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.

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).

Hepatoprotective, antioxidant, anti-inflammatory, and antiviral activities of silymarin against mayaro virus infection.

Infection caused by Mayaro virus (MAYV) is responsible for causing acute nonspecific fever, in which the majority of patients develop incapacitating and persistent arthritis/arthralgia. Mayaro fever is a neglected and underreported disease without treatment or vaccine, which has gained attention in recent years after the competence of Aedes aegypti to transmit MAYV was observed in the laboratory, coupled with the fact that cases are being increasingly reported outside of endemic forest areas, calling attention to the potential of an urban cycle arising in the near future. Thus, to mitigate the lack of information about the pathological aspects of MAYV, we previously described the involvement of oxidative stress in MAYV infection in cultured cells and in a non-lethal mouse model. Additionally, we showed that silymarin, a natural compound, attenuated MAYV-induced oxidative stress and inhibited MAYV replication in cells. The antioxidant and anti-MAYV effects prompted us to determine whether silymarin could also reduce oxidative stress and MAYV replication after infection in an immunocompetent animal model. We show that infected mice exhibited reduced weight gain, hepatomegaly, splenomegaly, anaemia, thrombocytopenia, leukopenia, increased liver transaminases, increased pro-inflammatory cytokines and liver inflammation, increased oxidative damage biomarkers, and reduced antioxidant enzyme activity. However, in animals infected and treated with silymarin, all these parameters were reversed or significantly improved, and the detection of viral load in the liver, spleen, brain, thigh muscle, and footpad was significantly reduced. This work reinforces the potent hepatoprotective, antioxidant, anti-inflammatory, and antiviral effects of silymarin against MAYV infection, demonstrating its potential against Mayaro fever disease.

IL-10 receptor blockade controls the in vitro infectivity of Leishmania infantum and promotes a Th1 activation in PBMC of dogs with visceral leishmaniasis.

An important strategy to reduce the risk of visceral leishmaniasis (VL) in humans is to control the infection and disease progression in dogs, the domestic reservoir of Leishmania infantum parasites. Certain therapeutic strategies that modulate the host immune response show great potential for the treatment of experimental VL, restoring the impaired effector functions or decreasing host excessive responses. It is known that the overproduction of interleukin-10 (IL-10) promotes parasite replication and disease progression in human VL as well as in canine visceral leishmaniasis (CVL). Thus, in the present study we investigated the potential of the anti-canine IL-10 receptor-blocking monoclonal antibody (Bloq IL-10R) to control and reduce in vitro infectivity of L. infantum and improve the ability of PBMC isolated from VL dogs to alter the lymphoproliferative response and intracytoplasmic cytokines. Overall, GFP+Leishmania showed lower capacity of in vitro infectivity in the presence of Bloq IL-10R. Moreover, addition of Bloq IL-10R in cultured PBMC enhanced T-CD4 and CD8 proliferative response and altered the intracytoplasmic cytokine synthesis, reducing CD4+IL-4+ cells and increasing CD8+IFN-γ+ cells after specific antigen stimulation in PBMC of dogs. Furthermore, we observed an increase of TNF-α levels in supernatant of cultured PBMC under IL-10R neutralizing conditions. Together, our findings are encouraging and reaffirm an important factor that could influence the effectiveness of immune modulation in dogs with VL and suggest that blocking IL-10R activity has the potential to be a useful approach to CVL treatment.

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.

Dogs immunized with LBSap vaccine displayed high levels of IL-12 and IL-10 cytokines and CCL4, CCL5 and CXCL8 chemokines in the dermis.

The complex interplay between cytokines and chemokines regulates innate and adaptive immune responses against pathogens; specifically, cytokine and chemokine expression drives activation of immune effector cells and their recruitment to tissue infection sites. Herein, we inoculated dogs with Leishmania braziliensis antigens plus saponin (the LBSap vaccine), as well as with the vaccine components, and then used real-time PCR to evaluate the kinetics of dermal expression of mRNAs of cytokines (IL-12, IFN-γ, TNF-α, IL-4, IL-13, TGF-ß and IL-10) and chemokines (CCL2, CCL4, CCL5, CCL21 and CXCL8) 1, 12, 24 and 48 h after inoculation. We also evaluated the correlation between cytokine and chemokine expression and dermal cellularity. The LBSap vaccine induced high levels of IL-12 and IL-10 expression at 12 and 24 h, respectively. Furthermore, we observed positive correlations between IL-12 and IL-13 expression, IFN-γ and IL-13 expression, and IL-13 and TGF-ß expression, suggesting that a mixed cytokine microenvironment developed after immunization with the vaccine. Inoculation with the saponin adjuvant alone induced a chemokine and cytokine expression profile similar to that observed in the LBSap group. CCL4 and CXCL8 chemokine expression was up regulated by the LBSap vaccine. CCL5 expression was initially highest in the LBSap group, but at 48 h, expression was highest in the LB group. Information about the kinetics of the immune response to this vaccine gained using this dog model will help to elucidate the mechanisms of and factors involved in a protective response against Leishmania infection and will aid in establishing rational approaches for the development of vaccines against canine visceral leishmaniasis.

Parasite burden in hamsters infected with two different strains of leishmania (Leishmania) infantum: "Leishman Donovan units" versus real-time PCR.

To develop and test new therapeutics and immune prophylaxis strategies for visceral leishmaniasis (VL), understanding tissue parasitism evolution after experimental infection with Leishmania infantum is important. Experimental infection in a hamster model (Mesocricetus auratus) reproduces several typical aspects of canine and human VL that are closely related to the inoculum's route. We quantified the parasitism in the liver and spleen of hamsters experimentally infected by various routes (intradermal, intraperitoneal, and intracardiac [IC]) and different strains of L. infantum (MHOM/BR/74/PP75 and Wild) and compared two different methodologies to evaluate tissue parasitism (Leishman Donovan units [LDU] and real-time qPCR). In addition, the quantification of specific total-IgG in the serum of uninfected and infected hamsters was determined by ELISA. The animals were followed for 1, 3, 6 and 9 months post-infection for survival analysis. We found that infection with the Wild strain by the IC route resulted in higher mortality. Positive antibody (IgG) responses were detected with higher peaks at 6 and 9 months in the IC group inoculated with PP75 strain. However, in animals infected with the Wild strain the IgG levels were elevated in all infected groups during all the time evaluated. We also observed by LDU analysis that the IC route lead to higher parasitism in the liver and spleen with both strains. Furthermore, qPCR showed higher sensitivity for identifying animals with low parasitic burden. In conclusion, qPCR can be useful for assessing parasitism in the spleen and liver of a hamster model infected with L. infantum independent of the route of infection, and this technique may become an essential tool for assessing parasite density in the hamster model after experimental treatment or immunization with potential vaccine candidates.