Oropouche virus infection induces ROS production and oxidative stress in liver and spleen of mice.

Oropouche virus (OROV) is the aetiological agent of Oropouche fever, the symptoms of which are common to most arboviruses, such as fever, headache, malaise, nausea and vomiting. More than half a million people have been infected with OROV since its isolation in 1955. Although Oropouche fever is classified as a neglected and emerging disease, to date, there are no antiviral drugs or vaccines available against the infection and little is known about its pathogenicity. Therefore, it is essential to elucidate the possible mechanisms involved in its pathogenesis. Since oxidative stress plays a pivotal role in the progression of various viral diseases, in this study, redox homeostasis in the target organs of OROV infection was evaluated using an animal model. Infected BALB/c mice exhibited reduced weight gain, splenomegaly, leukopenia, thrombocytopenia, anaemia, development of anti-OROV neutralizing antibodies, increased liver transaminases, and serum levels of pro-inflammatory cytokines tumour necrosis factor (TNF-α) and interferon-γ (IFN-γ). The OROV genome and infectious particles were detected in the liver and spleen of infected animals, with liver inflammation and an increase in the number and total area of lymphoid nodules in the spleen. In relation to redox homeostasis in the liver and spleen, infection led to an increase in reactive oxygen species (ROS) levels, increased oxidative stress biomarkers malondialdehyde (MDA) and carbonyl protein, and decreased activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). Taken together, these results help elucidate some important aspects of OROV infection that may contribute to the pathogenesis of Oropouche.

Immunochemotherapy for visceral leishmaniasis: combinatorial action of Miltefosine plus LBSapMPL vaccine improves adaptative Th1 immune response with control of splenic parasitism in experimental hamster model.

The control of human visceral leishmaniasis (VL) is hard since there are no vaccines available as well as the treatment is hampered by toxicity and resistant parasites. Furthermore, as human, and canine VL causes immunosuppression, the combination of drugs with immunostimulatory agents is interesting to upregulate the immunity, reducing side-effects, improving treatment approaches against disease. Herein, we assessed the immunochemotherapy using miltefosine along with a vaccine formulated by Leishmania braziliensis antigens + saponin + monophosphoryl lipid-A (LBSapMPL) in L. infantum-infected hamsters. Two months after infection, the animals received treatments, and after 15 days they were evaluated for the treatment effect. The potential anti-Leishmania effect of miltefosine + LBSapMPL-vaccine was revealed by a specific immune response activation reflecting in control of spleen parasitism using half the miltefosine treatment time. The treated animals also showed an increase of total and T-CD4 splenocytes producing IFN-γ and TNF-α and a decrease of interleukin-10 and anti-Leishmania circulating IgG. In addition, it was demonstrated that the control of spleen parasitism is related to the generation of a protective Th1 immune response. Hence, due to the combinatorial action of miltefosine with LBSapMPL-vaccine in immunostimulating and controlling parasitism, this immunochemotherapy protocol can be an important alternative option against canine and human VL.

Establishment of monoclonal antibodies to evaluate the cellular immunity in a hamster model of L infantum infection.

Syrian hamsters (Mesocricetus auratus) are largely used as a model for infectious diseases because it is very susceptible to several pathogens, including Leishmania spp. parasites. However, the research community faces limitations in its use due to the lack of immunological reagents and tools to study the immune system in this model. In this context, we proposed the validation of some important commercially anti-mouse mAbs (CD4, TNF-α, IFN-γ and IL-10) and how this could be useful to evaluate a specific cellular immune response in Leishmania-infected hamster using flow cytometry experiments. Our data demonstrated a cross-reactivity between these anti-mouse mAbs and hamster molecules that were herein studied. Beyond that, it was able to characterize the development of a specific cellular immune response through cytokine production in L infantum-infected hamsters when compared to uninfected ones. These data not only aid the usage of hamsters as experimental model to investigate various infectious diseases, but they contribute to the design of novel approaches to further investigate the immunological mechanisms associated to pathogen infections.

Recent advances and new strategies in Leishmaniasis diagnosis.

Leishmaniasis is a set of complex and multifaceted syndromes, with different clinical manifestations, caused by different species of the genus Leishmania spp. that can be characterized by at least four syndromes: visceral leishmaniasis (VL, also known as kala-azar), post-kala-azar dermal leishmaniasis (PKDL), cutaneous leishmaniasis (CL), and mucocutaneous leishmaniasis (MCL). Among the most serious clinical forms, VL stands out, which causes the death of around 59,000 people annually. Fast and accurate diagnosis in VL is essential to reduce the disease's morbidity and mortality. There are a large number of diagnostic tests for leishmaniasis, however they do cross-react with other protozoa and their sensitivity changes according to the clinical form of the disease. Thus, it is essential and necessary to provide a diagnosis that is sufficiently sensitive to detect asymptomatic infected individuals and specific to discriminate individuals with other infectious and parasitic diseases, thus enabling more accurate diagnostic tools than those currently used. In this context, the aim of this review is to summarize the conventional diagnostic tools and point out the new advances and strategies on visceral and cutaneous leishmaniasis diagnosis.

Recent advances and new strategies on leishmaniasis treatment.

Leishmaniasis is one of the most important tropical neglected diseases according to the World Health Organization. Even after more than a century, we still have few drugs for the disease therapy and their great toxicity and side effects put in check the treatment control program around the world. Moreover, the emergence of strains resistant to conventional drugs, co-infections such as HIV/Leishmania spp., the small therapeutic arsenal (pentavalent antimonials, amphotericin B and formulations, and miltefosine), and the low investment for the discovery/development of new drugs force researchers and world health agencies to seek new strategies to combat and control this important neglected disease. In this context, the aim of this review is to summarize new advances and new strategies used on leishmaniasis therapy addressing alternative and innovative treatment paths such as physical and local/topical therapies, combination or multi-drug uses, immunomodulation, drug repurposing, and the nanotechnology-based drug delivery systems.Key points• The treatment of leishmaniasis is a challenge for global health agencies.• Toxicity, side effects, reduced therapeutic arsenal, and drug resistance are the main problems.• New strategies and recent advances on leishmaniasis treatment are urgent.• Immunomodulators, nanotechnology, and drug repurposing are the future of leishmaniasis treatment.

Liver infusion tryptose (LIT): the best choice for growth, viability, and infectivity of Leishmania infantum parasites.

Leishmania spp. parasites have a complex biological cycle presenting basically two different morphological stages, the amastigote and promastigote forms. In vitro cultivation allows a more complete study of the biological aspects of these parasites, indicating better conditions for infection, immunoassay tests, drug evaluations, and vaccines. Thus, we evaluated the three most used culture media for Leishmania spp., Grace's insect cell culture medium (Grace's), liver infusion tryptose (LIT), and Schneider's insect medium (Schneider's), without supplementation or supplemented with fetal calf serum (FCS) and bovine serum albumin (Albumin) to evaluate the growth, viability, and infectivity of the L. infantum promastigotes. It was observed that promastigote forms have a better growth in LIT and Schneider's with or without FCS when compared to that in Grace's. The supplementation with albumin promoted greater viability of the parasites independent of the medium. For in vitro infection of J774.A1 macrophages using light microscopy and flow cytometry analyses, FCS-supplemented LIT and Grace's promoted higher percentage of infected macrophages and parasite load compared with Schneider's media. Taken together, our results demonstrated that the supplementation of LIT culture medium with FCS is the most suitable strategy to cultivate Leishmania infantum parasites enabling the maintenance of growth and infective parasites for research uses.

Multiplex flow cytometry serology to diagnosis of canine visceral leishmaniasis.

An accurate diagnosis of visceral leishmaniasis is an essential tool for control of the disease. While serologic methods are very useful, these conventional methodologies still present limitations in terms of sensitivity and specificity. The use of flow cytometry is a worldwide trend in the development of high-performance diagnostic methods. Herein, we describe a new flow cytometry serology test, characterized by the employment of the Cytometric Bead Array microspheres A4 and E4 coated with the recombinant antigens rLci1A and rLci2B respectively, to improve the serodiagnosis of canine visceral leishmaniasis. The tests were conducted in a wide variety of sera groups (n = 140), where the diagnostics development would be optimized accounting not just the ability to identify infected dogs with different clinical status, but also to exclude cross-reaction and differentiate vaccinated dogs from dogs infected. Serological testing of the antigenic system A4-rLci1A showed a sensitivity of 90.0% and specificity of 75%, while the E4-rLci2B testing demonstrated a sensitivity of 95.0% and specificity of 82.5%. The use of a multiplex assay of A4-rLci1A and E4-rLci2B, resulted in a diagnostic improvement, with a sensitivity of 95.0% and specificity of 91.2%. Our results show that this novel flow cytometry serology test is a viable tool for sensitive and specific serodiagnosis. Notably, the combination of distinct antigenic systems allows us to test for antibodies to multiple recombinant antigens from a single serum sample. This benefit emphasizes the importance of this methodology as an alternative in the serological diagnosis.

Comparative analysis of real-time PCR assays in the detection of canine visceral leishmaniasis.

Dogs are important hosts and reservoirs of leishmaniasis, a disease caused by protozoan parasites from the genus Leishmania, affecting ~12 million people worldwide. The detection of visceral leishmaniasis (VL) in dogs by real-time PCR (qPCR) may improve on diagnosis, but the different qPCR methods available for Leishmania DNA detection have not been established as routine in diagnostic tools and/or epidemiologic studies for canine VL. Here, we compared three qPCR assays (DNApol, Linj31, and LDON) in the detection of VL by Leishmania infantum in spleen (n = 48; 7), skin (n = 48; 7), and whole blood (n = 44; 7) samples from serologically positive and negative dogs, respectively. Overall, the DNApol performed better than the Linj31 and LDON assays in the detection of positive samples in all tissues tested, yielding from 66.7 to 100.0% of positivity for both skin and spleen samples. For spleen samples, we observed no statistically significant differences between positive detection by the LDON and DNApol assays. Whole blood samples yielded the lowest rates of positive detection, regardless of the qPCR assay used. In contrast, positive detection of Leishmania DNA was as efficient from skin samples using the DNApol assay as from spleen samples using either the DNApol or the LDON assay. Although qPCR assays from skin samples may not be practical for use in the field, our study suggests that the DNApol and LDON assays from skin samples could be used in future to evaluate canine VL treatment in veterinary clinics.

Mixed Formulation of Conventional and Pegylated Meglumine Antimoniate-Containing Liposomes Reduces Inflammatory Process and Parasite Burden in Leishmania infantum-Infected BALB/c Mice.

Pentavalent antimonial has been the first choice treatment for visceral leishmaniasis; however, it has several side effects that leads to low adherence to treatment. Liposome-encapsulated meglumine antimoniate (MA) arises as an important strategy for chemotherapy enhancement. We evaluated the immunopathological changes using the mixture of conventional and pegylated liposomes with MA. The mice were infected with Leishmania infantum and a single-dose treatment regimen. Comparison was made with groups treated with saline, empty liposomes, free MA, and a liposomal formulation of MA (Lipo MA). Histopathological analyses demonstrated that animals treated with Lipo MA showed a significant decrease in the inflammatory process and the absence of granulomas. The in vitro stimulation of splenocytes showed a significant increase of gamma interferon (IFN-γ) produced by CD8+ T cells and a decrease in interleukin-10 (IL-10) produced by CD4+ and CD8+ T cells in the Lipo MA. Furthermore, the Lipo MA group showed an increase in the IFN-γ/IL-10 ratio in both CD4+ and CD8+ T cell subsets. According to the parasite load evaluation using quantitative PCR, the Lipo MA group showed no L. infantum DNA in the spleen (0.0%) and 41.4% in the liver. In addition, we detected a low positive correlation between parasitism and histopathology findings (inflammatory process and granuloma formation). Thus, our results confirmed that Lipo MA is a promising antileishmanial formulation able to reduce the inflammatory response and induce a type 1 immune response, accompanied by a significant reduction of the parasite burden into hepatic and splenic compartments in treated animals.

The TcI and TcII Trypanosoma cruzi experimental infections induce distinct immune responses and cardiac fibrosis in dogs.

Trypanosoma cruzi infection may be caused by different strains with distinct discrete typing units (DTUs) that can result in variable clinical forms of chronic Chagas disease. The present study evaluates the immune response and cardiac lesions in dogs experimentally infected with different T. cruzi strains with distinct DTUs, namely, the Colombian (Col) and Y strains of TcI and TcII DTU, respectively. During infection with the Col strain, increased levels of alanine aminotransferase, erythrocytes, haematocrit and haemoglobin were observed. In addition, CD8+ T-lymphocytes isolated from the peripheral blood produced higher levels of interleukin (IL)-4. The latter suggests that during the acute phase, infection with the Col strain may remain unnoticed by circulating mononuclear cells. In the chronic phase, a significant increase in the number of inflammatory cells was detected in the right atrium. Conversely, infection with the Y strain led to leucopoenia, thrombopoenia, inversion of the ratio of CD4+/CD8+ T-lymphocytes and alterations in monocyte number. The Y strain stimulated the production of interferon-γ by CD4+ and CD8+ T-lymphocytes and IL-4 by CD8+ T-cells. In the chronic phase, significant heart inflammation and fibrosis were observed, demonstrating that strains of different DTUs interact differently with the host.

Diagnosis of Canine Visceral Leishmaniasis by Flow Cytometry Serology using the rMELEISH Multiepitope Antigen Coupled in a Functional Bead.

BACKGROUND: Visceral leishmaniasis (VL) is a zoonotic disease, with dogs being the main reservoir of the Leishmania infantum parasite. OBJECTIVE: To develop a new flow cytometry test to diagnosis canine VL (CVL) diagnosis. METHODS: The current study addresses a new flow cytometry test using beads coupled to the multiepitope antigen rMELEISH. RESULTS: In the study set of samples a sensitivity (87.1%) and specificity (89.9%) was observed. Considering the dogs' clinical status, 20/20 (100.0%) of the symptomatic sera tested positive, while 19/22 (86.4%) of the oligosymptomatic and 16/20 (80.0%) of asymptomatic were positive. In the non-infected control, all samples (0/30) tested as negative. In the cross-reaction control, the test was more efficient in dogs infected with L. braziliensis (2/10) and Trypanosoma cruzi (0/10), than those with Babesia canis (4/10) and Ehrlichia canis (4/10). Dogs immunized with different vaccines (Leishmune, Leish-Tec®, or LBSap) did not present serological reactivity. CONCLUSION: The flow cytometry serology through coupling the antigen rMELEISH in functional beads showed high accuracy in diagnosing CVL.

Ivabradine restores tonic cardiovascular autonomic control and reduces tachycardia, hypertension and left ventricular inflammation in post-weaning protein malnourished rats.

Malnutrition results in autonomic imbalance and heart hypertrophy. Overexpression of hyperpolarization-activated cyclic nucleotide-gated channels (HCN) in the left ventricles (LV) is linked to hypertrophied hearts and abnormal myocardium automaticity. Given that ivabradine (IVA) has emerging pleiotropic effects, in addition to the widely known bradycardic response, this study evaluated if IVA treatment could repair the autonomic control and cardiac damages in malnourished rats. AIM: Assess the impact of IVA on tonic cardiovascular autonomic control and its relationship with hemodynamics regulation, LV inflammation, and HCN gene expression in post-weaning protein malnutrition condition. MAIN METHODS: After weaning, male rats were divided into control (CG; 22 % protein) and malnourished (MG; 6 % protein) groups. At 35 days, groups were subdivided into CG-PBS, CG-IVA, MG-PBS and MG-IVA (PBS 1 ml/kg or IVA 1 mg/kg) received during 8 days. We performed jugular vein cannulation and electrode implant for drug delivery and ECG registration to assess tonic cardiovascular autonomic control; femoral cannulation for blood pressure (BP) and heart rate (HR) assessment; and LV collection to evaluate ventricular remodeling and HCN gene expression investigation. KEY FINDINGS: Malnutrition induced BP and HR increases, sympathetic system dominance, and LV remodeling without affecting HCN gene expression. IVA reversed the cardiovascular autonomic imbalance; prevented hypertension and tachycardia; and inhibited the LV inflammatory process and fiber thickening caused by malnutrition. SIGNIFICANCE: Our findings suggest that ivabradine protects against malnutrition-mediated cardiovascular damage. Moreover, our results propose these effects were not attributed to HCN expression changes, but rather to IVA pleiotropic effects on autonomic control and inflammation.

The Use of an Adjuvant System Improves Innate and Adaptive Immune Response When Associated with a Leishmania (Viannia) braziliensis Antigen in a Vaccine Candidate against L. (Leishmania) infantum Infection.

BACKGROUND: The adjuvants' optimal dose and the administration route can directly influence the epitope recognition patterns and profiles of innate response. We aimed to establish the effect and the optimal dose of adjuvant systems for proposing a vaccine candidate to be employed with Leishmania (Viannia) braziliensis. METHODS: We evaluated the adjuvants saponin (SAP), monophosphoryl lipid A (MPL) and resiquimod (R-848) isolated and combined as adjuvant systems in a lower dose corresponding to 25%, 33%, and 50% of each adjuvant total dose. Male outbred BALB/c mice were divided into 13 groups, SAP, MPL, and R-848 isolated, and the adjuvant systems SAP plus MPL (SM), SAP plus R-848 (SR), and MPL plus R-848 (MR). RESULTS: SM50 increased levels of all chemokines analyzed and TNF production, while it presented an increased inflammatory cell infiltrate in the skin with macrophage recruitment. Thus, we proposed a vaccine candidate employing L. (V.) braziliensis antigen associated with the SM adjuvant system against experimental L. (Leishmania) infantum challenge. We observed a significant increase in the frequency of cells expressing the central and effector memory CD4+ T cells phenotype in immunized mice with the LBSM50. In the liver, there was a decreased parasite load when mice received LBSM50. CONCLUSIONS: When combined with L. (V.) braziliensis antigen, SM50 increases TNF and IFN-γ, which generates central and effector memory CD4+ T cells. Therefore, using an adjuvant system can promote an effective innate immune response with the potential to compose future vaccines.

Immunoproteomics approach for the discovery of antigens applied to the diagnosis of canine visceral leishmaniasis.

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.

CD4+ T-lymphocytes from asymptomatic dogs infected with Leishmania infantum are able to activate macrophages for higher leishmanicidal ability in an in vitro co-culture experiment.

Dogs are the most common domestic reservoir of Leishmania infantum, making canine visceral leishmaniasis (CVL) a serious public health issue. Identifying new methodologies that can mimic lymphoid and myeloid competence in naturally infected dogs could lower costs and save time in preliminary screenings of potential immunotherapeutic agents and vaccines against CVL. For that, we established a cell-to-cell communication approach between lymphocytes and myeloid cells from healthy, asymptomatic (infected, without apparent clinical signs) and symptomatic (infected with apparent clinical signs) dogs. Peripheral blood mononuclear cells (PBMC) from these dogs were used as source of CD4+, CD8+ T lymphocytes and macrophages, that were posteriorly infected with L. infantum GFP+ promastigotes (green fluorescent protein). Macrophages co-cultured with purified lymphocytes were tested for the ability to control cellular parasitism, and their microbicidal function by producing nitric oxide (NO) and reactive oxygen species (ROS). The kind of T cell response within the co-culture was also evaluated, by assessing their ability to produce interferon-gamma (IFN-γ) and interleukin 4 (IL-4). The data suggests that T lymphocytes from symptomatic dogs are more prone to produce IL-4 than the ones from asymptomatic dogs. Macrophages from asymptomatic dogs also demonstrated a higher microbicidal potential, with increased levels of NO and ROS production, compared to symptomatic dogs, mainly in highly parasitized cells. Together, our results identify the ratio of IL-4/IFN-γ produced by CD4+ and CD8+ T cells, as well as, the ratio between parasite GFP signal/NO and ROS signal in macrophages as potential immunological biomarkers of failure and success of the screened agents. Our findings also propose a reliable methodology that can be used to follow the immune response in trials of potential drugs or vaccines targeting CVL.

The entrance route: Oral, mucous, cutaneous, or systemic has a marked influence on the outcome of Trypanosoma cruzi experimental infection.

In recent decades, the oral infection of Trypanosoma cruzi has gathered increased attention due to frequent outbreaks that can lead to more severe clinical signs than those usually found in the areas of vector transmission. This study addresses the main routes of infection using metacyclic trypomastigotes (MT) and blood trypomastigotes (BT). Herein, BALB/c mice were infected with the Colombian (TcI) strain via intraperitoneal (IP), oral, intragastric (IG), ocular (OC) and cutaneous (CT) routes with 106 culture-derived MT or BT. Parasitemia was intermittent and low in animals inoculated with MT, in contrast, high parasitemia levels were found in BT-mice. A tropism for the muscles was observed in oral or IG infection with BT. Differently, the parasite was widely distributed in the tissues of mice infected with MT. However, the intensity of the inflammation infiltrating the tissues was higher in oral or IG infection with BT. Animals inoculated with BT via the IG route had similar serum levels of IFN-γ and smaller IL-10 compared to those infected with MT via the IG route. TNF-α levels were higher in the serum from BT-animals, which could explain the higher intensity of heart inflammation in these animals. Our results suggest that the infective form and the route of infection differentially modulated the outcome of Trypanosoma cruzi mice infection.

Successive Pandemic Waves with Different Virulent Strains and the Effects of Vaccination for SARS-CoV-2.

One hundred years after the flu pandemic of 1918, the world faces an outbreak of a new severe acute respiratory syndrome, caused by a novel coronavirus. With a high transmissibility, the pandemic has spread worldwide, creating a scenario of devastation in many countries. By the middle of 2021, about 3% of the world population had been infected and more than 4 million people had died. Different from the H1N1 pandemic, which had a deadly wave and ceased, the new disease is maintained by successive waves, mainly produced by new virus variants and the small number of vaccinated people. In the present work, we create a version of the SIR model using the spatial localization of persons, their movements, and considering social isolation probabilities. We discuss the effects of virus variants, and the role of vaccination rate in the pandemic dynamics. We show that, unless a global vaccination is implemented, we will have continuous waves of infections.

Immunoprophylaxis using polypeptide chimera vaccines plus adjuvant system promote Th1 response controlling the spleen parasitism in hamster model of visceral leishmaniasis.

In recent years, several advances have been observed in vaccinology especially for neglected tropical diseases (NTDs). One of the tools employed is epitope prediction by immunoinformatic approaches that reduce the time and cost to develop a vaccine. In this scenario, immunoinformatics is being more often used to develop vaccines for NTDs, in particular visceral leishmaniasis (VL) which is proven not to have an effective vaccine yet. Based on that, in a previous study, two predicted T-cell multi-epitope chimera vaccines were experimentally validated in BALB/c mice to evaluate the immunogenicity, central and effector memory and protection against VL. Considering the results obtained in the mouse model, we assessed the immune response of these chimeras inMesocricetus auratushamster, which displays, experimentally, similar pathological status to human and dog VL disease. Our findings indicate that both chimeras lead to a dominant Th1 response profile, inducing a strong cellular response by increasing the production of IFN-γ and TNF-α cytokines associated with a decrease in IL-10. Also, the chimeras reduced the spleen parasite load and the weight a correlation between protector immunological mechanisms and consistent reduction of the parasitic load was observed. Our results demonstrate that both chimeras were immunogenic and corroborate with findings in the mouse model. Therefore, we reinforce the use of the hamster as a pre-clinical model in vaccination trials for canine and human VL and the importance of immunoinformatic to identify epitopes to design vaccines for this important neglected disease.

LBMPL Vaccine Therapy Induces Progressive Organization of the Spleen Microarchitecture, Improved Th1 Adaptative Immune Response and Control of Parasitism in Leishmania infantum Naturally Infected Dogs.

The spleen plays a central role in human and canine visceral leishmaniasis, where the activation of the immune response occurs in one of the tissues where Leishmania infantum reproduces. Therefore, this organ is both a target to understand the mechanisms involved in the parasite control and a parameter for assessing the therapeutic response. In this sense, this study aimed to evaluate the main histological, immunological and parasitological aspects in the spleen of symptomatic dogs naturally infected by L. infantum treated with the therapeutic vaccine LBMPL. For this, dogs were divided into four groups: dogs uninfected and untreated (NI group); L. infantum-infected dogs that were not treated (INT group); L. infantum-infected dogs that received treatment only with monophosphoryl lipid A adjuvant (MPL group); and L. infantum-infected dogs that received treatment with the vaccine composed by L. braziliensis promastigote proteins associated with MPL adjuvant (LBMPL group). Ninety days after the therapeutics protocol, the dogs were euthanized and the spleen was collected for the proposed evaluations. Our results demonstrated a reduction of hyperplasia of red pulp and follicular area of white pulp, increased mRNA expression of IFN-γ, TNF-α, IL-12 and iNOS, and decreased IL-10 and TGF-ß1, and intense reduction of splenic parasitism in dogs treated with the LBMPL vaccine. These results possibly suggest that the pro-inflammatory environment promoted the progressive organization of the splenic architecture favoring the cellular activation, with consequent parasite control. Along with previously obtained data, our results propose the LBMPL vaccine as a possible treatment strategy for canine visceral leishmaniasis (CVL).

Comparative evaluation of meglumine antimoniate encapsulated in a mixture of conventional and PEGylated liposomes and immunotherapy using an anti-canine IL-10 receptor-blocking monoclonal antibody on canine visceral leishmaniasis.

This study compared the therapeutic potential of the chemotherapy using meglumine antimoniate encapsulated in a mixture of conventional and PEGylated liposomes (Nano Sbv) and immunotherapy with anti-canine IL-10 receptor-blocking monoclonal antibody (Anti IL-10R) on canine visceral leishmaniasis (CVL). Twenty mongrel dogs naturally infected by L. infantum, displaying clinical signs of visceral leishmaniasis were randomly divided in two groups. In the first one, nine dogs received six intravenous doses of a mixture of conventional and PEGylated liposomes containing meglumine antimoniate at 6.5 mg Sb/kg/dose. In the second one, eleven dogs received two intramuscular doses of 4 mg of anti-canine IL-10 receptor-blocking monoclonal antibody. The animals were evaluated before (T0) and 30, 90, and 180 days after treatments. Our major results demonstrated that both treatments were able to maintain hematological and biochemical parameters, increase circulating T lymphocytes subpopulations, increase the IFN-γ producing T-CD4 lymphocytes, restore the lymphoproliferative capacity and improve the clinical status. However, although these improvements were observed in the initial post-treatment times, they did not maintain until the end of the experimental follow-up. We believe that the use of booster doses or the association of chemotherapy and immunotherapy (immunochemotherapy) is promising to improve the effectiveness of treating CVL for improving the clinical signs and possibly reducing the parasite burden in dogs infected with Leishmania infantum.