Therapy For Visceral Leishmaniasis Research Articles

Use of N-acetylcysteine as treatment adjuvant regulates immune response in visceral leishmaniasis: Pilot clinical trial and in vitro experiments.

This investigation aimed to assess the effect of N-acetylcysteine (NAC) as an adjuvant treatment to alleviate visceral leishmaniasis (VL). The present work includes both blinded randomized clinical intervention and experimental in vitro studies. The clinical trial included 60 patients with VL randomly allocated into two groups: a test group (n = 30) treated with meglumine antimoniate plus NAC (SbV + NAC) and a control group (n = 30) treated with meglumine antimoniate only (SbV). The primary outcome was clinical cure (absence of fever, spleen and liver sizes reduction, and hematological improvement) in 180 days. The cure rate did not differ between the groups; both groups had similar results in all readout indices. The immunological parameters of the patients treated with SbV + NAC showed higher sCD40L in sera during treatment, and the levels of sCD40L were negatively correlated with Interleukin-10 (IL-10) serum levels. In addition, data estimation showed a negative correlation between the sCD40L levels and the spleen size in patients with VL. For the in vitro experiments, peripheral blood mononuclear cells (PBMCs) or PBMC-derived macrophages from healthy donors were exposed to soluble Leishmania antigen (SLA) or infected with stationary promastigotes of Leishmania infantum in the presence or absence of NAC. Results revealed that NAC treatment of SLA-stimulated PBMCs reduces the frequency of monocytes producing IL-10 and lowers the frequency of CD4+ and CD8+ T cells expressing (pro-)inflammatory cytokines. Together, these results suggest that NAC treatment may modulate the immune response in patients with VL, thus warranting additional investigations to support its case use as an adjuvant to antimony therapy for VL.

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.

Insights into the molecular basis of some chalcone analogues as potential inhibitors of Leishmania donovani: An integrated in silico and in vitro study

Abstract Protozoal infections caused by species belonging to Leishmania donovani complex are responsible for the most severe form of leishmaniasis, especially in Sudan and other developing countries. Drugs commonly used for the treatment of the disease show varying levels of effectiveness and also have associated side effects. Thus, the present work highlights the synthesis of some chalcones to be used as potential anti-leishmanial agents. The activity of the synthesized chalcones has been evaluated against L. donovani. The ADMET profile of the synthesized compounds were tested using various integrated web-based tools. Moreover, in order to investigate the molecular mechanism of action, the chalcone compounds were docked into L. donovani trypanothione reductase (TR) using Autodock 4.0 and molecular dynamics were studies. Eight compounds showed the highest activity against the morphological forms. Among these compounds, chalcones 15 has shown the highest inhibitory effect with IC50 value of 1.1 µM. In addition, pharmacokinetic and toxicological investigations revealed its good oral bioavailability and low toxicity. Furthermore, chalcone 15 was found to interact with high affinity (−13.7 kcal/mol) with TR, an essential enzyme for the leishmanial parasite. Thus, this promising activity against L. donovani supports the use of chalcone 15 as a potential new therapy for visceral leishmaniasis.

Identification of some chalcone analogues as potential antileishmanial agents: An integrated in vitro and in silico evaluation

Chalcones, either natural or synthetic, are known to exhibit various biological activities. The present study aimed to evaluate the in vitro and in silico activities of some chalcone analogues as potential antleishmanial agents via inhibition of the trypanothione reductase enzyme (TR). Five chalcone analogues were synthesized using Claisen-Schmidt reaction and their activity has been evaluated against Leishmania donovani and presented as IC 50 values. Various integrated web-based technologies were used to assess the synthesized compounds' absorption, distribution, metabolism, excretion, and toxicity profile (ADMET). The binding affinity of the most potent chalcone for the selected target was then investigated using Auto-Dock 4.0. Additionally, the molecular dynamics was performed using WEBGRO. ( E )-1-(4-bromophenyl)-3-(4-hydroxyphenyl) prop-2-en-1-one (Chalcone 4) has shown the highest inhibitory effect with IC 50 value 0.03 ± 0.16 µM. In addition, the pharmacokinetic and toxicological investigations revealed its good oral bioavailability and low toxicity. Furthermore, chalcone 4 was found to interact with high affinity (−8.6 kcal/mole) with trypanothione reductase (TR), an essential enzyme for the leishmanial parasite. Molecular dynamics simulation revealed several interesting features responsible for the potency and stability of chalcone 4 as TR inhibitor. Thus, the promising activity against Leishmania donovani , compared to amphotericin B and other reported chalcones derivatives, proposes the use of chalcone 4 as a potential new therapy for visceral leishmaniasis.

Screening and identification of potential inhibitor for visceral leishmaniasis (VL) through computational analysis

AimThe aim of this investigation is to detect potential inhibitor for visceral leishmaniasis through computational analysis. BackgroundLeishmaniasis is categorized as a vector born pathogenic infection prevalent in tropical, subtropical, and in Mediterranean zones spread by intra-macrophage protozoa. The clinical syndrome of leishmaniasis is divided into the following type’s namely cutaneous leishmaniasis, mucocutaneous leishmaniasis, visceral leishmaniasis, and dermal leishmaniasis. Trypanothione synthetase is a key enzyme involving in glutathione biosynthesis as well as hydrolysis. Trypanothione is one of the promising drug targets for parasites. Parasites are inimitable with concern to their dependence on trypanothione to regulate intracellular thiol-redox balance in fighting against oxidative stress and biochemical anxiety. However, trypanothione synthetase was presumed as the target therapeutic alternate in VL therapy. ObjectiveThe important objective of this current investigation is to identify or analyze the potential inhibitor for V. leishmaniasis through computational approaches which include virtual screening, molecular docking, ADME prediction, and molecular dynamic simulation. MethodsAn investigation was performed to develop a 3D protein structure, using computational screening among associated similar structured proteins from popular compound database banks such as Specs, Maybridge, and Enamine, to detect novel staging with a series of validation for emerging innovative drugs molecules. Modeled protein ligand complex was further analyzed to know the binding ability of the complex. Molecular dynamics were performed to ascertain its stability at 50 ns. ResultsTrypanothione synthetase overall ability in the outcome of series of analysis. Among three database compounds screened, the compound from the Specs database exhibited the better protein-ligand docking scores and fulfilled the drug-like properties through ADMET analysis, and the docked complexes had better stability throughout the simulation. Besides, the other two database leads fulfilled the pharmacological properties, and the complexes were stable in the simulation. ConclusionBy analyzing the various compounds from different databases, we concluded that the Specs database compound exhibits potential activity against the target protein and is considered a promising inhibitor for trypanothione synthetase.

Lethality among individuals infected with visceral leishmaniasis in Brazil: a retrospective study (2007-2018).

Visceral leishmaniasis (VL) is a public health problem with a high prevalence and lethality in Brazil. This study aims to estimate the lethality and associated factors in individuals with VL and assess survival time, emphasizing VL/HIV coinfection. This retrospective study has 37,583 individuals notified and confirmed with VL in the Sistema de Informação de Agravos de Notificação (SINAN) between 2007 and 2018 (Brazil). Lethality was evaluated considering VL deaths, deaths from other causes (OC), and non-deaths. We performed a multinomial logistic regression, with non-death as the benchmark category. We conducted a survival analysis (Kaplan-Meier method), emphasizing VL/HIV coinfection. Most individuals were young, male, mixed race, low schooling level, and urban dwellers. The lethality rate was 10.2% (VL and OC deaths) and 7.8% (VL deaths). The prevalence of HIV infection was 8.81%. A higher likelihood of VL and OC deaths was observed in older age groups, females, and with a higher number of symptoms. A higher likelihood of OC deaths was identified in individuals with HIV. A lower likelihood of VL and OC deaths was observed for individuals on VL therapy. The mean survival time was longer for VL/HIV individuals, who had a lower survival rate than those with VL. The data point to the need for attention to the timely diagnosis of VL and HIV and adequate pharmacological treatment in this population.

Nanosize Carriers for Drug and Vaccine Delivery: Advances and Challenges

Nanomedicine has emerged as a lucrative option, attracting the attention of scientific and industrial stalwarts for its enormous potential and new business opportunities in the health sector. It encompasses physical, chemical, and biological interventions used for the transformation of bulk materials into nanomaterials (NMs) with particles size ~ 1-100 nm for highly specific medical applications. Therefore, its effects in diagnosis, efficacy, treatment and prevention of diseases may easily be foreseen. The credit goes to nanotechnology, which has emerged with the ability to manipulate the NMs concerning size, shape, composition as well as surface characteristics. Due to the advantages of their tiny size as well as novel properties, NMs are useful for loading more drugs with controlled release and specific targeting. Although full bloom of nanomedicine realization might take years, recent innovations in formulations of nanotechnology-based smart drug/vaccine delivery systems are beginning to change the landscape of future medicines. They are being designed to overcome biological barriers in the living systems by improving the delivery and efficacy of traditional therapeutics and reducing the toxicity by specificity to target cells/tissues. This review focuses on basic understanding and progress in the field of nanomedicine (especially nanocarriers-based drug and vaccine delivery), including nanoformulation of Amphotericin B with functionalized carbon nanotubes for the therapy of visceral leishmaniasis.

Coalition of Biological Agent (Melatonin) With Chemotherapeutic Agent (Amphotericin B) for Combating Visceral Leishmaniasis via Oral Administration of Modified Solid Lipid Nanoparticles.

In this study, 2-hydroxypropyl-β-cyclodextrin (HPβCD) grafted solid lipid nanoparticle (SLN)-based bioconjugate was synthesized and used for administering a combination of melatonin (Mel) and amphotericin B (AmB) orally for effective visceral leishmaniasis (VL) treatment. The formulations (HPCD-Mel-AmB SLN) were synthesized by the emulsion solvent evaporation method. HPCD-Mel-AmB SLN showed a high loading capacity and a high entrapment efficiency of AmB (% DL = 9.0 ± 0.55 and % EE = 87.9 ± 0.57) and Mel (% DL = 7.5 ± 0.51 and % EE = 63 ± 6.24). The cumulative percent release of AmB and Mel was 66.10 and 73.06%, respectively, up to 72 h. Time-dependent cellular uptake was noticed for HPCD-Mel-AmB SLN for 4 h. Further, HPCD-Mel-AmB SLN did not show any toxic effects on J774A.1 macrophages and Swiss albino mice. HPCD-Mel-AmB SLN (10 mg/kg ×5 days, p.o.) has significantly diminished (98.89%) the intracellular parasite load in liver tissues of L. donovani-infected BALB/c mice, subsequently highlighting the role of melatonin toward an effective strategy in combating leishmanial infection. Therefore, these results indicated that administration of HPCD-Mel-AmB SLN improve the therapeutic index of the first-line drug in addition to the introduction of biological agent and would be a promising therapeutic candidate for effective VL therapy. In the present study, the objective is to test the efficacy of the chemotherapeutic approach in combination with a biological immunomodulatory agent against leishmanial infection using in vitro and in vivo studies. This information suggests that melatonin could be an efficacious and potent antileishmanial agent.

Effect of rK39 testing in guiding treatment initiation and outcome in patients with visceral leishmaniasis in Ethiopia: A prospective cohort study.

BackgroundThe rapid diagnostic test (RDT) rK39 is currently being used for routine diagnosis of visceral leishmaniasis (VL) in East Africa. However, continuous monitoring of the performance of the assay, in particular its impact on the clinical decision in initiating anti-leishmanial treatment and outcomes remains needed as there are concerns about the diagnostic performance of this test.MethodsVL patients prospectively enrolled in a diagnostic trial and with rK39 RDT were included. We evaluated the effect of rK39 testing in guiding treatment initiation and outcome. On the basis of rK39 RDT test result as well as clinical case definition for VL and microscopy examination, the clinicians decide whether to initiate VL therapy or not. Poisson regression models were used to identify factors associated with a decision to initiate VL therapy. In addition, treatment outcomes of those who received VL therapy were compared to those who received non-VL treatment.ResultsOf 324 VL suspects enrolled, 184 (56.8%) were rK39+ and 140 (43.2%) were rK39‒. In addition, microscopy exam was done on tissue aspirates from a sub-population (140 individuals), which is 43.2% of the suspected cases, comprising of 117 (63.6%) rK39+ and only 23 (16.4%) rK39‒ cases. Of those with microscopy examination, only 87 (62.1%) were found positive. Among 184 (56.8%) patients without microscopy, 67 (36.4%) were rK39+, of whom 83 (65.9%) were positive by microscopy, 21 (16.7%) were negative by microscopy and 22 (17.5%) had no microscopy results. On the other hand, of those who did not receive VL treatment 58/189 (30.7%) were rK39+ and 131 (69.3%) were rK39‒. Of the rK39+ cases who did not receive VL therapy, only 1 (1.7%) patient was microscopy positive, 12 (20.7%) were negative and 45 (77.6%) patients had no microscopy result. Of the rK39‒ cases (n = 131) who did not receive VL treatment, 16 were microscopy negative and 115 without microscopy exams. Whereas positive rK39 result [adjusted Relative Risk (aRR) 0.69; 95% CI: 0.49–0.96, p = 0.029] and positive microscopy results (aRR 0.03; 95% CI: 0.00–0.24, p = 0.001) were independently associated with VL treatment, having confirmed diagnosis other than VL (aRR 1.64; 95% CI: 1.09–2.46, p = 0.018) was independently associated with initiation of non-VL therapy. The proportion of rK39+ patients who received non-VL treatment with no improvement outcome was significantly higher when compared to those who received VL treatment (24.1%, 95% CI: 14.62–37.16 vs. 11.9%, 95%CI: 7.26–18.93; p<0.0001).ConclusionThe study shows that a significant proportion of patients with rK39+ results were undertreated. Failure to do microscopy was associated with lack of improved clinical outcome. Including an additional simple point-of-care assay in the diagnostic work-up is urgently needed to correctly identify VL cases and to prevent morbidity and mortality associated with the disease.

Heterologous vaccine therapy associated with half course of Miltefosine promote activation of the proinflammatory response with control of splenic parasitism in a hamster model of visceral leishmaniasis

Visceral leishmaniasis (VL) is a serious and neglected disease present worldwide. Chemotherapy using pentavalent antimony (SbV) is the most practical and inexpensive strategy available for the VL treatment today, however, it has high toxicity. Alternatively, other drugs are used as viable leishmanicidal therapeutic options. Miltefosine is the only anti-leishmanial agent administered orally, however, it has been reducing its effectiveness. In this sense, there is no ideal therapy for VL since the drugs currently used trigger severe side effects causing discontinuation of treatment, which carries an imminent risk for the emergence of parasite resistance. With that, other therapeutic strategies are gaining prominence. Among them, immunotherapy and/or immunochemotherapy, which the activation/modulation of the immune system can redirect the host's immune response to an effective therapeutic result. Therefore, this work was designed to assess an immunochemotherapy protocol composed of half course of Miltefosine associated with LBSap vaccine (Milt+LBSap) using the hamster Mesocricetus auratus as an experimental model for VL treatment. When evaluating the main hematobiochemical, immunological and therapeutic efficacy parameters, it was demonstrated that the treatment with Milt+LBSap showed restoration of hematobiochemical condition and reduced serum levels of IgG-anti-Leishmania compared to animals infected non treated (INT). Beyond that, an increase in the number of CD4+ lymphocytes producers of IFN-γ in relation to INT or to animals treated with miltefosine during 28 days, and TNF-α increased compared to INT were observed. Also, it was found a reduction of IL-10-production in relation to INT, or animals that received LBSap vaccine only, or miltefosine, following by a reduction in the splenic parasitic burden. These results demonstrate that the immunochemotherapy protocol used can stimulate the immune response, inducing an expressive cellular response sufficient to control spleen parasitism, standing out as a promising proposal for the VL treatment.

Targeting intracellular Leishmania (L.) infantum with nitazoxanide entrapped into phosphatidylserine-nanoliposomes: An experimental study

Leishmaniasis is a parasitic neglected tropical disease and result in a broad spectrum of clinical manifestations, ranging from a single ulceration to a progressive and fatal visceral disease. Comprising a limited and highly toxic therapeutic arsenal, new treatments are urgently needed. Targeting delivery of drugs has been a promising approach for visceral leishmaniasis (VL). Phosphatidylserine-liposomes have demonstrated superior efficacy in VL, targeting intracellular parasites in host cells through macrophage scavenger receptors. In this work, we investigated the in vitro and in vivo efficacy of the antihelminthic drug nitazoxanide in a nanoliposomal formulation against Leishmania (L.) infantum. Physicochemical parameters of liposomes containing nitazoxanide (NTZ-LP) were determined by dynamic light scattering and small angle X-ray scattering. The efficacy of the formulation was verified in an intracellular amastigote model and in an experimental hamster model. Our findings showed that NTZ-LP was able to eliminate the amastigotes inside the host cell with an IC50 value of 16 μM. NTZ-LP was labelled a fluorescent probe and by spectrofluorimetry, we observed that the infected macrophages internalized similar levels of the drug to the uninfected cells. The confocal microscopy images confirmed the uptake and demonstrated a diffuse distribution of the NTZ-LP in the cytoplasm of Leishmania-infected macrophages, with the vesicles in a closer proximity to the parasites. For the in vivo efficacy, the liposomal NTZ-LP was administrated intraperitoneally to Leishmania-infected hamsters for 10 consecutive days at 2 mg/kg/day. By qPCR we demonstrated a reduction of the parasite burden by 82% and 50% in the liver (p < 0.05) and spleen (p < 0.05), respectively. NTZ (non-liposomal) was administered at 100 mg/kg/day per oral (p.o.) for the same period, but demonstrated no efficacy. This liposomal formulation ensured a targeting delivery of NTZ to the intracellular parasites, resulting in an good efficacy at a low dose in animals, and it may represent a new candidate therapy for VL.

A new multi-epitope peptide vaccine induces immune responses and protection against Leishmania infantum in BALB/c mice.

Visceral leishmaniasis (VL) is a tropical and subtropical disease which is endemic in more than eighty countries aroundthe world. Leishmania infantum is one of the main causative agents of VL disease. Currently, there is no approved-to-market vaccine for VL therapy. In this study, we evaluated cellular and humoral immune responses induced by our newly designed multi-epitope vaccine in BALB/c mice. Four antigenic proteins, including histone H1, sterol 24-c-methyltransferase (SMT), Leishmania-specific hypothetical protein (LiHy), and Leishmania-specific antigenic protein (LSAP) were chosen for the prediction of potential immunodominant epitopes. Moreover, to enhance vaccine immunogenicity, two toll-like receptors 4(TLR4) agonists, resuscitation-promoting factors of Mycobacterium tuberculosis (RpfE and RpfB), were employed as the built-in adjuvants. Immunization with the designed multi-epitope vaccine elicited a robust Th1-type immune response, compared to other groups, as shown by increased levels of IL-2, IFN-γ, TNF-α, and IgG2a. Furthermore, a significant decrease was observed in Th-2-type-related cytokines such as IL-4 in immunized mice. The designed construct also induced a significant reduction in parasite load (p < 0.0001), conferring protection against L. infantum challenge. This study could be promising in gaining insight towards the potential of peptide epitope-based vaccines as effective protective approaches against Leishmania species.

Visceral leishmaniasis in a patient with active HBV/HDV co-infection

Visceral leishmaniasis (VL) is an endemic infection in different regions of Italy and Europe caused by protozoan parasites of the genus Leishmania, transmitted to humans through sandflies bites. Reactivation after Solid Organ Transplantation was reported and could be a risk of organ rejection. A 48 years old woman was admitted to our hospital, complaining about low-grade fever, loss of weight and new onset pancytopenia in a known cirrhosis due to active HBV/HDV co-infection. Clinical, microbiological and anatomo-pathological elements were pivotal to define the diagnosis of VL and started an appropriate anti-infective treatment. After that she underwent liver transplantation and a therapy for VL was set. No signs of reactivation were reported in the 14 months of follow-up.

Evaluation of synergistic therapeutic effect of shark cartilage extract with artemisinin and glucantime on visceral leishmaniasis in BALB/c mice.

Objective(s):Because leishmaniasis is related to the impaired functioning of T-cells, the use of an immunomodulator can increase the efficacy of antileishmanial therapy in visceral leishmaniasis. In this study, we used shark cartilage extract with artemisinin and glucantime against visceral leishmaniasis in BALB/c mice, and evaluated the synergistic therapeutic effect. Materials and Methods:The culturing method and quantitative real-time PCR by using the kDNA gene was used to detect parasite loads in the spleen and liver. INF-γ and IL-4 cytokine levels and survival rates were assayed.Results:The drug therapy with target drugs reduced parasite burden in the spleen and liver significantly. Although parasite burden was lower in the artemisinin treated group than in the glucantime treated group (P<0.05). The mice survival rate records, throughout the experimental period, showed highly significant survival rates in the test groups compared to the control group (P<0.001). The results of cytokine assay in mice treated with glucantime-shark cartilage extract combination indicated significant increases of IFNγ and IL-4 (P<0.05). Although the increase of IFNγ was more notable than IL-4. The synergistic therapeutic effect is shown in all groups except in the group treated with shark cartilage extract-artemisinin combination. The IFN-γ in glucantime-shark cartilage extract combination treated group was higher than in other groups (P<0.05). The survival rate in this group was more than in other groups too (P<0.05).Conclusion:Combination therapy with shark cartilage extract as an immunomodulator can increase antileishmanial effects of antimony drugs in VL treatment.

MicroRNA expression profiling of dibenzalacetone (DBA) treated intracellular amastigotes of Leishmania donovani

BackgroundAmong different leishmanial infections, visceral leishmaniasis (VL) if not treated is the most severe form with high mortality rates. In India, it is caused by the protozoan parasite Leishmania donovani. The therapy of visceral leishmaniasis is limited due to high toxicity, resistance to existing drugs and increasing cases of Leishmania co-infections. Hence, there is a need to identify novel drug and targets to overcome these hindrances. MicroRNAs (miRNAs) are a class of small non-coding RNAs (∼22–24 nucleotide in length) that regulate gene expression in various biological processes. They play as intracellular mediators that are essential for different biological processes. ObjectivesThe aim of present study is to explore the leishmaniacidal role of trans-dibenzalacetone (DBA, a synthetic monoketone analog of curcumin) on the expression profile of miRNA in intracellular amastigotes of Leishmania donovani. MethodsSmall RNA libraries of samples (macrophages-infected with Leishmania amastigotes; and infected macrophages treated with DBA) were prepared by using Illumina Trueseq Small RNA kit. ResultsUsing miRDIP database, we identified target gene of differentially expressed miRNAs (target miRNAs: hsa-mir-15b, hsa-mir-671, hsa-mir-151a and has-mir-30c) which was confirmed by real time stem-loop PCR. Ten KEGG pathways were significantly enriched with these target miRNA genes and they mainly relate to mitogen-activated protein kinases (MAPK) pathway. We have previously established the antiproliferative and apoptotic effect of trans-dibenzalacetone (DBA, a synthetic monoketone analog of curcumin) on the Leishmania donovani parasites. In the present study, using GFP-ATG8 gene as a marker for tracking putative autophagosomes, we confirmed that autophagic vacuolization may lead to autophagic cell death in the DBA-treated parasites. Our results demonstrated that curcumin analog DBA has a role to play in regulating the balance between autophagy and apoptosis. ConclusionsWe conclude that curcumin analog DBA triggers imbalance between two known phenotypes of cell death viz apoptosis and autophagy.

Using focused pharmacovigilance for ensuring patient safety against antileishmanial drugs in Bangladesh\u2019s National Kala-azar Elimination Programme

BackgroundAdverse effects of antileishmanial drugs can affect patients’ quality of life and adherence to therapy for visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL). In Bangladesh, there are 26 treatment centers that manage leishmaniasis cases coming from 100 endemic upazilas (subdistricts) of 26 districts (these include VL, PKDL, treatment failure, and relapse VL and cutaneous leishmaniasis cases). This study aimed to investigate the feasibility of using focused pharmacovigilance for VL (VLPV) in Bangladesh’s National Kala-azar Elimination Programme for the early detection and prevention of expected and unexpected adverse drug reactions (ADRs).MethodsThis activity has been going on since December 2014. Activity area includes secondary public hospital or Upazila health complex (UHC) in hundred sub districts and Surya Kanta Kala-azar Research Center (SKKRC) in Mymensingh District, a specialized center for management of complicated VL and PKDL cases. Communicable Disease Control (CDC) of the Directorate General of Health Services (DGHS) assigned twenty five of hundred UHCs and SKKRC (total 26) as treatment centers depending on their suitable geographical location. This was implemented for better management of VL cases with Liposomal Amphotericin B (AmBisome®) to ensure patient convenience and proper utilization of this expensive donated drug. A VLPV expert committee and a UHC VLPV team were established, an operational manual and pharmacovigilance report forms were developed, training and refresher training of health personnel took place at UHCs and at the central level, collected information such as patient data including demographics, treatment history and response, adverse events were analyzed. This report includes information for the period from December 2014 to December 2016.ResultsFrom December 2014 to December 2016, 1327 leishmaniasis patients were treated and 1066 (80%) were available for VLPV. Out of these, 57, 33, 9, and 1% were new VL, PKDL, VL relapse, and other cases, respectively. Liposomal amphotericin B was mostly used (82%) for case management, followed by miltefosine (20%) and paromomycin (3%). Out of the 1066 patients, 26% experienced ADRs. The most frequent ADR was fever (17%, 176/1066), followed by vomiting (5%, 51/1066). Thirteen serious adverse events (SAEs) (eight deaths and five unexpected SAEs) were observed. The expert committee assessed that three of the deaths and all unexpected SAEs were possibly related to treatment. Out of the five unexpected SAEs, four were miltefosine-induced ophthalmic complications and the other was an AmBisome®-induced avascular necrosis of the nasal alae. The Directorate General of the Drug Administration entered the ADRs into the World Health Organization Uppsala Monitoring Centre (WHO-UMC) VigiFlow database.ConclusionsThis study found that VLPV through NKEP is feasible and should be continued as a routine activity into the public health system of Bangladesh to ensure patient safety against anti-leishmanial drugs.

Miltefosine-resistant Leishmania infantum strains with an impaired MT/ROS3 transporter complex retain amphotericin B susceptibility.

Increasing numbers of miltefosine treatment failures in visceral leishmaniasis therapy and reports of miltefosine resistance in the Indian subcontinent resulted in the recommendation to use liposomal amphotericin B as first-line therapy. Cross-resistance between miltefosine and amphotericin B has recently been documented, suggesting a role of mutations in the miltefosine transporter, a complex encoded by the MT and ROS3 genes. This study aimed to further explore the putative role of MT/ROS3 defects in the molecular basis of amphotericin B cross-resistance. The susceptibility profiles of different miltefosine-resistant Leishmania infantum strains with well-characterized mutations in the transporter complex and the corresponding episomally restored susceptible parasite lines were determined using both the routine extracellular promastigote assay and the intracellular amastigote assay. In vitro amastigote and promastigote susceptibility testing of the two miltefosine-resistant and the episomally reconstituted L. infantum lines revealed full susceptibility to amphotericin B, despite the variable miltefosine susceptibility profile. Mutations present in either the MT and/or ROS3 gene are not sufficient to elicit higher tolerance to amphotericin B. Additional synergistic adaptations may be responsible for the miltefosine/amphotericin B cross-resistance described earlier.

Immunotherapeutic Potential of Eugenol Emulsion in Experimental Visceral Leishmaniasis.

BackgroundThe therapy of visceral leishmaniasis (VL) is limited by resistance, toxicity and decreased bioavailability of the existing drugs coupled with dramatic increase in HIV-co-infection, non-availability of vaccines and down regulation of cell-mediated immunity (CMI). Thus, we envisaged combating the problem with plant-derived antileishmanial drug that could concomitantly mitigate the immune suppression of the infected hosts. Several plant-derived compounds have been found to exert leishmanicidal activity via immunomodulation. In this direction, we investigated the antileishmanial activity of eugenol emulsion (EE), complemented with its immunomodulatory and therapeutic efficacy in murine model of VL.Methodology/Principal FindingsOil-in-water emulsion of eugenol (EE) was prepared and size measured by dynamic light scattering (DLS). EE exhibited significant leishmanicidal activity with 50% inhibitory concentration of 8.43±0.96 μg ml-1 and 5.05±1.72 μg ml─1, respectively against the promastigotes and intracellular amastigotes of Leishmania donovani. For in vivo effectiveness, EE was administered intraperitoneally (25, 50 and 75 mg/kg b.w./day for 10 days) to 8 week-infected BALB/c mice. The cytotoxicity of EE was assessed in RAW 264.7 macrophages as well as in naive mice. EE induced a significant drop in hepatic and splenic parasite burdens as well as diminution in spleen and liver weights 10 days post-treatment, with augmentation of 24h-delayed type hypersensitivity (DTH) response and high IgG2a:IgG1, mirroring induction of CMI. Enhanced IFN-γ and IL-2 levels, with fall in disease-associated Th2 cytokines (IL-4 and IL-10) detected by flow cytometric bead-based array, substantiated the Th1 immune signature. Lymphoproliferation and nitric oxide release were significantly elevated upon antigen revoke in vitro. The immune-stimulatory activity of EE was further corroborated by expansion of IFN-γ producing CD4+ and CD8+ splenic T lymphocytes and up-regulation of CD80 and CD86 on peritoneal macrophages. EE treated groups exhibited induction of CD8+ central memory T cells as evidenced from CD62L and CD44 expression. No biochemical alterations in hepatic and renal enzymes were observed.ConclusionsOur results demonstrate antileishmanial activity of EE, potentiated by Th1 immunostimulation without adverse side effects. The Th1 immune polarizing effect may help to alleviate the depressed CMI and hence complement the leishmanicidal activity.

The anti-tubercular drug delamanid as a potential oral treatment for visceral leishmaniasis.

There is an urgent requirement for safe, oral and cost-effective drugs for the treatment of visceral leishmaniasis (VL). We report that delamanid (OPC-67683), an approved drug for multi-drug resistant tuberculosis, is a potent inhibitor of Leishmania donovani both in vitro and in vivo. Twice-daily oral dosing of delamanid at 30 mg kg(-1) for 5 days resulted in sterile cures in a mouse model of VL. Treatment with lower doses revealed a U-shaped (hormetic) dose-response curve with greater parasite suppression at 1 mg kg(-1) than at 3 mg kg(-1) (5 or 10 day dosing). Dosing delamanid for 10 days confirmed the hormetic dose-response and improved the efficacy at all doses investigated. Mechanistic studies reveal that delamanid is rapidly metabolised by parasites via an enzyme, distinct from the nitroreductase that activates fexinidazole. Delamanid has the potential to be repurposed as a much-needed oral therapy for VL.

Differential Role of Leptin as an Immunomodulator in Controlling Visceral Leishmaniasis in Normal and Leptin-Deficient Mice.

Visceral leishmaniasis (VL) is caused by the protozoan parasite Leishmania donovani. There are no vaccines and available drugs against leishmaniasis are toxic. Immunomodulators that specifically boost the anti-microbial activities of the immune cells could alleviate several of these limitations. Therefore, finding novel immunomodulators for VL therapy is a pressing need. This study is aimed to evaluate the immunomodulatory role of leptin, an adipocyte-derived hormone capable of regulating the immune response, in L. donovani-infected mice. We observed that recombinant leptin treatment reduced splenic parasite burden compared with non-treated infected normal mice. Decrease in parasite burden correlated with an induction of innate immune response in antigen-presenting cells that showed an increase in nitric oxide, enhanced pro-inflammatory cytokine (interferon gamma [IFNγ], interleukin12 [IL]12, and IL1β) response in the splenocytes, indicating host-protecting Th1 response mediated by leptin. Moreover, in infected normal mice, leptin treatment induced IFNγ production from both CD4+ and CD8+ T cells, compared with non-treated infected mice. Alternatively, leptin-deficient (Ob/Ob) mice had higher splenic and liver parasite burden compared with the infected normal mice. However, leptin treatment failed to reduce the splenic parasite burden and improve a host-protective cytokine response in these mice. In addition, in contrast to dendritic cells (DCs) from a normal mouse, Ob/Ob mouse–derived DCs showed a defect in the induction of innate immune response on Leishmania infection that could not be reversed by leptin treatment. Therefore, our findings reveal that leptin has a differential immunomodulatory effect in controlling VL in normal and Ob/Ob mice.