An introduction to dynamic nucleoporins in Leishmania species: Novel targets for tropical-therapeutics.

As an ailment, leishmaniasis is still an incessant challenge in neglected tropical diseases and neglected infections of poverty worldwide. At present, the diagnosis and treatment to combat Leishmania tropical infections are not substantial remedies and require advanced & specific research. Therefore, there is a need for a potential novel target to overcome established medicament modalities' limitations in pathogenicity. In this review, we proposed a few ab initio findings in nucleoporins of nuclear pore complex in Leishmania sp. concerning other infectious protists. So, through structural analysis and dynamics studies, we hypothesize the nuclear pore molecular machinery & functionality. The gatekeepers Nups, export of mRNA, mitotic spindle formation are salient features in cellular mechanics and this is regulated by dynamic nucleoporins. Here, diverse studies suggest that Nup93/NIC96, Nup155/Nup144, Mlp1/Mlp2/Tpr of Leishmania Species can be a picked out marker for diagnostic, immune-modulation, and novel drug targets. In silico prediction of nucleoporin-functional interactors such as NUP54/57, RNA helicase, Ubiquitin-protein ligase, Exportin 1, putative T-lymphocyte triggering factor, and 9 uncharacterized proteins suggest few more noble targets. The novel drug targeting to importins/exportins of Leishmania sp. and defining mechanism of Leptomycin-B, SINE compounds, Curcumins, Selinexor can be an arc-light in therapeutics. The essence of the review in Leishmania's nucleoporins is to refocus our research on noble molecular targets for tropical therapeutics. Supplementary Information: The online version contains supplementary material available at 10.1007/s12639-022-01515-0.

Therapeutic Potential of Quercetin-Loaded Nanoemulsion against Experimental Visceral Leishmaniasis: In Vitro/Ex Vivo Studies and Mechanistic Insights.

Visceral leishmaniasis (VL) is one of the most fatal and neglected tropical diseases caused by Leishmania donovani (L. donovani). The applications of currently available chemotherapy (amphotericin B, miltefosine, and others) in VL treatment have been limited due to their poor bioavailability, unfavorable toxicity profile, and prolonged parenteral dosing. Quercetin (QT), a potent natural antioxidant, is a prominent target when conducting investigations on alternative therapies against L. donovani infections. However, the therapeutic applications of QT have been restricted due to its low solubility and bioavailability. In the present study, we developed and evaluated the antileishmanial activity (ALA) of quercetin-loaded nanoemulsion (QTNE) against L. donovani clinical strains. In vitro anti-promastigote assay results demonstrated that QTNE (IC50 6.6 µM, 48 h) significantly inhibited the growth of parasites more efficiently than the pure QT suspension in a dose- and time-dependent manner. Results of the anti-amastigote assay revealed that the infected macrophages (%) of QTNE were significantly more than those of the pure QT suspension at all concentrations (6.6, 26.4, and 52.8 µM; p < 0.05, p < 0.01 compared to the control). Moreover, the results of in vitro and ex vivo studies assisted in determining the mechanistic insights associated with the ALA of QTNE. The overall findings suggested that QTNE exhibited potential ALA by enhancing the intracellular ROS and nitric oxide levels, inducing distortion of membrane integrity and phosphatidylserine release (AV/PI), rupturing the parasite DNA (late apoptosis/necrosis process), and upregulating the immunomodulatory effects (IFN-γ and IL-10 levels). Additionally, QTNE showed superior biocompatibility against all of the treated healthy cells (PBMCs, PECs, and BMCs) as compared to the control. In conclusion, QTNE acts as a potential antileishmanial agent targeting both promastigote and intracellular amastigote forms of L. donovani, which thus opens a new avenue for the use of QTNE in VL therapy.

A randomized, open-label study to evaluate the efficacy and safety of liposomal amphotericin B (AmBisome) versus miltefosine in patients with post-kala-azar dermal leishmaniasis.

BACKGROUND: Treatment of post-kala-azar dermal leishmaniasis cases is of paramount importance for kala-azar elimination; however, limited treatment regimens are available as of now. AIM: To compare the effectiveness of liposomal amphotericin B vs miltefosine in post-kala-azar dermal leishmaniasis patients. METHODOLOGY: This was a randomized, open-label, parallel-group study. A total of 100 patients of post kala azar dermal leishmaniasis, aged between 5 and 65 years were recruited, 50 patients in each group A (liposomal amphotericin B) and B (miltefosine). Patients were randomized to receive either liposomal amphotericin B (30 mg/kg), six doses each 5 mg/kg, biweekly for 3 weeks or miltefosine 2.5 mg/kg or 100 mg/day for 12 weeks. All the patients were followed at 3rd, 6th and 12th months after the end of the treatment. RESULTS: In the liposomal amphotericin B group, two patients were lost to follow-up, whereas four patients were lost to follow-up in the miltefosine group. The initial cure rate by "intention to treat analysis" was 98% and 100% in liposomal amphotericin B and miltefosine group, respectively. The final cure rate by "per protocol analysis" was 74.5% and 86.9% in liposomal amphotericin B and miltefosine, respectively. Twelve patients (25.5%) in the liposomal amphotericin B group and six patients (13%) in the miltefosine group relapsed. None of the patients in either group developed any serious adverse events. LIMITATIONS: Quantitative polymerase chain reaction was not performed at all the follow-up visits and sample sizes. CONCLUSION: Efficacy of miltefosine was found to be better than liposomal amphotericin B, hence, the use of miltefosine as first-line therapy for post-kala-azar dermal leishmaniasis needs to be continued. However, liposomal amphotericin B could be considered as one of the treatment options for the elimination of kala-azar from the Indian subcontinent.

In vitro and in silico analysis of L. donovani enoyl acyl carrier protein reductase - A possible drug target.

The emergence of increased resistance to the available drugs has created a situation that demands to find out more specific molecular drug targets for Leishmaniasis. The enoyl acyl carrier protein reductase (ENR), a regulatory enzyme in type II fatty acid synthesis, was confirmed as a novel drug target and triclosan as its specific inhibitor in many microorganisms. In this study, the triclosan was tested for the leishmanicidal property against Leishmania donovani (L. donovani) and the results of in vitro and ex vivo drug assays on promastigotes and amastigotes showed that triclosan possessed antileishmanial activity with a half minimal inhibitory concentration (IC50) of 30 µM. Consequently, adopting in silico approach, we have tested the triclosan's ability to bind with the L. donovani enoyl acyl carrier protein reductase (LdENR). The 3D structure of LdENR was modelled, triclosan and cofactors were docked in LdENR model and molecular dynamic simulations were performed to observe the protein-ligands interactions, stability, compactness and binding energy calculation of the ligands-LdENR complexes. The observation showed that triclosan stably interacted with LdENR in presence of both the cofactors (NADPH and NADH), however, simulation results favor NADH as a preferred co-factor for LdENR. These results support that the reduction of L. donovani growth in the in vitro and ex vivo drug assays may be due to the interaction of triclosan with LdENR, which should be confirmed through enzymatic assays. The results of this study suggest that LdENR could be a potential drug target and triclosan as a lead for Leishmaniasis.Communicated by Ramaswamy H. Sarma.

Synthesis, characterization, and mechanistic studies of a gold nanoparticle-amphotericin B covalent conjugate with enhanced antileishmanial efficacy and reduced cytotoxicity.

BACKGROUND: Amphotericin B (AmB) as a liposomal formulation of AmBisome is the first line of treatment for the disease, visceral leishmaniasis, caused by the parasite Leishmania donovani. However, nephrotoxicity is very common due to poor water solubility and aggregation of AmB. This study aimed to develop a water-soluble covalent conjugate of gold nanoparticle (GNP) with AmB for improved antileishmanial efficacy and reduced cytotoxicity. METHODS: Citrate-reduced GNPs (~39 nm) were functionalized with lipoic acid (LA), and the product GNP-LA (GL ~46 nm) was covalently conjugated with AmB using carboxyl-to-amine coupling chemistry to produce GNP-LA-AmB (GL-AmB ~48 nm). The nanoparticles were characterized by dynamic light scattering, transmission electron microscopy (TEM), and spectroscopic (ultraviolet-visible and infrared) methods. Experiments on AmB uptake of macrophages, ergosterol depletion of drug-treated parasites, cytokine ELISA, fluorescence anisotropy, flow cytometry, and gene expression studies established efficacy of GL-AmB over standard AmB. RESULTS: Infrared spectroscopy confirmed the presence of a covalent amide bond in the conjugate. TEM images showed uniform size with smooth surfaces of GL-AmB nanoparticles. Efficiency of AmB conjugation was ~78%. Incubation in serum for 72 h showed <7% AmB release, indicating high stability of conjugate GL-AmB. GL-AmB with AmB equivalents showed ~5-fold enhanced antileishmanial activity compared with AmB against parasite-infected macrophages ex vivo. Macrophages treated with GL-AmB showed increased immunostimulatory Th1 (IL-12 and interferon-γ) response compared with standard AmB. In parallel, AmB uptake was ~5.5 and ~3.7-fold higher for GL-AmB-treated (P<0.001) macrophages within 1 and 2 h of treatment, respectively. The ergosterol content in GL-AmB-treated parasites was ~2-fold reduced compared with AmB-treated parasites. Moreover, GL-AmB was significantly less cytotoxic and hemolytic than AmB (P<0.01). CONCLUSION: GNP-based delivery of AmB can be a better, cheaper, and safer alternative than available AmB formulations.

Immunomodulation induced through ornithine decarboxylase DNA immunization in Balb/c mice infected with Leishmania donovani.

We report here a Leishmania donovani ornithine decarboxylase (Ld-ODC) gene used as a DNA vaccine against visceral leishmaniasis in a murine Balb/c mouse model. This study also evaluated the possible mechanism of action directed by this candidate. We found a Th1 immune response after immunization using an Ld-ODC DNA vaccine, with results based on the rearrangement of TCR-V-α-2, proliferation of Carboxy fluorescein Succinimidyle ester positive T cells, which were able to produce cytokines such as TNF-α, IFN-γ, IL-12 and IL-2, but not IL-4, IL-5, IL-6 and IL-10, and modulations of the STAT-1 and p38 MAP kinase signaling pathways. The results were corroborated with the reduction in the amastigote proliferation and parasite killing in spleens after infection in vitro. We conclude this study suggesting that the Ld-ODC DNA construct could be a new vaccine candidate against visceral leishmaniasis.

Leishmania donovani resistant to Ambisome or Miltefosine exacerbates CD58 expression on NK cells and promotes trans-membrane migration in association with CD2.

Visceral leishmaniasis (VL) is a disease that is associated with compromised immunity and drug un-responsiveness as well as with the emergence of drug resistance in Leishmania donovani (Ld). Ld down-modulates cellular immunity by manipulating signaling agents, including a higher expression of the adhesion molecule CD58. The expression of CD58 and CD2 on natural killer (NK) cells facilitates intercellular adhesion and signaling. The influence of drug-resistant Ld on the expression of CD58 and CD2 was addressed in this study. The mean florescence intensity (MFI) of CD58 but not of CD2 was twofold higher on CD56+ cells during VL, but was down-regulated after treatment. In addition, MFI of CD58 on CD56+ cells was further exacerbated in VL subjects who had relapsed after Ambisome or Miltefosine treatment. The same pattern of CD58 expression was also obtained upon stimulation of healthy peripheral blood mononuclear cells with Miltefosine- or Ambisome-resistant Ld. The ratio of CD56+CD58+IFN-γ+/CD56+CD58+IL-10+ cells was reduced by 6.98-fold after stimulation with Ld. Further, an antagonist to CD58 or its counter-receptor CD2 down-regulated CD56+ NK cell recruitment across a polycarbonate trans-membrane at Ld infection sites. This study reports that factors associated with drug resistance in Ld probably promote higher expression of CD58 on CD56+ cells and their migration to the infection site in association with CD2.

Immunomodulation mediated through Leishmania donovani protein disulfide isomerase by eliciting CD8+ T-cell in cured visceral leishmaniasis subjects and identification of its possible HLA class-1 restricted T-cell epitopes.

Protein disulphide isomerase (PDI) is one of the key enzymes essential for the survival of Leishmania donovani in the host. Our study suggested that PDI is associated with the generation of Th1-type of cellular responses in treated Visceral leishmaniasis (VL) subjects. The stimulation of Peripheral blood mononuclear cells (PBMCs) with recombinant Protein Disulphide Isomerase upregulated the reactive oxygen species generation, Nitric oxide release, IL12 and IFN-γ production indicating its pivotal role in protective immune response. Further, a pre-stimulation of PBMCs with Protein disulphide isomerase induced a strong IFN-γ response through CD8+ T cells in treated VL subjects. These findings also supported through the evidence that this antigen was processed and presented by major histocompatibility complex class I (MHC-1) dependent pathway and had an immunoprophylactic potential which can induce CD8+ T cell protective immune response in MHC class I dependent manner against VL. To find out the possible epitopes that might be responsible for CD8+ T cell specific IFN-γ response, computational approach was adopted. Six novel promiscuous epitopes were predicted to be highly immunogenic and can be presented by 32 different HLA allele to CD8+ T cells. Further investigation will explore more about their immunological relevance and usefulness as vaccine candidates.

Leishmania donovani phosphoproteins pp41 and pp29 re-establishes host protective immune response in visceral leishmaniasis.

As phospho proteins are reported to be involved in virulence and survival, the ability of Leishmania to inhibit macrophage effector functions may result from a direct interference of leishmanial molecules with macrophage signal transduction pathways. Several such proteins such as pp63, pp41 and pp29 have also been identified as a Th1 stimulatory protein in the Leishmania donovani. In the present study, the immunogenicity of a cocktail of pp63+pp41+pp29 was assessed by estimation of serum antibody titre, nitric oxide(NO) production, estimation of Th1 cytokine(IFN-γ) as well as Th2 cytokines(IL-4), and determination of parasite load in L. donovani infected mice. In the group immunized with antigenic cocktail there was a sharp rise in antibody titer up to Day 20 which reduced considerably by Day 50. Groups of mice vaccinated with pp63, pp41, pp29 and the antigenic cocktail expressed 10-fold, 16-fold, 22-fold and 25-fold increase respectively in NO production by splenocytes. The animal groups immunized with pp63, pp41, pp29 and the antigenic cocktail showed reduced parasite load in the liver and spleen, as well as increased IFN-gamma production in the spleen. Furthermore immunized animals remained with a normal hematological profile, whereas L. donovani in unimmunized mice lead to significant anemia.

Evaluation of rK-39 strip test using urine for diagnosis of visceral leishmaniasis in an endemic region of India.

The definitive diagnosis of visceral leishmaniasis (VL) requires invasive procedures for demonstration of parasites in tissue smear or culture. These procedures need expertise and laboratory supports and cannot be performed in the field. The aim of the present study was to evaluate the existing rK-39 immunochromatographic nitrocellulose strips test (ICT) with some modification in human urine for diagnosis of VL. The test was performed on both sera and urine samples on the same 786 subjects (365 confirmed VL and 421 control subjects). The sensitivity of the rK-39 ICT in serum was 100%, whereas the specificity was 93.8%, 100%, and 96.2% in healthy controls from endemic, non-endemic, and other infectious diseases, respectively. However, in urine samples, the test showed 96.1% sensitivity and 100% specificity. Considering sensitivity and feasibility of the test in the field, rK-39 ICT using urine samples can be an alternative to conventional invasive VL diagnosis.

Agar barrier method for removal of yeast and other fungal contamination from Leishmania promastigotes culture.

The present study explains a novel method of Leishmania promastigotes culture decontamination. The method is based on motility of Leishmania promastigotes across agar barrier which facilitates decontamination from yeast and other non motile contamination. This is inexpensive, easy, rapid and reliable physical method and is able to save valuable isolates in culture.