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TLR9 and MyD88 are crucial for the maturation and activation of dendritic cells by paromomycin-miltefosine combination therapy in visceral leishmaniasis.

ABSTRACT: BACKGROUND AND PURPOSE: The combination of paromomycin-miltefosine is a successful anti-leishmanial therapy in visceral leishmaniasis (VL). This encouraged us to study its effect on Toll-like receptor (TLR)-mediated immunomodulation of dendritic cells (DC), as DC maturation and activation is crucial for anti-leishmanial activity. EXPERIMENTAL APPROACH: In silico protein-ligand interaction and biophysical characterization of TLR9-drug interaction was performed. Interaction assays of HEK293 cells with different concentrations of miltefosine and/or paromomycin were performed, and NF-?B promoter activity measured. The role of TLR9 and MyD88 in paromomycin/miltefosine-induced maturation and activation of DCs was evaluated through RNA interference techniques. The effect of drugs on DCs was measured in terms of counter-regulatory production of IL-12 over IL-10, and characterized by chromatin immunoprecipitation assay at the molecular level. KEY RESULTS: Computational and biophysical studies revealed that paromomycin/miltefosine interact with TLR9. Both drugs, as a monotherapy/combination, induced TLR9-dependent NF-?B promoter activity through MyD88. Moreover, the drug combination induced TLR9/MyD88-dependent functional maturation of DCs, evident as an up-regulation of co-stimulatory markers, enhanced antigen presentation by increasing MHC II expression, and increased stimulation of naive T-cells to produce IFN-?. Both drugs, by modifying histone H3 at the promoter level, increased the release of IL-12, but down-regulated IL-10 in a TLR9-dependent manner. CONCLUSIONS AND IMPLICATIONS: These results provide the first evidence that the combination of paromomycin-miltefosine critically modifies the maturation, activation and development of host DCs through a mechanism dependent on TLR9 and MyD88. This has implications for evaluating the success of other combination anti-leishmanial therapies that act by targeting host DCs.

SUBMITTER: Das S

PROVIDER: S-EPMC3952803 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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TLR9 and MyD88 are crucial for the maturation and activation of dendritic cells by paromomycin-miltefosine combination therapy in visceral leishmaniasis.

Das Sushmita S Rani Mukta M Rabidas Vidyanand V Pandey Krishna K Sahoo Ganesh Chandra GC Das Pradeep P

British journal of pharmacology 20140301 5

<h4>Background and purpose</h4>The combination of paromomycin-miltefosine is a successful anti-leishmanial therapy in visceral leishmaniasis (VL). This encouraged us to study its effect on Toll-like receptor (TLR)-mediated immunomodulation of dendritic cells (DC), as DC maturation and activation is crucial for anti-leishmanial activity.<h4>Experimental approach</h4>In silico protein-ligand interaction and biophysical characterization of TLR9-drug interaction was performed. Interaction assays of ...[more]

PMID: 24670148

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Project description:BackgroundAmBisome therapy for VL has an excellent efficacy and safety profile and has been adopted as a first-line regimen in Bangladesh. Second-line treatment options are limited and should preferably be given in short course combinations in order to prevent the development of resistant strains. Combination regimens including AmBisome, paromomycin and miltefosine have proved to be safe and effective in the treatment of VL in India. In the present study, the safety and efficacy of these same combinations were assessed in field conditions in Bangladesh.MethodsThe safety and efficacy of three combination regimens: a 5 mg/kg single dose of AmBisome + 7 subsequent days of miltefosine (2.5 mg/kg/day), a 5 mg/kg single dose of AmBisome + 10 subsequent days of paromomycin (15 mg/kg/day) and 10 days of paromomycin (15 mg/kg/day) + miltefosine (2.5 mg/kg/day), were compared with a standard regimen of AmBisome 15 mg/kg given in 5 mg/kg doses on days 1, 3 and 5. This was a phase III open label, individually randomized clinical trial. Patients from 5 to 60 years with uncomplicated primary VL were recruited from the Community Based Medical College Bangladesh (CBMC,B) and the Upazila Health Complexes of Trishal, Bhaluka and Fulbaria (all located in Mymensingh district), and randomly assigned to one of the treatments. The objective was to assess safety and definitive cure at 6 months after treatment.Results601 patients recruited between July 2010 and September 2013 received either AmBisome monotherapy (n = 158), AmBisome + paromomycin (n = 159), AmBisome + miltefosine (n = 142) or paromomycin + miltefosine (n = 142). At 6 months post- treatment, final cure rates for the intention-to-treat population were 98.1% (95%CI 96.0-100) for AmBisome monotherapy, 99.4% (95%CI 98.2-100) for the AmBisome + paromomycin arm, 94.4% (95%CI 90.6-98.2) for the AmBisome + miltefosine arm, and 97.9% (95%CI 95.5-100) for paromomycin + miltefosine arm. There were 12 serious adverse events in the study in 11 patients that included 3 non-study drug related deaths. There were no relapses or PKDL up to 6 months follow-up. All treatments were well tolerated with no unexpected side effects. Adverse events were most frequent during treatment with miltefosine + paromomycin, three serious adverse events related to the treatment occurred in this arm, all of which resolved.ConclusionNone of the combinations were inferior to AmBisome in both the intention-to-treat and per-protocol populations. All the combinations demonstrated excellent overall efficacy, were well tolerated and safe, and could be deployed under field conditions in Bangladesh. The trial was conducted by the International Centre for Diarrhoeal Disease Research (ICDDR,B) and the Shaheed Suhrawardy Medical College (ShSMC), Dhaka, in collaboration with the trial sites and sponsored by the Drugs for Neglected Diseases initiative (DNDi).Trial registrationClinicalTrials.gov NCT01122771.

Project description:Immuno-modulators in combination with antileishmanial drug miltefosine is a better therapeutic approach for treatment of Visceral Leishmaniasis (VL) as it not only reduces the dose of miltefosine but also shortens the treatment regimen. However, immunological mechanisms behind the perceived benefits of this combination therapy have not been investigated in detail. In the present study, we hypothesized that potential use of drugs that target the host in addition to the parasite might represent an alternative strategy for combination therapy. We investigated immune responses generated in Leishmania donovani infected animals (hamsters and mice) treated with combination of CpG-ODN-2006 and miltefosine at short dose regimen. Infected animals were administered CpG-ODN-2006 (0.4 mg/kg, single dose), as free and liposomal form, either alone or in combination with miltefosine for 5 consecutive days and parasite clearance was evaluated at day 4 and 7 post treatment. Animals that received liposomal CpG-ODN-2006 (lipo-CpG-ODN-2006) and sub-curative miltefosine (5 mg/kg) showed the best inhibition of parasite multiplication (?97%) which was associated with a biased Th1 immune response in. Moreover, compared to all the other treated groups, we observed increased mRNA expression levels of pro-inflammatory cytokines (IFN-?, TNF-? and IL-12) and significantly suppressed levels of Th2 cytokines (IL-10 and TGF-?) on day 4 post treatment in animals that underwent combination therapy with lipo-CpG-ODN-2006 and sub-curative miltefosine. Additionally, same therapy also induced heightened iNOS mRNA levels and NO generation, increased IgG2 antibody level and strong T-cell response in these hamsters compared with all the other treated groups. Collectively, our results suggest that combination of lipo-CpG-ODN-2006 and sub-curative miltefosine generates protective T-cell response in an animal model of visceral leishmaniasis which is characterized by strong Th1 biased immune response thereby underlining our hypothesis that combination therapy, at short dose regimen can be used as a novel way of treating visceral leishmaniasis.

Project description:Miltefosine unresponsive and relapse cases of visceral leishmaniasis (VL) are increasingly being reported. However, there has been no laboratory confirmed reports of miltefosine resistance in VL. Here, we report two laboratory confirmed cases of VL from India.Two patients with VL were referred to us with suspected VL. The first patient was a native of the VL endemic state of Bihar, but residing in Delhi, a VL non-endemic area. He was treated with broad-spectrum antibiotics and antipyretics but was unresponsive to treatment. The second patient was from Jharkhand state in eastern India (adjoining Bihar), another endemic state for VL. He was refractory to anti-leishmanial treatment, which included administration of miltefosine. Following investigation, both patients were serologically positive for VL, and blood buffy coat from both patients grew Leishmania donovani. The isolates derived from both cases were characterized for their drug susceptibility, genetically characterised, and SNPs typed for LdMT and LdROS gene expression. Both patients were successfully treated with amphotericin B.The in vitro drug susceptibility assays carried out on both isolates showed good IC50 values to amphotericin B (0.1?±?0.0004 ?g/ml and 0.07?±?0.0019 ?g/ml). One isolate was refractory to SbIII with an IC50 of > 200 ?M while the second isolate was sensitive to SbIII with an IC50 of 36.70?±?3.2 ?M. However, in both the isolates, IC50 against miltefosine was more than 10-fold higher (> 100 ?M) than the standard strain DD8 (6.8?±?0.1181 ?M). Furthermore, genetic analyses demonstrated single nucleotide polymorphisms (SNPs) (354Tyr?Phe and 1078Phe?Tyr) in the LdMT gene of the parasites.Here, we document two laboratory confirmed cases of miltefosine resistant VL from India. Our finding highlights the urgent need to establish control measures to prevent the spread of these strains. We also propose that LdMT gene mutation analysis could be used as a molecular marker of miltefosine resistance in L. donovani.

Project description:In 2010, WHO recommended a new first-line treatment for visceral leishmaniasis (VL) in Eastern Africa. The new treatment, a combination of intravenous (IV) or intramuscular (IM) sodium stibogluconate (SSG) and IM paromomycin (PM) was an improvement over SSG monotherapy, the previous first-line VL treatment in the region. To monitor the new treatment's safety and effectiveness in routine clinical practice a pharmacovigilance (PV) programme was developed.A prospective PV cohort was developed. Regulatory approval was obtained in Sudan, Kenya, Uganda and Ethiopia. Twelve sentinel sites sponsored by the Ministries of Health, Médecins Sans Frontières (MSF) and Drugs for Neglected Diseases initiative (DNDi) participated. VL patients treated using the new treatment were consented and included in a common registry that collected demographics, baseline clinical characteristics, adverse events, serious adverse events and treatment outcomes. Six-monthly periodic safety update reports (PSUR) were prepared and reviewed by a PV steering committee.Overall 3126 patients were enrolled: 1962 (62.7%) from Sudan, 652 (20.9%) from Kenya, 322 (10.3%) from Ethiopia and 190 (6.1%) from Uganda. Patients were mostly male children (68.1%, median age 11 years) with primary VL (97.8%). SSG-PM initial cure rate was 95.1%; no geographical differences were noted. HIV/VL co-infected patients and patients older than 50 years had initial cure rates of 56 and 81.4%, respectively, while 1063 (34%) patients had at least one adverse event (AE) during treatment and 1.92% (n = 60) had a serious adverse event (SAE) with a mortality of 1.0% (n = 32). There were no serious unexpected adverse drug reactions.This first regional PV programme in VL supports SSG-PM combination as first-line treatment for primary VL in Eastern Africa. SSG-PM was effective and safe except in HIV/VL co-infected or older patients. Active PV surveillance of targeted safety, effectiveness and key VL outcomes such us VL relapse, PKDL and HIV/VL co-infection should continue and PV data integrated to national and WHO PV databases.

Dataset Information

TLR9 and MyD88 are crucial for the maturation and activation of dendritic cells by paromomycin-miltefosine combination therapy in visceral leishmaniasis.

Publications

TLR9 and MyD88 are crucial for the maturation and activation of dendritic cells by paromomycin-miltefosine combination therapy in visceral leishmaniasis.

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