and IgG2a antibodies. Immunological assays correlated with significant reductions in the parasite load in the spleens, livers, bone marrows and draining lymph nodes from vaccinated mice, when compared to values found in the controls. The micellar composition showed slightly better immunological and parasitological data, as compared to rLiHyC/S. Results suggest that rLiHyC associated with adjuvants could be considered for future studies as a vaccine candidate against VL.
Assuntos
Leishmania infantum , Vacinas contra Leishmaniose , Leishmaniose Visceral , Saponinas , Animais , Antígenos de Protozoários , Interferon gama , Interleucina-12 , Camundongos , Camundongos Endogâmicos BALB C , Micelas , Proteínas RecombinantesRESUMO
Visceral leishmaniasis (VL) is a neglected tropical disease found in tropical and subtropical regions in the world. The therapeutics used for the treatment against disease presents problems, mainly related to drug toxicity, route of administration, high cost and/or by emergence of resistant strains. In this context, the search for alternative antileishmanial candidates is desirable. Recently, a naphthoquinone derivative namely 2-(2,3,4-tri-O-acetyl-6-deoxy-ß-L-galactopyranosyloxy)-1,4-naphthoquinone or Flau-A showed an effective in vitro biological action against Leishmania infantum. In the present study, the efficacy of this naphthoquinone derivative was evaluated in an in vivo infection model. BALB/c mice (n = 12 per group) were infected and later received saline or were treated with empty micelles (B/Mic), free Flau-A or it incorporated in Poloxamer 407-based micelles (Flau-A/Mic). The products were administered subcutaneously in the infected animals, which were then euthanized one (n = 6 per group) and 15 (n = 6 per group) days post-therapy, when immunological and parasitological evaluations were performed. Results showed that animals treated with Flau-A or Flau-A/Mic produced significantly higher levels of antileishmanial IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and IgG2a isotype antibody, when compared to data found in the control (saline and B/Mic) groups; which showed significantly higher levels of parasite-specific IL-4, IL-10 and IgG1 antibody. In addition, animals receiving free Flau-A or Flau-A/Mic presented also significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, when compared to the controls. A low hepatic and renal toxicity was also found. Overall, Flau-A/Mic showed better immunological and parasitological results, when compared to the use of free molecule. In conclusion, preliminary data suggest that this composition could be considered in future studies as promising therapeutic candidate against VL.
Assuntos
Antiprotozoários/química , Antiprotozoários/uso terapêutico , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Naftoquinonas/química , Naftoquinonas/uso terapêutico , Animais , Antiprotozoários/farmacologia , Feminino , Leishmania infantum/genética , Leishmania infantum/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Micelas , Naftoquinonas/farmacologia , Carga Parasitária , Reação em Cadeia da Polimerase em Tempo Real , Baço/parasitologiaRESUMO
Apoptosis is associated with resolution of inflammation. However, apoptosis may also occur in active inflammation, balancing inflammatory recruitment instead of a resolution event. To test that hypothesis, we measured apoptosis and chemokines expression, involved in recruitment of inflammatory cells. Clinical affected and subclinically infected dogs with canine leishmaniosis (CanL) and uninfected controls were assessed. Apoptosis in renal tissue (glomeruli, tubules, and inflammatory infiltrate) and cellularity in inflammatory foci were quantified. Messenger RNA of CCL5, CCL4, MCP-1, MCP-2, Caspase (Casp) 3, Casp 8, Casp 9, Bax, Bcl2 and Fas were quantified by qRT PCR. Clinical affected dogs showed more intense inflammation and higher cellularity in the inflammatory infiltrates than subclinically infected ones, which were higher than controls. Glomerular and tubular cells showed higher apoptotic index in clinical affected dogs when compared to controls. Apoptosis within the inflammatory infiltrates was higher in clinical affected dogs. Bax/Bcl2 ratio and CCL4 showed higher expression in kidney from clinical affected when compared to subclinically infected dogs. Casp 3/CCL4 ratio expression were higher in subclinically infected dogs than in the clinical affected group. Additionally, results suggest that Casp 3/CCL4 ratio is balancing towards an inflammatory recruitment and CCL4 and Bax/Bcl2 ratio expression is associated with active inflammation in clinical affected CanL. Data demonstrate that apoptosis was not always correlated with resolution of inflammation, when a morphometric and a molecular evaluation were performed concomitantly. In kidneys of Leishmania infected dogs, apoptosis and chemokines may be balancing inflammatory recruitment. In conclusion, Bax/Bcl2 ratio, chemokines, Casp 8, Casp 3 and Fas were associated with renal apoptosis, active inflammation and increased inflammatory recruitment observed in clinical affected animals, influencing the clinical presentation of leishmaniosis.
Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Animais , Apoptose , Quimiocinas/genética , Doenças do Cão/parasitologia , Cães/parasitologia , Inflamação/veterinária , Rim/parasitologia , Rim/patologia , Leishmaniose Visceral/veterináriaRESUMO
Leishmania braziliensis is responsible for most cases of human tegumentary leishmaniasis (HTL) and has caused a wide range of clinical manifestations, including cutaneous (CL) and mucosal leishmaniasis (ML). The diagnosis is based on criteria that consider epidemiological data, clinical findings, and laboratory tests and is hard to establish. For laboratory tests, none of the assays available can be considered gold standards for disease detection. In addition, the Montenegro skin test, essential to supporting infectologists in the clinical management of the disease, is no longer available in Brazil. Thus, the aim of this study was to develop new targets to be used in diagnostic tests for HTL. In the first step, we carried out two-dimensional gel electrophoresis, followed by mass spectrometry, combined with heat map analysis and immunoproteomics approach, and disclosed eight proteins expressed in the amastigote stage specifically recognized by serum from CL and ML patients. A chimeric protein was designed based on the combination of thirteen linear B-cell epitopes, identified by immunoinformatics analysis, from L. braziliensis proteins. Our results showed that the strategy used in this work was successful in developing an antigen to be used in immunological assays (100.0% sensitivity and specificity) in the detection of HTL cases and in comparison with results obtained from an ELISA using soluble L. braziliensis antigen (SLb-Antigen) and immunofluorescence assay (Bio-Manguinhos/FIOCRUZ). The present technology opens the door for its use in field exams by means of an immunochromatographic test, which will be even more helpful in regions without laboratory structures.Key points⢠Rational strategy to develop antigens.⢠Integration between immunoproteomic and immunoinformatics analysis.⢠Chimeric protein shows high performance in HTL diagnosis.
Assuntos
Leishmania braziliensis , Leishmaniose Cutânea , Ensaio de Imunoadsorção Enzimática , Humanos , Leishmaniose Cutânea/diagnóstico , Proteômica , Proteínas Recombinantes de FusãoRESUMO
Treatment against visceral leishmaniasis (VL) is mainly hampered by drug toxicity, long treatment regimens and/or high costs. Thus, the identification of novel and low-cost antileishmanial agents is urgent. Acarbose (ACA) is a specific inhibitor of glucosidase-like proteins, which has been used for treating diabetes. In the present study, we show that this molecule also presents in vitro and in vivo specific antileishmanial activity against Leishmania infantum. Results showed an in vitro direct action against L. infantum promastigotes and amastigotes, and low toxicity to mammalian cells. In addition, in vivo experiments performed using free ACA or incorporated in a Pluronic® F127-based polymeric micelle system called ACA/Mic proved effective for the treatment of L. infantum-infected BALB/c mice. Treated animals presented significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes when compared to the controls, as well as the development of antileishmanial Th1-type humoral and cellular responses based on high levels of IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and IgG2a isotype antibodies. In addition, ACA or ACA-treated animals suffered from low organ toxicity. Treatment with ACA/Mic outperformed treatments using either Miltefosine or free ACA based on parasitological and immunological evaluations performed one and 15 days post-therapy. In conclusion, data suggest that the ACA/Mic is a potential therapeutic agent against L. infantum and merits further consideration for VL treatment.
Assuntos
Acarbose/farmacologia , Acarbose/uso terapêutico , Imunidade , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/imunologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/imunologia , Animais , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Reposicionamento de Medicamentos , Feminino , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Micelas , Carga Parasitária , Fosforilcolina/análogos & derivados , Fosforilcolina/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Resultado do TratamentoRESUMO
The pathogenesis of Canine leishmaniosis (CanL) is associated with altered cytokine expression and parasitic tissue shows a lot of inflammation. The aim of this study was to assess the renal inflammation and cytokine expression in eight symptomatic and eight asymptomatic Leishmania- infected dogs, and seven uninfected control dogs. Kidney fragments were stained with hematoxylin and eosin for morphometric evaluation. mRNA expression levels of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin (IL)-2, IL-4, IL-10, and IL-12 were assessed in the kidney fragments using quantitative real time-polymerase chain reaction. Inflammation, quantified by the average area of the infiltrated immune cells, was greater in symptomatic dogs than in those asymptomatic, whereas asymptomatic dogs exhibited higher inflammation than the control dogs (p > 0.05, Tukey's test). Expression levels of IFN-γ, TNF-α, IL-4, IL-10, and IL-12 were upregulated in symptomatic dogs and downregulated in asymptomatic dogs compared with those of the uninfected group. Furthermore, IL-4 showed higher expression in symptomatic dogs than in asymptomatic ones (p < 0.05, Mann-Whitney test), which was directly associated with clinical manifestations (p < 0.05, Chi-square test). However, IL-12 was predominantly expressed in symptomatic dogs, shifting the balance from IL-12/IL-4 to IL-12, which elicits a change in the inflammatory response. Leishmania was not found in the renal tissues in any one of the studied groups. Our data suggests that the balance between IL-12 and IL-4 plays an important role in the regulation of inflammation in renal tissue and clinical presentations in CanL.
Assuntos
Doenças do Cão/imunologia , Inflamação/veterinária , Interleucina-12/genética , Interleucina-4/genética , Rim/imunologia , Leishmaniose/imunologia , Leishmaniose/veterinária , Animais , Doenças do Cão/parasitologia , Cães , Feminino , Regulação da Expressão Gênica/imunologia , Inflamação/parasitologia , Interleucina-12/imunologia , Interleucina-4/imunologia , Leishmania infantum/imunologia , MasculinoRESUMO
AIMS: Treatment for visceral leishmaniasis (VL) is hampered by the toxicity and/or high cost of drugs, as well as by emergence of parasite resistance. Therefore, there is an urgent need for new antileishmanial agents. METHODS AND RESULTS: In this study, the antileishmanial activity of a diprenylated flavonoid called 5,7,3,4'-tetrahydroxy-6,8-diprenylisoflavone (CMt) was tested against Leishmania infantum and L amazonensis species. Results showed that CMt presented selectivity index (SI) of 70.0 and 165.0 against L infantum and L amazonensis promastigotes, respectively, and of 181.9 and 397.8 against respective axenic amastigotes. Amphotericin B (AmpB) showed lower SI values of 9.1 and 11.1 against L infantum and L amazonensis promastigotes, respectively, and of 12.5 and 14.3 against amastigotes, respectively. CMt was effective in the treatment of infected macrophages and caused alterations in the parasite mitochondria. L infantum-infected mice treated with miltefosine, CMt alone or incorporated in polymeric micelles (CMt/Mic) presented significant reductions in the parasite load in distinct organs, when compared to the control groups. An antileishmanial Th1-type cellular and humoral immune response were developed one and 15 days after treatment, with CMt/Mic-treated mice presenting a better protective response. CONCLUSION: Our data suggest that CMt/Mic could be evaluated as a chemotherapeutic agent against VL.
Assuntos
Antiprotozoários/administração & dosagem , Leishmaniose Visceral/tratamento farmacológico , Animais , Antiprotozoários/química , Antiprotozoários/farmacologia , Feminino , Flavonoides/administração & dosagem , Flavonoides/química , Flavonoides/farmacologia , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/crescimento & desenvolvimento , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/crescimento & desenvolvimento , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Micelas , Carga ParasitáriaRESUMO
Leishmania infantum pyridoxal kinase (PK) protein was characterized after an immunoproteomics screening performed with the sera from patients suffering visceral leishmaniasis (VL). Since it was recognized by sera of mammalian hosts infected by a viscerotropic Leishmania species, PK could emerge as a new vaccine candidate against disease, due to its antigenicity and immunogenicity. In this context, in the present study, the effects of the immunization using PK were evaluated when administered as a DNA plasmid (pDNAA3/PK) or recombinant protein (rPK) plus saponin. The immune response elicited by both vaccination regimens reduced in significant levels the parasite load in spleen, liver, draining lymph nodes and bone marrow, being associated with the development of Th1-type immune response, which was characterized by high levels of IFN-γ, IL-12, GM-CSF, and specific IgG2a antibody, besides low production of IL-4, IL-10, and protein and parasite-specific IgG1 antibodies. CD8+ T cells were more important in the IFN-γ production in the pDNAA3/PK group, while CD4+ T cells contributed more significantly to production of this cytokine in the rPK/Saponin group. In addition, increased IFN-γ secretion, along with low levels of IL-10, were found when PBMCs from VL patients after treatment and healthy individuals were stimulated with the protein. In conclusion, when administered either as a DNA plasmid or recombinant protein plus adjuvant, PK can direct the immune response towards a Th1-type immune profile, protecting mice against L. infantum challenge; therefore, it can be seen as a promising immunogen against human VL.
Assuntos
Antígenos de Protozoários/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/imunologia , Piridoxal Quinase/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Humanos , Leishmania infantum/imunologia , Camundongos , Proteínas Recombinantes/imunologia , Vacinas de DNA/imunologiaRESUMO
The treatment against visceral leishmaniasis (VL) presents problems, mainly related to the toxicity and/or high cost of the drugs. In this context, a prophylactic vaccination is urgently required. In the present study, a Leishmania protein called LiHyE, which was suggested recently as an antigenic marker for canine and human VL, was evaluated regarding its immunogenicity and protective efficacy in BALB/c mice against Leishmania infantum infection. In addition, the protein was used to stimulate peripheral blood mononuclear cells (PBMCs) from VL patients before and after treatment, as well as from healthy subjects. Vaccination results showed that the recombinant (rLiHyE) protein associated with liposome or saponin induced effective protection in the mice, since significant reductions in the parasite load in spleen, liver, draining lymph nodes, and bone marrow were found. The parasitological protection was associated with Th1-type cell response, since high IFN-γ, IL-12, and GM-CSF levels, in addition to low IL-4 and IL-10 production, were found. Liposome induced a better parasitological and immunological protection than did saponin. Experiments using PBMCs showed rLiHyE-stimulated lymphoproliferation in treated patients' and healthy subjects' cells, as well as high IFN-γ levels in the cell supernatant. In conclusion, rLiHyE could be considered for future studies as a vaccine candidate against VL.