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).

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.

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.

Sesquiterpene lactones from Lychnophora species: Antinociceptive, anti-inflammatory, and antioxidant pathways to treat acute gout.

ETHNOPHARMACOLOGICAL RELEVANCE: Lychnophora trichocarpha and Lychnophora passerina are species used in folk medicine to treat inflammation, pain, and rheumatism. Previous studies have demonstrated the anti-inflammatory effect of ethanol extracts of these species and identified that sesquiterpene lactones contribute to this activity. AIM OF THE STUDY: Gout is an acute inflammatory arthritis caused by the deposition of monosodium urate (MSU) crystals in joints. Inflammation in joints induces oxidative stress in defense cells, releasing pro-inflammatory mediators. This study has three objectives: (1) to demonstrate the effects of sesquiterpene lactones lychnopholide and eremantholide C isolated from L. trichocarpha and goyazensolide isolated from L. passerina on arthritis induced by MSU crystals in C57BL6 mice; (2) to determine whether or not these compounds can inhibit the migration of neutrophils and the release of TNF-α and IL-1ß cytokines in the inflammation region; and (3) to evaluate the effects of sesquiterpene lactones on the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) in the cartilage of C57BL/6 mice with gouty arthritis. MATERIALS AND METHODS: The anti-inflammatory, antinociceptive, and antioxidant activities of sesquiterpene lactones in C57BL/6 mice with MSU crystal-induced arthritis were evaluated. In our experimental model, the mice were injected with MSU crystals in the tibiofemoral joint to induce arthritis and then treated with indomethacin, vitamin C, and sesquiterpene lactones. Nociception was evaluated before and after inflammation induction and treatments, neutrophil migration, IL-1ß and TNF-α concentrations, and SOD and CAT activities. RESULTS: Sesquiterpene lactones exerted an anti-inflammatory effect by inhibiting neutrophil migration and TNF-α production. These compounds also demonstrated antinociceptive and antioxidant activities. CONCLUSION: Lychnopholide, eremantholide C, and goyazensolide improved the inflammation induced by MSU crystals by inhibiting the migration of neutrophils to the inflamed area and by blocking the release of the pro-inflammatory cytokine TNF-α. In addition, sesquiterpene lactones reduced oxidative stress by activating SOD and CAT. These results suggest that sesquiterpene lactones have anti-gout activity through the inflammation, pain, and oxidative stress pathways.

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.

Antiproliferative and toxicogenomic effects of resveratrol in bladder cancer cells with different TP53 status.

The antitumor activity of resveratrol, a polyphenolic compound found mainly in grapes, has been studied in several types of cancer. In bladder cancer, its antiproliferative effects have already been demonstrated; however, its mechanism of action is not completely understood. The aim of this study was to evaluate resveratrol antitumor activity (12.5, 25, 50, 100, 150, 200, and 250 µM) and its possible mechanisms of action in bladder tumor cells with different TP53 gene status (RT4, grade 1, TP53 wild type; 5637-grade 2 and T24-grade 3, TP53 mutated). Cell proliferation, clonogenic survival, morphological changes, cell cycle progression, apoptosis rates, genotoxicity, global methylation, immunocytochemistry for p53 and PCNA and relative expression profiles of the AKT, mTOR, RASSF1A, HOXB3, SRC, PLK1, and DNMT1 were evaluated. Resveratrol decreased cell proliferation and induced DNA damage in all cell lines. Regarding the long-term effects, resveratrol reduced the number of colonies in all cell lines; however, TP53 wild type cells were more resistant. Increased rates of apoptosis were found in the TP53 wild type cells and this was accompanied by AKT, mTOR, and SRC downregulation. In addition, the resveratrol antiproliferative effects in wild type TP53 cells were accompanied by modulation of the DNMT1 gene. In the TP53 mutated cells, cell cycle arrest at S phase with PLK1 downregulation was observed. Additionally, there was modulation of the HOXB3/RASSF1A pathway and nuclear PCNA reduction in the highest-grade cells. In conclusion, resveratrol has antiproliferative activity in bladder tumor cells; however, the mechanisms of action are dependent on TP53 status. Environ. Mol. Mutagen., 60:740-751, 2019. © 2019 Wiley Periodicals, Inc.

Functionalized rifampicin-loaded nanostructured lipid carriers enhance macrophages uptake and antimycobacterial activity.

Tuberculosis is an infectious bacterial disease that causes millions of deaths worldwide. Current treatment recommended by WHO is effective, however it is an extensive and arduous process associated to severe adverse effects, which induces a low patient compliance and the emerging of multidrug resistant tuberculosis. Thus, as a main goal of this study, rifampicin nanoparticles were surface functionalized with a tuftsin-modifed peptide to selectively recognize receptors located on infected alveolar macrophages, enhancing nanoparticles uptake by these cells and improving antimycobacterial activity. A tuftsin-based modified peptide was synthesized and successfully attached to nanoparticles interface (NP-pRIF). In parallel, nanoparticles without peptide were also developed for comparison (NP-RIF). Physicochemical characterization demonstrated that stable and monodisperse nanodelivery systems were obtained, with a controlled drug release profile and non-cytotoxic potential. Moreover, nanoparticles containing peptide were significantly more internalized by macrophages than nanoparticles without peptide over a wide range of time. Both nanoparticles were 2-fold more effective against M. tuberculosis than free rifampicin, suggesting NP-pRIF as a promising strategy for the management of tuberculosis treatment.

The Schistosomiasis SpleenOME: Unveiling the Proteomic Landscape of Splenomegaly Using Label-Free Mass Spectrometry.

Schistosomiasis is a neglected parasitic disease that affects millions of people worldwide and is caused by helminth parasites from the genus Schistosoma. When caused by S. mansoni, it is associated with the development of a hepatosplenic disease caused by an intense immune response to the important antigenic contribution of adult worms and to the presence of eggs trapped in liver tissue. Although the importance of the spleen for the establishment of immune pathology is widely accepted, it has received little attention in terms of the molecular mechanisms operating in response to the infection. Here, we interrogated the spleen proteome using a label-free shotgun approach for the potential discovery of molecular mechanisms associated to the peak of the acute phase of inflammation and the development of splenomegaly in the murine model. Over fifteen hundred proteins were identified in both infected and control individuals and 325 of those proteins were differentially expressed. Two hundred and forty-two proteins were found upregulated in infected individuals while 83 were downregulated. Functional enrichment analyses for differentially expressed proteins showed that most of them were categorized within pathways of innate and adaptive immunity, DNA replication, vesicle transport and catabolic metabolism. There was an important contribution of granulocyte proteins and antigen processing and presentation pathways were augmented, with the increased expression of MHC class II molecules but the negative regulation of cysteine and serine proteases. Several proteins related to RNA processing were upregulated, including splicing factors. We also found indications of metabolic reprogramming in spleen cells with downregulation of proteins related to mitochondrial metabolism. Ex-vivo imunophenotyping of spleen cells allowed us to attribute the higher abundance of MHC II detected by mass spectrometry to increased number of macrophages (F4/80+/MHC II+ cells) in the infected condition. We believe these findings add novel insights for the understanding of the immune mechanisms associated with the establishment of schistosomiasis and the processes of immune modulation implied in the host-parasite interactions.

Immunological profile of resistance and susceptibility in naturally infected dogs by Leishmania infantum.

Visceral leishmaniasis has a great impact on public health, and dogs are considered the main domestic reservoir of Leishmania infantum, the causal parasite. In this study, 159 animals naturally infected by L. infantum from an endemic area of Brazil were evaluated through an analysis of cellular responses, using flow cytometry, and of the hematological parameters. The results confirmed that disease progression is associated with anemia and reductions in eosinophils, monocytes and lymphocytes. The investigation of the immune response, based on the immunophenotypic profile of peripheral blood, showed declines in the absolute numbers of T lymphocytes CD5(+) and their subsets (CD4(+) and CD8(+)) and a drop of B lymphocytes in asymptomatic seropositive (AD-II) and symptomatic seropositive (SD) dogs. Neutrophils, when stimulated with soluble antigen of L. infantum, showed higher synthesis of interferon (IFN)-γ(+) in AD-II and SD groups, with decreased production of interleukin (IL)-4(+) in asymptomatic seronegative dogs positive for L. infantum infection based on polymerase chain reaction testing (AD-I group). In the AD-II and SD groups, subpopulations of stimulated lymphocytes (CD4(+) and CD8(+)) also exhibited greater synthesis of IFN-γ(+) and IL-4(+) in culture. These results suggest that the animals of the AD-II and SD groups exhibited a mixed immune response (Type 1 and 2) and the AD-I group presenting an immune profile very similar to normal control animals.

Shotgun proteomics to unravel the complexity of the Leishmania infantum exoproteome and the relative abundance of its constituents.

The exoproteome of some Leishmania species has revealed important insights into host-parasite interaction, paving the way for the proposal of novel disease-oriented interventions. The focus of the present investigation constituted the molecular profile of the L. infantum exoproteome revealed by a shotgun proteomic approach. Promastigotes under logarithmic phase of growth were obtained and harvested by centrifugation at different time points. Cell integrity was evaluated through the counting of viable parasites using propidium iodide labeling, followed by flow cytometry analysis. The 6h culture supernatant, operationally defined here as exoproteome, was then conditioned to in solution digestion and the resulting peptides submitted to mass spectrometry. A total of 102 proteins were identified and categorized according to their cellular function. Their relative abundance index (emPAI) allowed inference that the L. infantum exoproteome is a complex mixture dominated by molecules particularly involved in nucleotide metabolism and antioxidant activity. Bioinformatic analyses support that approximately 60% of the identified proteins are secreted, of which, 85% possibly reach the extracellular milieu by means of non-classic pathways. At last, sera from naturally infected animals, carriers of differing clinical forms of Canine Visceral Leishmaniasis (CVL), were used to test the immunogenicity associated to the L. infantum exoproteome. Western blotting experiments revealed that this sub-proteome was useful at discriminating symptomatic animals from those exhibiting other clinical forms of the disease. Collectively, the molecular characterization of the L. infantum exoproteome and the preliminary immunoproteomic assays opened up new research avenues related to treatment, prognosis and diagnosis of CVL.

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.

A killed Leishmania vaccine with sand fly saliva extract and saponin adjuvant displays immunogenicity in dogs.

A vaccine against canine visceral leishmaniasis (CVL), comprising Leishmania braziliensis promastigote protein, sand fly gland extract (SGE) and saponin adjuvant, was evaluated in dog model, in order to analyse the immunogenicity of the candidate vaccine. The vaccine candidate elicited strong antigenicity in dogs in respect of specific SGE and Leishmania humoral immune response. The major saliva proteins recognized by serum from immunized dogs exhibited molecular weights of 35 and 45 kDa, and were related to the resistance pattern against Leishmania infection. Immunophenotypic analysis revealed increased circulating CD21+ B-cells and CD5+ T-cells, reflected by higher counts of CD4+ and CD8+ T-cells. The observed interaction between potential antigen-presenting cells (evaluated as CD14+ monocytes) and lymphocyte activation status indicated a relationship between innate and adaptive immune responses. The higher frequency in L. chagasi antigen-specific CD8+ T-lymphocytes, and their positive association with intense cell proliferation, in addition to the progressively higher production of serum nitric oxide levels, showed a profile compatible with anti-CVL vaccine potential. Further studies on immunological response after challenge with L. chagasi may provide important information that will lead to a better understanding on vaccine trial and efficacy.

Immunogenicity of a killed Leishmania vaccine with saponin adjuvant in dogs.

Cellular and humoral immune responses of dogs to a candidate vaccine, composed of Leishmania braziliensis promastigote protein plus saponin as adjuvant, have been investigated as a pre-requisite to understanding the mechanisms of immunogenicity against canine visceral leishmaniasis (CVL). The candidate vaccine elicited strong antigenicity related to the increases of anti-Leishmania IgG isotypes, together with higher levels of lymphocytes, particularly of circulating CD8(+) T-lymphocytes and Leishmania chagasi antigen-specific CD8(+) T-lymphocytes. As indicated by the intense cell proliferation and increased nitric oxide production during in vitro stimulation by L. chagasi soluble antigens, the candidate vaccine elicited an immune activation status potentially compatible with effective control of the etiological agent of CVL.