Placement of a subcutaneous ureteral bypass in a Miniature Pinscher with presumed xanthine urolithiasis as a result of allopurinol treatment.

INTRODUCTION: This case report describes the long-term success of a subcutaneous ureteral bypass device in a dog for treatment of a ureteral obstruction. The suspected xanthine urolithiasis was secondary to treatment with allopurinol for leishmaniasis. The dog presented initially with lethargy, anuria and abdominal pain. Mild azotemia was found on biochemical analysis and abdominal ultrasound revealed bilateral ureteral obstruction. A subcutaneous ureteral bypass was subsequently placed using a standard surgical technique. The dog recovered uneventfully and the azotemia resolved within days. Follow-up examinations were performed every trimester for over three years and no complications like obstruction of the bypass tubes, urinary tract infection or azotemia were recognized during this follow-up period. Allopurinol was replaced with domperidone as long-term treatment against Leishmaniasis which resulted in a mild increase of the leishmania serum antibody titer. The subcutaneous ureteral bypass placement was successful and safe in this dog and is a valuable alternative in cases of ureteral obstruction also in dogs.

INTRODUCTION: Ce rapport de cas décrit le succès à long terme d'une dérivation urétérale sous-cutanée chez un chien pour le traitement d'une obstruction urétérale. L'urolithiase xanthique suspectée était secondaire à un traitement à l'allopurinol contre la leishmaniose. Le chien a d'abord présenté une léthargie, une anurie et des douleurs abdominales. L'analyse biochimique a révélé une légère azotémie et l'échographie abdominale a révélé une obstruction urétérale bilatérale. Une dérivation urétérale sous-cutanée a été mise en place selon une technique chirurgicale standard. Le chien s'est rétabli sans incident et l'azotémie a disparu en quelques jours. Des examens de suivi ont été effectués tous les trimestres pendant plus de trois ans et aucune complication telle qu'une obstruction du tube de dérivation, une infection urinaire ou une azotémie n'a été constatée au cours de cette période de suivi. L'allopurinol a été remplacé par de la dompéridone dans le cadre d'un traitement à long terme contre la leishmaniose, ce qui a entraîné une légère augmentation du titre des anticorps sériques contre la leishmaniose. La mise en place d'une dérivation urétérale sous-cutanée s'est avérée efficace et sûre chez ce chien et constitue une alternative intéressante en cas d'obstruction urétérale, y compris chez les chiens.

Role of Macrophage PIST Protein in Regulating Leishmania major Infection.

PDZ protein interacting specifically with Tc10 or PIST is a mammalian trans-Golgi resident protein that regulates subcellular sorting of plasma membrane receptors. PIST has recently emerged as a key player in regulating viral pathogenesis. Nevertheless, the involvement of PIST in parasitic infections remains unexplored. Leishmania parasites infiltrate their host macrophage cells through phagocytosis, where they subsequently multiply within the parasitophorous vacuole (PV). Host cell autophagy has been found to be important in regulating this parasite infection. Since PIST plays a pivotal role in triggering autophagy through the Beclin 1-PI3KC3 pathway, it becomes interesting to identify the status of PIST during Leishmania infection. We found that while macrophage cells are infected with Leishmania major (L. major), the expression of PIST protein remains unaltered; however, it traffics from the Golgi compartment to PV. Further, we identified that in L. major-infected macrophage cells, PIST associates with the autophagy regulatory protein Beclin 1 within the PVs; however, PIST does not interact with LC3. Reduction in PIST protein through siRNA silencing significantly increased parasite burden, whereas overexpression of PIST in macrophages restricted L. major infectivity. Together, our study reports that the macrophage PIST protein is essential in regulating L. major infectivity.

Leishmaniasis in otorhinolaryngology: an emerging disease and its relationship with anti-tumor necrosis factor-alpha drugs.

PURPOSE: To investigate the clinical manifestations, management and outcomes of Leishmania lesions in the ear-nose-throat (ENT) region, and its relationship with tumor necrosis factor (TNF)-α blocking drugs. METHODS: Single-center retrospective observational study. Patients diagnosed with cutaneous and mucosal leishmaniasis in the otorhinolaryngologic area at a tertiary referral center over a period of 8 years. RESULTS: Three cases of Leishmania lesions in the ear and two in the nose were encountered at our institution. All patients were under treatment with TNF-α blocking drugs. Diagnosis was challenging, and it was important to have a clinical suspicion in order to use accurate detection techniques. All patients received systemic treatment and achieved a complete resolution of the lesions. CONCLUSIONS: With the increasing use of biologic treatments like TNF-α blockers, this type of infection will be increasingly frequent in endemic areas and also worldwide. It is important to include leishmaniasis in the differential diagnosis of inflammatory/infectious lesions in the ENT region.

FVB/NJ strain as a mouse model for cutaneous leishmaniasis by Leishmania (L.) amazonensis.

BACKGROUND: Leishmaniases encompass a spectrum of neglected diseases caused by parasites of the genus Leishmania, grouped in two forms: tegumentary and visceral leishmaniasis. OBJECTIVES: In this study, we propose Friend Virus B NIH Jackson (FVB/NJ) mouse strain as a new experimental model of infection with Leishmania (Leishmania) amazonensis, the second most prevalent agent of tegumentary leishmaniasis in Brazil. METHODS AND FINDINGS: We performed in vitro infections of FVB/NJ macrophages and compared them with BALB/c macrophages, showing that BALB/c cells have higher infection percentages and a higher number of amastigotes/cell. Phagocytosis assays indicated that BALB/c and FVB/NJ macrophages have similar capacity to uptake parasites after 5 min incubations. We also investigated promastigotes' resistance to sera from FVB/NJ and BALB/c and observed no difference between the two sera, even though FVB/NJ has a deficiency in complement components. Finally, we subcutaneously infected FVB/NJ and BALB/c mice with 2 × 106 parasites expressing luciferase. Analysis of lesion development for 12 weeks showed that FVB/NJ and BALB/c mice have similar lesion profiles and parasite burdens. MAIN CONCLUSIONS: This work characterises for the first time the FVB/NJ mouse as a new model for tegumentary leishmaniasis caused by Leishmania (L.) amazonensis.

The cytosolic hyperoxidation-sensitive and -robust Leishmania peroxiredoxins cPRX1 and cPRX2 are both dispensable for parasite infectivity.

Typical two-cysteine peroxiredoxins (2-Cys-PRXs) are H2O2-metabolizing enzymes whose activity relies on two cysteine residues. Protists of the family Trypanosomatidae invariably express one cytosolic 2-Cys-PRX (cPRX1). However, the Leishmaniinae sub-family features an additional isoform (cPRX2), almost identical to cPRX1, except for the lack of an elongated C-terminus with a Tyr-Phe (YF) motif. Previously, cytosolic PRXs were considered vital components of the trypanosomatid antioxidant machinery. Here, we shed new light on the properties, functions and relevance of cPRXs from the human pathogen Leishmania infantum. We show first that LicPRX1 is sensitive to inactivation by hyperoxidation, mirroring other YF-containing PRXs participating in redox signaling. Using genetic fusion constructs with roGFP2, we establish that LicPRX1 and LicPRX2 can act as sensors for H2O2 and oxidize protein thiols with implications for signal transduction. Third, we show that while disrupting the LicPRX-encoding genes increases susceptibility of L. infantum promastigotes to external H2O2in vitro, both enzymes are dispensable for the parasites to endure the macrophage respiratory burst, differentiate into amastigotes and initiate in vivo infections. This study introduces a novel perspective on the functions of trypanosomatid cPRXs, exposing their dual roles as both peroxidases and redox sensors. Furthermore, the discovery that Leishmania can adapt to the absence of both enzymes has significant implications for our understanding of Leishmania infections and their treatment. Importantly, it questions the conventional notion that the oxidative response of macrophages during phagocytosis is a major barrier to infection and the suitability of cPRXs as drug targets for leishmaniasis.

In vitro evaluation of antileishmanial activity of Boswellia serrata essential oil nanoliposome.

BACKGROUND: Leishmaniasis poses a significant health risk. OBJECTIVES: This study aimed to evaluate the effects of Boswellia serrata (B. serrata) essential oil nanoliposomes on Leishmania tropica (L. tropica) in vitro. METHODS: A mixture of B. serrata essential oil, phosphatidylcholine and Tween 80 were used to prepare B. serrata essential oil nanoliposomes, followed by drying, hydration and size characterisation. The promastigotes of L. tropica were cultured in Roswell Park Memorial Institute medium (RPMI-1640) containing streptomycin, penicillin and fetal bovine serum. Different concentrations of B. serrata essential and nanoliposomes were tested for their antileishmanial properties by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide tests (MTT). RESULTS: Results of Dynamic Light Scattering (DLS) for B. serrata nanoliposomes indicate that they are successful at producing nanoliposomes with dimensions of 74.8 nm. At 1 µg/mL dose, B. serrata essence caused 17 ± 1.73% mortality, while B. serrata nanoliposomes induced 26 ± 1.15% mortality. B. serrata essence achieved a mortality of 55 ± 2.88% at 10 µg/mL, whereas B. serrata nanoliposomes demonstrated a mortality of 63.66±0.88% at 10 µg/mL. Furthermore, there was a significant difference between similar concentrations of B. serrata and B. serrata nanoliposomes. The LC50 of B. serrata essential oil is 7.26 µg/mL in the 95% confidence interval (12.13-5.25). The LC90 value of B. serrata essential oil is 129.37 µg/mL in the 95% confidence interval (50.07-852.58). The LC50 value of B. serrata nanoliposome is 4.20 µg/mL in the 95% confidence interval (6.13-3.10). LC90 value for B. serrata nanoliposome is calculated as 91.89 µg/mL in the 95% confidence interval (37.09-583.29). CONCLUSIONS: In vitro experiments have shown that B. serrata oil and the nanoliposome suppress the proliferation of L. tropica promastigotes, which suggests it may be a promising option for treating leishmaniasis.

Leishmania amazonensis infection regulates oxidate stress in hyperglycemia and diabetes impairing macrophage's function and immune response.

Leishmaniasis is a group of infectious diseases caused by protozoa of the Leishmania genus and its immunopathogenesis results from an unbalanced immune response during the infection. Diabetes is a chronic disease resulting from dysfunction of the body's production of insulin or the ability to use it properly, leading to hyperglycemia causing tissue damage and impairing the immune system. AIMS: The objective of this work was to evaluate the effects of hyperglycemia and diabetes during Leishmania amazonensis infection and how these conditions alter the immune response to the parasite. METHODS: An in vitro hyperglycemic stimulus model using THP-1-derived macrophages and an in vivo experimental diabetes with streptozotocin (STZ) in C57BL/6 mice was employed to investigate the impact of diabetes and hyperglicemia in Leishmania amazonensis infection. RESULTS: We observed that hyperglycemia impair the leishmanicidal capacity of macrophages derived from THP-1 cells and reverse the resistance profile that C57BL/6 mice have against infection by L. amazonensis, inducing more exacerbated lesions compared to non-diabetic animals. In addition, the hyperglycemic stimulus favored the increase of markers related to the phenotype of M2 macrophages. The induction of experimental diabetes in C57BL/6 mice resulted in a failure in the production of nitric oxide (NO) in the face of infection and macrophages from diabetic animals failed to process and present Leishmania antigens, being unable to activate and induce proliferation of antigen-specific lymphocytes. CONCLUSION: Together, these data demonstrate that diabetes and hyperglycemia can impair the cellular immune response, mainly of macrophages, against infection by parasites of the genus Leishmania.

Annexin A1 improves immune responses and control of tissue parasitism during Leishmania amazonensis infection in BALB/c mice.

Leishmaniases, a group of diseases caused by the species of the protozoan parasite Leishmania, remains a significant public health concern worldwide. Host immune responses play a crucial role in the outcome of Leishmania infections, and several mediators that regulate inflammatory responses are potential targets for therapeutic approaches. Annexin A1 (AnxA1), an endogenous protein endowed with anti-inflammatory and pro-resolving properties, has emerged as a potential player. We have shown that during L. braziliensis infection, deficiency of AnxA1 exacerbates inflammatory responses but does not affect parasite burden. Here, we have investigated the role of AnxA1 in L. amazonensis infection, given the non-healing and progressive lesions characteristic of this infectious model. Infection of AnxA1 KO BALB/c mice resulted in increased lesion size and tissue damage associated with higher parasite burdens and enhanced inflammatory response. Notably, therapeutic application of the AnxA1 peptidomimetic Ac2-26 improves control of parasite replication and increases IL-10 production in vivo and in vitro, in both WT and AnxA1 KO mice. Conversely, administration of WRW4, an inhibitor of FPR2/3, resulted in larger lesions and decreased production of IL-10, suggesting that the effects of AnxA1 during L. amazonensis infection are associated with the engagement of these receptors. Our study illuminates the role of AnxA1 in L. amazonensis infection, demonstrating its impact on the susceptibility phenotype of BALB/c mice. Furthermore, our results indicate that targeting the AnxA1 pathway by using the Ac2-26 peptide could represent a promising alternative for new treatments for leishmaniasis.

Leishmania spp. diagnosis and therapeutic management in a cat from urban area in Ibagué (Colombia).

BACKGROUND: Leishmania spp., a protozoan transmitted by sandflies, widely affects humans and dogs in Colombia, nevertheless feline leishmaniasis (FeL) remains understudied. OBJECTIVE: This study reports a case of feline leishmaniasis in Colombia and its therapeutic management. METHODS: Complete blood count, renal and hepatic serum biochemistry, nodular lesion cytology, FeLV/FIV snap test, abdominal ultrasound, and molecular diagnosis of Leishmania spp. 16 s rRNA gene amplification by real-time-PCR (qPCR), ITS-1 and hsp70 gene by endpoint-PCR and Sanger sequencing were performed. RESULTS: The patient was negative for FIV/FeLV and showed leukocytosis, lymphocytosis, thrombocytopenia, neutrophilia, monocytosis, hypergammaglobulinemia, increased gamma-glutamyl-transferase, cortical nephrocalcinosis, diffuse heterogeneous splenic parenchyma, and cholangitis. Nodular lesion cytology, qPCR and Sanger sequencing confirmed the diagnosis of Leishmania spp. The patient was treated with allopurinol and miltefosine. After treatment, clinical signs disappeared. CONCLUSION: Clinical examination, cytology, and molecular tests allowed a rapid and sensitive FeL diagnosis. Allopurinol and miltefosine improved the clinical condition of the cat.

Using meta-analysis and machine learning to investigate the transcriptional response of immune cells to Leishmania infection.

BACKGROUND: Leishmaniasis is a parasitic disease caused by the Leishmania protozoan affecting millions of people worldwide, especially in tropical and subtropical regions. The immune response involves the activation of various cells to eliminate the infection. Understanding the complex interplay between Leishmania and the host immune system is crucial for developing effective treatments against this disease. METHODS: This study collected extensive transcriptomic data from macrophages, dendritic, and NK cells exposed to Leishmania spp. Our objective was to determine the Leishmania-responsive genes in immune system cells by applying meta-analysis and feature selection algorithms, followed by co-expression analysis. RESULTS: As a result of meta-analysis, we discovered 703 differentially expressed genes (DEGs), primarily associated with the immune system and cellular metabolic processes. In addition, we have substantiated the significance of transcription factor families, such as bZIP and C2H2 ZF, in response to Leishmania infection. Furthermore, the feature selection techniques revealed the potential of two genes, namely G0S2 and CXCL8, as biomarkers and therapeutic targets for Leishmania infection. Lastly, our co-expression analysis has unveiled seven hub genes, including PFKFB3, DIAPH1, BSG, BIRC3, GOT2, EIF3H, and ATF3, chiefly related to signaling pathways. CONCLUSIONS: These findings provide valuable insights into the molecular mechanisms underlying the response of immune system cells to Leishmania infection and offer novel potential targets for the therapeutic goals.

Swarna Bhasma Induces Antigen-Presenting Abilities of Macrophages and Helps Antigen Experienced CD4+ T Cells to Acquire Th1 Phenotypes Against Leishmania donovani Antigens.

In leishmaniasis, the protective immunity is largely mediated by proinflammatory cytokine producing abilities of T cells and an efficient parasite killing by phagocytic cells. Notwithstanding a substantial progress that has been made during last decades, the mechanisms or factors involved in establishing protective immunity against Leishmania are not identified. In ancient Indian literature, metallic "bhasma," particularly that of "swarna" or gold (fine gold particles), is indicated as one of the most prominent metal-based therapeutic medicine, which is known to impart protective and curative properties in various health issues. In this work, we elucidated the potential of swarna bhasma (SB) on the effector properties of phagocytes and antigen-activated CD4+ T cells in augmenting the immunogenicity of L. donovani antigens. The characterization of SB revealing its shape, size, composition, and measurement of cytotoxicity established the physiochemical potential for its utilization as an immunomodulator. The activation of macrophages with SB enhanced their capacity to produce nitric oxide and proinflammatory cytokines, which eventually resulted in reduced uptake of parasites and their proliferation in infected cells. Further, in Leishmania-infected animals, SB administration reduced the generation of IL-10, an anti-inflammatory cytokine, and enhanced pro-inflammatory cytokine generation by antigen activated CD4+ T cells with increased frequency of double (IFNγ+/TNFα+) and triple (IFNγ+TNFα+IL-2+) positive cells and abrogated disease pathogeneses at the early days of infection. Our results also suggested that cow-ghee (A2) emulsified preparation of SB, either alone or with yashtimadhu, a known natural immune modulator which enhances the SB's potential in enhancing the immunogenicity of parasitic antigens. These findings suggested a definite potential of SB in enhancing the effector functions of phagocytes and CD4+ T cells against L. donovani antigens. Therefore, more studies are needed to elucidate the mechanistic details of SB and its potential in enhancing vaccine-induced immunity.

The ß-Carboline Harmine Has a Protective Immunomodulatory Role in Nonhealing Cutaneous Leishmaniasis.

Cutaneous leishmaniasis affects 1 million people worldwide annually. Although conventional treatments primarily target the parasite, there is growing interest in host immune modulation. In this study, we investigated the impact of synthetic ß-carboline harmine (ACB1801), previously shown to be immunoregulatory in cancer, on the pathology caused by a drug-resistant Leishmania major strain causing persistent cutaneous lesions. Exposure to ACB1801 in vitro had a modest impact on parasite burden within host macrophages. Moreover, it significantly increased major histocompatibility complex II and costimulatory molecule expression on infected dendritic cells, suggesting an enhanced immune response. In vivo, ACB1801 monotherapy led to a substantial reduction in lesion development and parasite burden in infected C57BL/6 mice, comparable with efficacy of amphotericin B. Transcriptomics analysis further supported ACB1801 immunomodulatory effects, revealing an enrichment of TNF-α, IFN-γ, and major histocompatibility complex II antigen presentation signatures in the draining lymph nodes of treated mice. Flow cytometry analysis confirmed an increased frequency (1.5×) of protective CD4+IFN-γ+TNF-α+ T cells and a decreased frequency (2×) in suppressive IL-10+FoxP3- T cells at the site of infection and in draining lymph nodes. In addition, ACB1801 downregulated the aryl hydrocarbon receptor signaling, known to enhance immunosuppressive cytokines. Thus, these results suggest a potential use for ACB1801 alone or in combination therapy for cutaneous leishmaniasis.

Development of a novel squalene/α-tocopherol-based self-emulsified nanoemulsion incorporating Leishmania peptides for induction of antigen-specific immune responses.

Vaccination has emerged as the most effective strategy to confront infectious diseases, among which is leishmaniasis, that threat public health. Despite laborious efforts there is still no vaccine for humans to confront leishmaniasis. Multi-epitope protein/peptide vaccines present a number of advantages, however their use along with appropriate adjuvants that may also act as antigen carriers is considered essential to overcome subunit vaccines' low immunogenicity. In the present study, a stable self-emulsified nanoemulsion was developed and double-adjuvanted with squalene and α-tocopherol. The prepared nanoemulsion droplets exhibited low cytotoxicity in a certain range of concentrations, while they were efficiently taken up by macrophages and dendritic cells in vitro as well as in vivo in secondary lymphoid organs. To further characterize nanoformulation's potent antigen delivery capability, three multi-epitope Leishmania peptides were incorporated into the nanoemulsion. Peptide encapsulation resulted in dendritic cells' functional differentiation characterized by elevated levels of maturation markers and intracellular cytokine production. Intramuscular administration of the nanoemulsion incorporating Leishmania peptides induced antigen-specific spleen cell proliferation as well as elicitation of CD4+ central memory cells, supporting the potential of the developed nanoformulation to successfully act also as an antigen delivery vehicle and thus encouraging further preclinical studies on its vaccine candidate potency.

Exploring microalgal and cyanobacterial metabolites with antiprotozoal activity against Leishmania and Trypanosoma parasites.

Neglected tropical diseases (NTD) like Leishmaniasis and trypanosomiasis affect millions of people annually, while currently used antiprotozoal drugs have serious side effects. Drug research based on natural products has shown that microalgae and cyanobacteria are a promising platform of biochemically active compounds with antiprotozoal activity. These unicellular photosynthetic organisms are rich in polyunsaturated fatty acids, pigments including phycocyanin, chlorophylls and carotenoids, polyphenols, bioactive peptides, terpenes, alkaloids, which have proven antioxidant, antimicrobial, antiviral, antiplasmodial and antiprotozoal properties. This review provides up-to-date information regarding ongoing studies on substances synthesized by microalgae and cyanobacteria with notable activity against Leishmania spp., Trypanosoma cruzi, and Trypanosoma brucei, the causative agents of Leishmaniasis, Chagas disease, and human African trypanosomiasis, respectively. Extracts of several freshwater or marine microalgae have been tested on different strains of Leishmania and Trypanosoma parasites. For instance, ethanolic extract of Chlamydomonas reinhardtii and Tetraselmis suecica have biological activity against T. cruzi, due to their high content of carotenoids, chlorophylls, phenolic compounds and flavonoids that are associated with trypanocidal activity. Halophilic Dunaliella salina showed moderate antileishmanial activity that may be attributed to the high ß-carotene content in this microalga. Peptides such as almiramides, dragonamides, and herbamide that are biosynthesized by marine cyanobacteria Lyngbya majuscula were found to have increased activity in micromolar scale IC50 against L. donovani, T. Cruzi, and T. brucei parasites. The cyanobacterial peptides symplocamide and venturamide isolated from Symploca and Oscillatoria species, respectively, and the alkaloid nostocarbonile isolated from Nostoc have shown promising antiprotozoal properties and are being explored for pharmaceutical and medicinal purposes. The discovery of new molecules from microalgae and cyanobacteria with therapeutic potential against Leishmaniasis and trypanosomiasis may address an urgent medical need: effective and safe treatments of NTDs.

Interactions between Leishmania parasite and sandfly: a review.

Leishmaniasis transmission cycles are maintained and sustained in nature by the complex crosstalk of the Leishmania parasite, sandfly vector, and the mammalian hosts (human, as well as zoonotic reservoirs). Regardless of the vast research on human host-parasite interaction, there persists a substantial knowledge gap on the parasite's development and modulation in the vector component. This review focuses on some of the intriguing aspects of the Leishmania-sandfly interface, beginning with the uptake of the intracellular amastigotes from an infected host to the development of the parasite within the sandfly's alimentary canal, followed by the transmission of infective metacyclic stages to another potential host. Upon ingestion of the parasite, the sandfly hosts an intricate repertoire of immune barriers, either to evade the parasite or to ensure its homeostatic coexistence with the vector gut microbiome. Sandfly salivary polypeptides and Leishmania exosomes are co-egested with the parasite inoculum during the infected vector bite. This has been attributed to the modulation of the parasite infection and subsequent clinical manifestation in the host. While human host-based studies strive to develop effective therapeutics, a greater understanding of the vector-parasite-microbiome and human host interactions could help us to identify the targets and to develop strategies for effectively preventing the transmission of leishmaniasis.

Leishmania infection upregulates and engages host macrophage Argonaute 1, and system-wide proteomics reveals Argonaute 1-dependent host response.

Leishmania donovani, an intracellular protozoan parasite, is the causative agent of visceral leishmaniasis, the most severe form of leishmaniasis in humans. It is becoming increasingly clear that several intracellular pathogens target host cell RNA interference (RNAi) pathways to promote their survival. Complexes of Argonaute proteins with small RNAs are core components of the RNAi. In this study, we investigated the potential role of host macrophage Argonautes in Leishmania pathogenesis. Using Western blot analysis of Leishmania donovani-infected macrophages, we show here that Leishmania infection selectively increased the abundance of host Argonaute 1 (Ago1). This increased abundance of Ago1 in infected cells also resulted in higher levels of Ago1 in active Ago-complexes, suggesting the preferred use of Ago1 in RNAi in Leishmania-infected cells. This analysis used a short trinucleotide repeat containing 6 (TNRC6)/glycine-tryptophan repeat protein (GW182) protein-derived peptide fused to Glutathione S-transferase as an affinity matrix to capture mature Ago-small RNAs complexes from the cytosol of non-infected and Leishmania-infected cells. Furthermore, Ago1 silencing significantly reduced intracellular survival of Leishmania, demonstrating that Ago1 is essential for Leishmania pathogenesis. To investigate the role of host Ago1 in Leishmania pathogenesis, a quantitative whole proteome approach was employed, which showed that expression of several previously reported Leishmania pathogenesis-related proteins was dependent on the level of macrophage Ago1. Together, these findings identify Ago1 as the preferred Argonaute of RNAi machinery in infected cells and a novel and essential virulence factor by proxy that promotes Leishmania survival.

Immunosuppressants alter the immune response associated with Glucantime® treatment for Leishmania infantum infection in a mouse model.

Background: Immunosuppression is a major risk factor for the development of visceral leishmaniasis (VL). The number of patients receiving immunosuppressant drugs such as TNF antagonist (anti-TNF) and methotrexate (MTX) is increasing. In these patients, VL is more severe, their response to treatment poorer, and they are at higher risk of relapse, a consequence (largely) of the poor and inappropriate immune response they develop. Objectives: To examine the effect of immunosuppressive treatment on the host immune response and thus gain insight into the reduced efficacy of pentavalent antimonials in these patients. Experiments were performed using BALB/c mice immunosuppressed with anti-TNF or MTX, infected with Leishmania infantum promastigotes, and then treated with Glucantime® at clinical doses. Results: Immunosuppression with both agents impeded parasite elimination from the spleen and bone marrow. Low pro-inflammatory cytokine production by CD4+ and CD8+ T cells was detected, along with an increase in PD-1 and IL-10 expression by B and T cells in the immunosuppressed groups after treatment. Conclusion: The immunosuppressed mice were unable to develop specific cellular immunity to the parasite, perhaps explaining the greater risk of VL relapse seen in pharmacologically immunosuppressed human patients.

Cellular mediators in human leishmaniasis: Critical determinants in parasite killing or disease progression.

Data on cellular immunity mediators in the early phase of human leishmaniasis are still limited and controversial. In order to mimic the changes of humoral mediators during the early phase of human natural infection, some Th1, Th2, Treg, and Breg cytokines, MCP-1, and the nitric oxide (NO) from human PBMC, stimulated by Leishmania infantum, Leishmania major, Leishmania donovani and Leishmania tropica infective metacyclic promastigotes, were determined. After 4 h of L. major, L. donovani, and L. tropica challenge, TNFα, IL-1ß, IL-6 levels were significantly higher than negative control cultures with saline (SF) instead of Leishmania promastigotes, unlike L. infantum-stimulated TNFα and L. major-stimulated IL-1ß. We obtained higher levels of IL-4 and IL-10 cytokines after stimulation of human PBMCs by L. infantum and L. donovani, compared to those observed after the challenge of PBMCs by L. major and L. tropica. Regarding IL-35, such cytokine levels were significantly increased following infection with L. infantum and L. donovani, in contrast to L. major and L. tropica. Up to our knowledge, we are the first to study the effect of four different species of Leishmania on IL-35 levels in human cells. Our study highlights how several Leishmania species can up-regulate different groups of cytokines (Th1, Th2, Treg and Breg) and modulate NO release in a different way. This original aspect can be explained by different Leishmania cell products, such as LPG, obtained from different strains/species of live parasites. Our findings would contribute to the development of new therapeutics or vaccination strategies.

Adjuvantation of whole-killed Leishmania vaccine with anti-CD200 and anti-CD300a antibodies potentiates its efficacy and provides protection against wild-type parasites.

One of the major reasons behind the limited success of vaccine candidates against all forms of leishmaniasis is the inability of parasitic antigens to induce robust cell-mediated immunity and immunological memory. Here we find, for the first time, that the adjuvantation of whole-killed Leishmania vaccine (Leishvacc) with anti-CD200 and anti-CD300a antibodies enhances CD4+ T cells mediated immunity in vaccinated mice and provides protection against wild-type parasites. The antibody adjuvantation, either alone or with a TLR4 agonist monophosphoryl A (MPL-A), induced the production of pro-inflammatory cytokines viz., IFN-γ, TNF-α, and IL-2 by antigen experienced CD4+ T cells, and also enhanced their rate of conversion into their memory phenotypes against Leishvacc antigens. The antibody adjuvanted vaccine also promoted the generation of IgG2a-mediated protective humoral immunity in vaccinated mice. Further, the mice vaccinated with antibodies adjuvanted vaccine showed strong resilience against metacyclic forms of L. donovani parasites as we observed reduced clinical features such as splenomegaly, hepatomegaly, granulomatous tissues in the liver, and parasitic load in their spleen. The findings of this study demonstrate that the anti-CD200 and anti-CD300a antibodies have potential to increase the protective efficacy of the whole-killed Leishmania vaccine, and opens up a new gateway to diversify the roles of immune checkpoints in vaccine development against leishmaniasis.