Leishmania donovani infection induce Extracellular signal-regulated kinase ½ (ERK½) mediated lipid droplet generation in macrophages.

Recently unfolded mechanisms showed lipid droplet helps in pathogen survival and paralyzes host immune response. In the present study, we showed the extent of lipid droplet(LD) generation in Leishmania donovani infection, the signaling involved, and their function concerning pathogenicity. RAW 264.7 and J774A.1 cells were used to infect with L. donovani and then flow cytometry and confocal microscopy were used to detect lipid droplet generation and subsequent assays. In this study, we showed that L. donovani AG83 (AG83/MHOM/1983) triggers lipid droplet formation in macrophages in a time-dependent manner. We provide novel insight into the signaling molecules which is responsible for LD accumulation. Interestingly, LPG deficient attenuated Leishmania strain UR6 (UR6/MHOM/1978) failed to fuel LD generation. But inhibition of phagosome maturation drastically stimulates LD accumulation in UR6 infected MΦs. Aspirin treatment in AG83 infected MΦs does not only lower LD load but also favors phagolysosome biogenesis and corrects cytokine balance. Employing strategies to circumvent halt in phagosome maturation using drugs that manipulate lipid droplet generation could be used as a therapeutic tool to resist parasite growth in the early hour of infection.

The catalytic core of Leishmania donovani RECQ helicase unwinds a wide spectrum of DNA substrates and is stimulated by replication protein A.

RecQ helicases are superfamily 2 (SF2) DNA helicases that unwind a wide spectrum of complex DNA structures in a 3' to 5' direction and are involved in maintaining genome stability. RecQ helicases from protozoan parasites have gained significant interest in recent times because of their involvement in cellular DNA repair pathways, making them important targets for drug development. In this study, we report biophysical and biochemical characterization of the catalytic core of a RecQ helicase from hemoflagellate protozoan parasite Leishmania donovani. Among the two putative RecQ helicases identified in L. donovani, we cloned, overexpressed and purified the catalytic core of LdRECQb. The catalytic core was found to be very efficient in unwinding a wide variety of DNA substrates like forked duplex, 3' tailed duplex and Holliday junction DNA. Interestingly, the helicase core also unwound blunt duplex with slightly less efficiency. The enzyme exhibited high level of DNA-stimulated ATPase activity with preferential stimulation by forked duplex, Holliday junction and 3' tailed duplex. Walker A motif lysine mutation severely affected the ATPase activity and significantly affected unwinding activity. Like many other RecQ helicases, L. donovani RECQb also possesses strand annealing activity. Unwinding of longer DNA substrates by LdRECQb catalytic core was found to be stimulated in the presence of replication protein A (LdRPA-1) from L. donovani. Detailed biochemical characterization and comparison of kinetic parameters indicate that L. donovani RECQb shares considerable functional similarity with human Bloom syndrome helicase.

A Chimera of Th1 Stimulatory Proteins of Leishmania donovani Offers Moderate Immunotherapeutic Efficacy with a Th1-Inclined Immune Response against Visceral Leishmaniasis.

Immunotherapy, a treatment based on host immune system activation, has been shown to provide a substitute for marginally effective conventional chemotherapy in controlling visceral leishmaniasis (VL), the deadliest form of leishmaniasis. As the majority of endemic inhabitants exhibit either subclinical or asymptomatic infection which often develops into the active disease state, therapeutic intervention seems to be an important avenue for combating infections by stimulating the natural defense system of infected individuals. With this perspective, the present study focuses on two immunodominant Leishmania (L.) donovani antigens (triosephosphate isomerase and enolase) previously proved to be potent prophylactic VL vaccine candidates, for generating a recombinant chimeric antigen. This is based on the premise that in a heterogeneous population, a multivalent antigen vaccine would be required for an effective response against leishmaniasis (a complex parasitic disease). The resulting molecule rLdT-E chimeric protein was evaluated for its immunogenicity and immunotherapeutic efficacy. A Th1 stimulating adjuvant BCG was employed with the protein which showed a remarkable 70% inhibition of splenic parasitic multiplication positively correlated with boosted Th1 dominant immune response against lethal L. donovani challenge in hamsters as evidenced by high IFN-γ and TNF-α and low IL-10. In addition, immunological analysis of antibody subclass presented IgG2-based humoral response besides considerable delayed-type hypersensitivity and lymphocyte proliferative responses in rLdT-E/BCG-treated animals. Our observations indicate the potential of the chimera towards its candidature for an effective vaccine against Leishmania donovani infection.

Diversity and Within-Host Evolution of Leishmania donovani from Visceral Leishmaniasis Patients with and without HIV Coinfection in Northern Ethiopia.

Visceral leishmaniasis (VL) is a fatal disease and a growing public health problem in East Africa, where Ethiopia has one of the highest VL burdens. The largest focus of VL in Ethiopia is driven by high prevalence in migrant agricultural workers and associated with a high rate of coinfection with HIV. This coinfection makes VL more difficult to treat successfully and is associated with a high rate of relapse, with VL/HIV patients frequently experiencing many relapses of VL before succumbing to this infection. We present genome-wide data on Leishmania donovani isolates from a longitudinal study of cohorts of VL and VL/HIV patients reporting to a single clinic in Ethiopia. Extensive clinical data allow us to investigate the influence of coinfection and relapse on the populations of parasites infecting these patients. We find that the same parasite population is responsible for both VL and VL/HIV infections and that, in most cases, disease relapse is caused by recrudescence of the population of parasites that caused primary VL. Complex, multiclonal infections are present in both primary and relapse cases, but the infrapopulation of parasites within a patient loses genetic diversity between primary disease presentation and subsequent relapses, presumably due to a population bottleneck induced by treatment. These data suggest that VL/HIV relapses are not caused by genetically distinct parasite infections or by reinfection. Treatment of VL does not lead to sterile cure, and in VL/HIV, the infecting parasites are able to reestablish after clinically successful treatment, leading to repeated relapse of VL. IMPORTANCE Visceral leishmaniasis (VL) is the second largest cause of deaths due to parasite infections and a growing problem in East Africa. In Ethiopia, it is particularly associated with migrant workers moving from regions of nonendemicity for seasonal agricultural work and is frequently found as a coinfection with HIV, which leads to frequent VL relapse following treatment. Insight into the process of relapse in these patients is thus key to controlling the VL epidemic in Ethiopia. We show that there is little genetic differentiation between the parasites infecting HIV-positive and HIV-negative VL patients. Moreover, we provide evidence that relapses are caused by the initially infecting parasite population and that treatment induces a loss of genetic diversity in this population. We propose that restoring functioning immunity and improving antiparasitic treatment may be key in breaking the cycle of relapsing VL in VL/HIV patients.

Peptide-mediated leishmaniasis management strategy: Tachyplesin emerges as an effective anti-leishmanial peptide against Leishmania donovani.

Visceral leishmaniasis is one of the neglected tropical diseases caused by an intracellular parasite, Leishmania donovani. Drug resistance, adverse side effects and long treatment regimes are important limitations in achieving the effective elimination of visceral leishmaniasis. In the absence of any vaccine, chemotherapy remains a viable treatment for leishmaniasis. For effective killing of leishmania parasite, the drug molecule needs to cross the cell membrane. In the present study, marine membrane-active peptide Tachyplesin has been used against Leishmania donovani. Further, the mechanism of action and importance of cysteine amino acids of Tachyplesin in anti-leishmanial activity has been assessed. The cargo-carrying ability of Tachyplesin in L. donovani has been established. Thus, dual-use of Tachyplesin as an anti-leishmanial peptide as well as a cargo delivery vehicle makes the marine peptide an attractive therapeutic target against visceral leishmaniasis.

Leishmaniasis: where are we and where are we heading?

Leishmaniasis is a zoonotic disease in humans caused by the bite of a parasite-infected sandfly. The disease, widely referred to as "poor man's disease," affects millions of people worldwide. The clinical manifestation of the disease depends upon the species of the parasite and ranges from physical disfigurement to death if left untreated. Here, we review the past, present, and future of leishmaniasis in detail. The life cycle of Leishmania sp., along with its epidemiology, is discussed, and in addition, the line of therapeutics available for treatment currently is examined. The current status of the disease is critically evaluated, keeping emerging threats like human immunodeficiency virus (HIV) coinfection and post kala-azar dermal leishmaniasis (PKDL) into consideration. In summary, the review proposes a dire need for new therapeutics and reassessment of the measures and policies concerning emerging threats. New strategies are essential to achieve the goal of leishmaniasis eradication in the next few decades.

Leishmania donovani Metacyclic Promastigotes Impair Phagosome Properties in Inflammatory Monocytes.

Leishmaniasis, a debilitating disease with clinical manifestations ranging from self-healing ulcers to life-threatening visceral pathologies, is caused by protozoan parasites of the Leishmania genus. These professional vacuolar pathogens are transmitted by infected sand flies to mammalian hosts as metacyclic promastigotes and are rapidly internalized by various phagocyte populations. Classical monocytes are among the first myeloid cells to migrate to infection sites. Recent evidence shows that recruitment of these cells contributes to parasite burden and the establishment of chronic disease. However, the nature of Leishmania-inflammatory monocyte interactions during the early stages of host infection has not been well investigated. Here, we aimed to assess the impact of Leishmania donovani metacyclic promastigotes on antimicrobial responses within these cells. Our data showed that inflammatory monocytes are readily colonized by L. donovani metacyclic promastigotes, while infection with Escherichia coli is efficiently cleared. Upon internalization, metacyclic promastigotes inhibited superoxide production at the parasitophorous vacuole (PV) through a mechanism involving exclusion of NADPH oxidase subunits gp91phox and p47phox from the PV membrane. Moreover, we observed that unlike phagosomes enclosing zymosan particles, vacuoles containing parasites acidify poorly. Interestingly, whereas the parasite surface coat virulence glycolipid lipophosphoglycan (LPG) was responsible for the inhibition of PV acidification, impairment of the NADPH oxidase assembly was independent of LPG and GP63. Collectively, these observations indicate that permissiveness of inflammatory monocytes to L. donovani may thus be related to the ability of this parasite to impair the microbicidal properties of phagosomes.

Interplay Between Sialic Acids, Siglec-E, and Neu1 Regulates MyD88- and TRIF-Dependent Pathways for TLR4-Activation During Leishmania donovani Infection.

TLR4 activates two distinct signaling pathways involving adaptors MyD88 and TRIF to produce proinflammatory cytokines and type-I interferon respectively. How Leishmania donovani suppresses these pathways is not well studied. We earlier reported, TLR4 is hypersialylated due to reduced membrane-bound neuraminidase (Neu1) on infected-macrophages. We hypothesized that such enhanced sialoglycoconjugates on host cells may modulate the interactions with siglecs- which are the inhibitory receptors. Here, we examined the impact of such sialylation on overall TLR4 activation both in murine cell line J774A.1 and primary bone marrow derived macrophages (BMDM). Supporting this hypothesis, we demonstrated siglec-E engages hypersialylated TLR4 during infection. Such sialic acids-siglec-E interaction enhanced siglec-E phosphorylation that mediated its strong association with SHP1/SHP2 and also upregulated their phosphorylation in both types of macrophages. Pre-treatment of parasites and host cells with neuraminidase reduced SHP1/SHP2 phosphorylation and triggered TLR4 activation respectively through enhanced nuclear translocation of p-65. Moreover, a reciprocal interplay between Neu1 and siglec-E differentially regulates MyD88- and TRIF-pathways through sialic acids on TLR4 as their common substrate during infection. Correspondingly, Neu1 overexpression enhanced MyD88-signaling while still suppressing TRIF-activation. However, silencing siglec-E specifically activated TRIF-signaling. Pro-inflammatory cytokines corresponding to MyD88 and TRIF pathways were also upregulated respectively. Additionally, Neu1 overexpression or siglec-E silencing prevented TLR4 ubiquitination and subsequent degradation by Triad3A. Neu1-overexpression and siglec-E-silencing together followed by infection activated both MyD88 and TRIF-signaling through their enhanced TLR4-association. This elevated the MyD88-specific cytokines and TRIF-mediated IRF3 and IFN-ß genes, thus upregulating the pro-inflammatory cytokines and nitric oxide levels and reduced anti-inflammatory cytokines. All these significantly inhibited parasite survival in macrophages thus demonstrating a previously unidentified dualistic regulation of TLR4signaling pathways activation through sialic acids by interplay of Neu1 and siglec-E during Leishmania infection.

IFN-γ+ CD4+T cell-driven prophylactic potential of recombinant LDBPK_252400 hypothetical protein of Leishmania donovani against visceral leishmaniasis.

Visceral leishmaniasis (VL) is a potentially fatal parasitic disease causing high morbidity and mortality in developing countries. Vaccination is considered the most effective and powerful tool for blocking transmission and control of diseases. However, no vaccine is available so far in the market for humans. In the present study, we characterized the hypothetical protein LDBPK_252400 of Leishmania donovani (LdHyP) and explored its prophylactic behavior as a potential vaccine candidate against VL. We found reduced hepato-splenomegaly along with more than 50% parasite reduction in spleen and liver after vaccination in mice. Protection in vaccinated mice after the antigen challenge correlated with the stimulation of antigen specific IFN-γ expressing CD4+T cell (~4.6 fold) and CD8+T cells (~2.1 fold) in vaccinated mice in compared to infected mice, even after 2-3 months of immunization. Importantly, antigen-mediated humoral immunity correlated with high antigen specific IgG2/IgG1 responses in vaccinated mice. In vitro re-stimulation of splenocytes with LdHyP enhances the expression of TNF-α, IFN-γ, IL-12 and IL-10 cytokines along with lower IL-4 cytokine and IL-10/IFN-γ ratio in vaccinated mice. Importantly, we observed ~3.5 fold high NO production through activated macrophages validates antigen mediated cellular immunity induction, which is critical in controlling infection progression. These findings suggest that immunization with LdHyP mount a very robust immunity (from IL-10 towards TFN-γ mediated responses) against L. donovani infection and could be explored further as a putative vaccine candidate against VL.

Prevalence of asymptomatic visceral leishmaniasis in human and dog, Benishangul Gumuz regional state, Western Ethiopia.

BACKGROUND: The Benishangul-Gumuz region is an important development corridor in Ethiopia. Large-scale projects such as the Great Renaissance Dam, mining and agriculture have entailed huge environmental modifications and settlement pattern changes. There is no detailed epidemiological information on visceral leishmaniasis (VL) in the region. MATERIALS AND METHODS: A cross-sectional study was carried out to assess the epidemiology and risk factors associated with Leishmania infection. A leishmanin skin test (LST) was done for 1342 participants, and for 253 of them rK39 and DAT were carried out. Thirty-six dogs owned by households with LST-positive member(s) were rK39 and DAT tested. A pretested questionnaire was used to capture individual and household characteristics. RESULTS: Of the 89.2% (1197/1342) who availed themselves of the LST reading, 6.0% were positive. The rk39 and DAT positivity among the 253 tested were 3.2% and 5.9%, respectively. In dogs, positivity rates by rK39 and DAT were 13.9% and 5.6%, respectively. Of the household and individual risk factors, presence of a dog in the household (P = 0.005), male sex (0.003), residence woreda (0.000) and occupation (0.023) showed a strong positive association with LST positivity. Individuals who lived in households that had dogs were 2.6 times more likely to be LST positive (AOR = 2.6; 95% CI = 1.54, 4.40). Being female decreased the probability of being LST positive by 0.38 times (AOR = 0.38; 95% CI = 0.20, 0.72). People living in Guba and Kurmuk had 4.7 (AOR = 4.74, 95% CI 1.83, 12.31) and 5.9 (AOR = 5.85, 95% CI 2.27, 15.09) times more risk of being infected. CONCLUSIONS: We demonstrated the presence of active VL transmission in the areas. Thus, we underline the need to establish the responsible vector(s) and reservoir(s) for comprehensive early containment plans to prevent potentially harmful public health and economic consequences.

Leishmania donovani infection suppresses Allograft Inflammatory Factor-1 in monocytes and macrophages to inhibit inflammatory responses.

Macrophages and monocytes are important for clearance of Leishmania infections. However, immune evasion tactics employed by the parasite results in suppressed inflammatory responses, marked by deficient macrophage functions and increased accumulation of monocytes. This results in an ineffective ability to clear parasite loads. Allograft Inflammatory Factor-1 (AIF1) is expressed in myeloid cells and serves to promote immune responses. However, AIF1 involvement in monocyte and macrophage functions during parasitic infections has not been explored. This study now shows that Leishmania donovani inhibits AIF1 expression in macrophages to block pro-inflammatory responses. Mice challenged with the parasite had markedly reduced AIF1 expression in splenic macrophages. Follow-up studies using in vitro approaches confirmed that L. donovani infection in macrophages suppresses AIF1 expression, which correlated with reduction in pro-inflammatory cytokine production and increased parasite load. Ectopic overexpression of AIF1 in macrophages provided protection from infection, marked by robust pro-inflammatory cytokine production and efficient pathogen clearance. Further investigations found that inhibiting AIF1 expression in bone marrow cells or monocytes impaired differentiation into functional macrophages. Collectively, results show that AIF1 is a critical regulatory component governing monocyte and macrophage immune functions and that L. donovani infection can suppress the gene as an immune evasion tactic.

Spatial Point Pattern Analysis Identifies Mechanisms Shaping the Skin Parasite Landscape in Leishmania donovani Infection.

Increasing evidence suggests that in hosts infected with parasites of the Leishmania donovani complex, transmission of infection to the sand fly vector is linked to parasite repositories in the host skin. However, a detailed understanding of the dispersal (the mechanism of spread) and dispersion (the observed state of spread) of these obligatory-intracellular parasites and their host phagocytes in the skin is lacking. Using endogenously fluorescent parasites as a proxy, we apply image analysis combined with spatial point pattern models borrowed from ecology to characterize dispersion of parasitized myeloid cells (including ManR+ and CD11c+ cells) and predict dispersal mechanisms in a previously described immunodeficient model of L. donovani infection. Our results suggest that after initial seeding of infection in the skin, heavily parasite-infected myeloid cells are found in patches that resemble innate granulomas. Spread of parasites from these initial patches subsequently occurs through infection of recruited myeloid cells, ultimately leading to self-propagating networks of patch clusters. This combination of imaging and ecological pattern analysis to identify mechanisms driving the skin parasite landscape offers new perspectives on myeloid cell behavior following parasitism by L. donovani and may also be applicable to elucidating the behavior of other intracellular tissue-resident pathogens and their host cells.

A Brief Report and Mini-Review of Visceral Leishmaniasis-associated Hemophagocytic Lymphohistiocytosis in Children.

We present the case of a 7-year-old boy who fulfilled the diagnostic criteria for hemophagocytic lymphohistiocytosis (HLH). Prompt visualization of his bone marrow confirmed the diagnosis of visceral leishmaniasis (VL). He responded well to treatment with liposomal amphotericin-B. The patient had a false-negative enzyme-linked immunosorbent assay for Leishmania infantum and a false-positive immunoglobulin M test for Epstein Barr virus (EBV). Because age at presentation is similar in children with VL and familial HLH for whom EBV is the usual trigger, ruling out VL is extremely important because nonspecific serologic tests for EBV can lead to the inappropriate diagnosis of EBV-driven primary HLH and to the administration of unnecessary immunochemotherapy.

Rab5b function is essential to acquire heme from hemoglobin endocytosis for survival of Leishmania.

Previously, we showed that Rab5a and Rab5b differentially regulate fluid-phase and receptor-mediated endocytosis in Leishmania, respectively. To unequivocally demonstrate the role of Rab5b in hemoglobin endocytosis in Leishmania, we generated null-mutants of Rab5b parasites by sequentially replacing both copies of LdRab5b with the hygromycin and neomycin resistance gene cassettes. LdRab5b-/- null-mutant parasite was confirmed by qPCR analysis of genomic DNA using LdRab5b specific primers. LdRab5b-/- cells showed severe growth defect indicating essential function of LdRab5b in parasite. To characterize the role of Rab5b in Hb endocytosis in parasites, LdRab5b-/- cells were rescued by exogenous addition of hemin in growth medium. Our results showed that LdRab5b-/- cells are relatively smaller in size. Ultrastructural analysis revealed the presence of relatively enlarged flagellar pocket and bigger intracellular vesicles in these cells in comparison to control cells. Both promastigotes and amastigotes of Rab5b null-mutant parasites were unable to internalize Hb but fluid phase endocytosis of different markers was not affected. However, complementation of LdRab5b:WT in LdRab5b-/- cells (LdRab5b-/-:pRab5b:WT) rescued Hb internalization in these cells. Interestingly, LdRab5b-/- cells showed significantly less Hb-receptor on cell surface in comparison to control cells indicating a block in HbR trafficking. Finally, we showed that LdRab5b-/- parasites can infect the macrophages but are unable to survive after 96 h of infection in comparison to control cells. However, supplementation of hemin in the growth medium significantly rescued LdRab5b-/-Leishmania survival in macrophage indicating that LdRab5b function is essential for the acquisition of heme from internalized Hb for the survival of Leishmania.

Leishmanial CpG DNA nanovesicles: A propitious prophylactic approach against visceral leishmaniasis.

Unmethylated CpG motifs with phosphothioate backbone trigger TLR9 to elicit innate immune response characterized by the production of Th1 cytokines. The use of CpG DNA as an adjuvant has established its role in potentiating the humoral and cell mediated vaccine specific immune response. However, none of the synthetic oligodeoxynucleotides (ODNs) know and used till date are associated with the parasite itself. Our group identified a novel CG rich sequence of 14 base pairs from Leishmania donovani genome (Ld CpG ODN) and established it as a TLR9 agonist. The present study was designed to ascertain the adjuvanticity of Ld CpG ODN with soluble leishmanial antigen in experimental model of L. donovani. During the study Schizophyllan (SPG), a fungal polymer was used for encapsulating Ld CpG ODN for efficient endosomal delivery. The synthesized nanovehicles were of nearly 100 nm and localized within endosomes as confirmed by confocal microscopy. Immunization studies displayed the superior ability of synthesized nanovehicles co-administered with parasite antigen in augmenting innate immune response in comparison to ODN, nanoparticles or soluble antigen alone. The response included generation of ROS, NO and iNOS expression followed by proinflammatory cytokine milieu with reduced parasitic load within liver, spleen and bone marrow. These immune-tailored particles in combination with parasitic antigens elicited significant generation of cell mediated response owing to the presence of high levels of CD8+ T-cells and lymphocyte proliferation. Moreover, vaccination regime with synthesized adjuvant also activated humoral immunity by escalating the levels of IgG2 followed by reduced levels of anti-leishmanial IgG and IgG1 antibodies. The findings support the efficacy of Ld CpG ODN as a potential adjuvant against visceral leishmaniasis.

Neutrophils and Visceral Leishmaniasis: Impact on innate immune response and cross-talks with macrophages and dendritic cells.

Neutrophils with their array of microbicidal activities are the first innate immune cells to guard against infection. They are also most crucial for the host's initial defense against Leishmania parasites which cause clinically diverse diseases ranging from self-healing cutaneous leishmaniasis (CL) to a more severe visceral form, visceral leishmaniasis (VL). Neutrophils are recruited in large numbers at the infection site after bite of sandfly, which is the vector for the disease. The initial interaction of neutrophils with the parasites may modulate the subsequent innate and adaptive immune responses and hence affect the disease outcome. The purpose of this review is to comprehensively appraise the role of neutrophils during the early stages of Leishmania infection with a focus on the visceral form of the disease. In the past decade, new insights regarding the role of neutrophils in VL have surfaced which have been extensively elaborated in the present review. In addition, since much of the information regarding neutrophil-Leishmania early interaction has accumulated through studies on mouse models of CL, these studies are also revisited. We begin by reviewing the factors which drive the recruitment of neutrophils at the site of injection by the sandfly. We then discuss the studies delineating the molecular mechanisms involved in the uptake of the Leishmania parasite by neutrophils and how the parasite subverts their microbicidal functions. In the end, the interaction of infected neutrophils with macrophages and dendritic cells is summarized.

Synergic effect of eugenol oleate with amphotericin B augments anti-leishmanial immune response in experimental visceral leishmaniasis in vitro and in vivo.

Present treatment regimen on visceral leishmaniasis has multiple limitations including severe side effects, toxicity, and resistance of Leishmania strains. Amphotericin B is a well-established pharmacologically approved drug; however, mainly toxicity is a foremost issue with that drug. Recently, our group identified eugenol oleate as an anti-leishmanial immunomodulatory compound. The important objectives of this present study was to evaluate the possible synergistic effect of eugenol oleate with amphotericin B to reduce the toxicity of this approved drug. Results obtained from this study signified that combination of eugenol oleate and amphotericin B showed indifferent combinatorial effect against promastigotes with xΣFIC 1.015, while, moderate synergistic activity with xΣFIC 0.456 against amastigotes. It was also notable that eugenol oleate (2.5 µM) with low concentrations of amphotericin B (0.3125 µM) showed 96.45% parasite reduction within L. donovani-infected murine macrophages. Furthermore, eugenol oleate and amphotericin B significantly (p < 0.01) enhanced the nitrite generation, and pro-inflammatory cytokines (IL-12, IFN-γ and TNF-α) in infected macrophages in vitro and in BALB/c mice in vivo. Eugenol oleate (10 mg/Kg b. wt.) with amphotericin B (1 mg/Kg b.wt.) significantly (p < 0.01) controlled the parasite burden in liver by 96.2% and in spleen by 93.12%. Hence, this study strongly suggested the synergic potential of eugenol oleate with low concentration of amphotericin B in experimental visceral leishmaniasis through anti-leishmanial immune response.

First Serological Study Revealing High Humoral Response and Evidence for Antigenic Heterogeneity in Leishmania donovani Induced CL in Sri Lanka.

Posing a threat to the ongoing leishmaniasis elimination efforts in the Indian subcontinent, L. donovani-induced cutaneous leishmaniasis (CL) has been recently reported in many countries. Sri Lanka reports a large focus of human cutaneous leishmaniasis (CL) caused by Leishmania donovani, a usually visceralizing parasite. Enhanced case detection, early treatment, and in-depth understanding of sequalae are required to contain the spread of disease. Visceralizing potential of dermotropic strains has not been fully ruled out. Sri Lankan strains have shown a poor response to established serological assays. The present concern was to develop an in-house serological assay and to determine the seroprevalence of CL for identifying visceralizing potential and its usefulness in enhancing case detection. Crude cell lysate of dermotropic L. donovani promastigotes-based indirect enzyme-linked immunosorbent assay (ELISA) was previously optimized. Assay was evaluated using sera from 200 CL patients, 50 endemic and 50 nonendemic healthy controls, 50 patients with other skin diseases, and 50 patients with other systemic diseases. Seroprevalence and clinicoepidemiological associations were analyzed. Assay was compared with light microscopy (LM) and in vitro culturing (IVC). Cost comparison was carried out. Seroprevalence of CL was 82.0%. The assay had 99.5% specificity, and all healthy controls were negative at 0.189 cut-off. Positive and negative predictive values were 99.4% and 84.7%, respectively. Positivity obtained in ELISA was comparable to LM and higher than that of IVC. Cost per patient was 3.0 USD for both ELISA and LM and 6.0 USD for IVC. Infections occurring in all age groups and both genders demonstrated >75.0% of seropositivity. Patients had lesions with different durations/types/sizes showed >70.0% of seropositivity. Study identified a high seroprevalence of L. donovani-induced CL for the first time, indicating potential for visceralization or transient serological response. This can be used as a second line test in LM-negative CL cases to enhance clinical case detection. Further studies are warranted to examine in-depth correlations, antigen profiles, comparison with other established serological tools, and usefulness in the detection of asymptomatic cases. (National patent LK/P/1/19697).

In-situ proliferation contributes to the accumulation of myeloid cells in the spleen during progressive experimental visceral leishmaniasis.

Visceral leishmaniasis (VL) is characterized by expansion of myeloid cells in the liver and spleen, which leads to a severe splenomegaly associated with higher risk of mortality. This increased cellularity is thought to be a consequence of recruitment of cells to the viscera. We studied whether the local proliferation of splenic myeloid cells contributes to increased splenic cellularity. We found that a monocyte-like population of adherent splenic cells from Leishmania donovani-infected hamsters had enhanced replicative capacity ex vivo and in vivo (BrdU incorporation, p<0.0001). In vitro assays demonstrated that proliferation was more pronounced in the proinflammatory M1 environment and that intracellular infection prevented proliferation. Secondary analysis of the published splenic transcriptome in the hamster model of progressive VL revealed a gene expression signature that included division of tumoral cells (Z = 2.0), cell cycle progression (Z = 2.3), hematopoiesis (Z = 2.8), proliferation of stem cells (Z = 2.5) and overexpression of proto-oncogenes. Regulators of myeloid cell proliferation were predicted in-silico (CSF2, TLR4, IFNG, IL-6, IL-4, RTK signaling, and STAT3). The in-silico prediction was confirmed with chemical inhibitors of PI3K/AKT, MAPK and STAT3 which decreased splenic myeloid cell division ex vivo. Hamsters infected with L. donovani treated with a STAT3 inhibitor had reduced in situ splenic myeloid proliferation (p = 0.03) and parasite burden. We conclude that monocyte-like myeloid cells have increased STAT3-dependent proliferation in the spleen of hamsters with visceral leishmaniasis and that inhibition of STAT3 reduces myeloid cell proliferation and parasite burden.

Integrative genomic, proteomic and phenotypic studies of Leishmania donovani strains revealed genetic features associated with virulence and antimony-resistance.

BACKGROUND: Leishmaniasis is a neglected tropical disease affecting millions of people worldwide. Emerging drug resistance of Leishmania species poses threaten to the effective control and elimination programme of this neglected tropical disease. METHODS: In this work, we conducted drug-resistance testing, whole genome resequencing and proteome profiling for a recently reported clinical isolate with supposed drug resistance (HCZ), and two reference sensitive strains (DD8 and 9044) of Leishmania donovani, to explore molecular mechanisms underlying drug resistance in this parasite. RESULTS: With reference to DD8 and 9044 strains, HCZ isolate showed higher-level virulence and clear resistance to antimonials in promastigote culture, infected macrophages and animal experiment. Pairwise genomic comparisons revealed genetic variations (86 copy number variations, 271 frameshift mutations in protein-coding genes and two site mutations in non-coding genes) in HCZ isolate that were absent from the reference sensitive strains. Proteomic analysis indicated different protein expression between HCZ isolate and reference strains, including 69 exclusively detected proteins and 82 consistently down-/upregulated molecules in the HCZ isolate. Integrative analysis showed linkage of 12 genomic variations (gene duplication, insertion and deletion) and their protein expression changes in HCZ isolate, which might be associated with pathogenic and antimony-resistant phenotype. Functional annotation analyses further indicated that molecules involved in nucleotide-binding, fatty acid metabolism, oxidation-reduction and transport might play a role in host-parasite interaction and drug-resistance. CONCLUSIONS: This comprehensive integrative work provided novel insights into the genetic basis underlying virulence and resistance, suggesting new aspects to be investigated for a better intervention against L. donovani and associated diseases.