Little is known about CD8 T cells in human visceral leishmaniasis (VL) and it is unclear if these cells have a protective, pathological and/or suppressive function. In experimental VL CD8 T cells have been shown to contribute to parasite control and play an important role in vaccine-generated immunity. To better understand the role of CD8 T cells in human VL, we examined molecules associated with anergy and cytotoxic T lymphocytes (CTL) in peripheral blood mononuclear cells (PBMC) and splenic aspirates (SA), and in CD8 cells derived from these tissues. Gene and surface marker expression suggest that splenic CD8 cell predominantly display an anergic phenotype, whereas CD8-PBMC have features of both anergic cells and CTLs. CD8 cells contribute to the baseline IFNγ levels in whole blood (WB) and SA cultures, but not to the Leishmania induced IFNγ release that is revealed using WB cultures. Blockade of CTLA-4 or PD1 had no effect on IFNγ production or parasite survival in SA cultures. Following cure, CD8 T cells contribute to the Leishmania induced IFNγ production observed in Leishmania stimulated cell cultures. We suggest CD8 T cells are driven to anergy/exhaustion in human VL, which affect their ability to contribute to protective immune responses.
Visceral leishmaniasis; CD8 T cell; IL-10; PD1; CTLA-4; spleen; PBMC
Background. Long regimens for the treatment of post-kala-azar dermal leishmaniasis (PKDL) result in noncompliance. A safe, effective, and acceptable regimen for the treatment of PKDL is still to be developed. Miltefosine has been found to be effective in the treatment of Visceral Leishmaniasis (VL). Hence, its efficacy was tested in patients of PKDL. Methods. In this exploratory study, 33 patients with PKDL aged 10 years and above were administered miltefosine (50 mg for those weighing <25 kg or 100 mg in divided doses for those ≥25 kg and 2.5 mg per kg for children) for 12 weeks and followed up for one year to find out the efficacy. Results. Out of 33 patients, 3 patients withdrew consent. Treatment was stopped due to adverse effect in 1 patient. 28 (96.6%) got cured with complete disappearance of lesion while 1 patient (3.4%) failed treatment by protocol analysis. Conclusion. Miltefosine was found to be effective and safe in the treatment of PKDL.
Visceral leishmaniasis (VL), also called Kala Azar (KA) or black fever in India, claims around 20,000 lives every year. Chemotherapy remains one of the most important tools in the control of VL. Current chemotherapy for Kala Azar in India relies on a rather limited arsenal of drugs including sodium antimony gluconate and amphotericin B in addition to the very expensive drug miltefosine. Pentavalent antimonials have been used for more than half a century in the therapy of leishmaniasis as it is relatively safe and inexpensive, however, the spread of resistance to this drug is forcing clinicians in India to abandon this treatment. Consequently, improvement of antimonial chemotherapy has become a major challenging area of study by leishmaniacs worldwide. The alarming emergence of resistance to the commonly used antleishmanial drug, sodium antimony gluconate, in India, has led us to elucidate the resistance mechanism(s) in clinical isolates. Studies on laboratory mutants have shown that resistance to antimonials is highly dependent on thiol levels. The parasite evades cytotoxic effects of antimonial therapy by enhanced efflux of drug upon conjugation with thiols, through overexpressed membrane proteins belonging to the superfamily of ABC transporters.
We have carried out functional studies to determine the activity of the efflux pumps in antimonial resistant clinical isolates collected from disease endemic areas in India and also carried out molecular characterization of thiol levels in these parasites.
Overexpression of the gene coding for γ glutamylcysteine synthetase was observed in these resistant clinical isolates thereby establishing that thiols represent the key determinants of antimonial resistance. The SbIII/thiol conjugates can be sequestered by ABC transporter multidrug resistance protein A (MRPA) into intracellular organelles or can be directly pumped out by an uncharacterized transporter.
Our studies investigating antimonial resistance in different L. donovani clinical isolates suggest that over functioning of MRP plays a role in generation of antimony resistance phenotype in some L. donovani clinical isolates.
Leishmania donovani; Drug resistance; Drug efflux; Thiols; Clinical isolates
Studies employing serological, DTH or conventional PCR techniques suggest a vast proportion of Leishmania infected individuals living in regions endemic for Visceral Leishmaniasis (VL) remain asymptomatic. This study was designed to assess whether quantitative PCR (qPCR) can be used for detection of asymptomatic or early Leishmania donovani infection and as a predictor of progression to symptomatic disease.
The study included 1469 healthy individuals living in endemic region (EHC) including both serology-positive and -negative subjects. TaqMan based qPCR assay was done on peripheral blood of each subject using kDNA specific primers and probes.
A large proportion of EHC 511/1469 (34.78%) showed qPCR positivity and 56 (3.81% of 1469 subjects) had more than 1 calculated parasite genome/ml of blood. However, the number of individuals with parasite load above 5 genomes/ml was only 20 (1.36% of 1469). There was poor agreement between serological testing and qPCR (k = 0.1303), and 42.89% and 31.83% EHC were qPCR positive in seropositive and seronegative groups, respectively. Ten subjects had developed to symptomatic VL after 12 month of their follow up examination, of which eight were initially positive according to qPCR and among these, five had high parasite load.
Thus, qPCR can help us to detect significant early parasitaemia, thereby assisting us in recognition of potential progressors to clinical disease. This test could facilitate early intervention, decreased morbidity and mortality, and possibly interruption of disease transmission.
Anthroponotic VL caused by Leishmania donovani in the Indian subcontinent accounts for 70% of the world burden of VL. Among the estimated 100,000 cases of VL acquired annually in India, 90% occur in the state of Bihar. Leishmania infection can result in either symptomatic or asymptomatic infection. L. donovani infection can also manifest as post-kala azar dermal leishmaniasis, a chronic cutaneous form thought to provide the reservoir for anthroponotic transmission of VL in regions endemic for this parasite species. We hypothesized that, in areas endemic for L. donovani, asymptomatic infections might also play a crucial role in disease transmission. This study describes use of quantitative PCR (qPCR) to determine the infection status in individuals living in an endemic region of India. We hypothesized that parasite load estimation by qPCR of peripheral blood cells among healthy individuals living in the endemic region might reveal the true frequency of infections through direct evidence of parasitemia. We reasoned this test would detect both asymptomatic non-progressors as well as asymptomatic individuals who will progress to fully symptomatic VL. Serologic testing by ELISA or DAT showed poor agreement with molecular detection of parasite DNA by qPCR, suggesting the tests differentiate between infection and immune response. Amongst ten healthy individuals who progressed to VL, only six were serologically positive whereas eight were initially qPCR positive, among whom five had high parasite loads in their blood. Thus, deployment of qPCR technique to estimate the presence and level of parasitemia in healthy individuals from Leishmania endemic regions may contribute to early case detection, thereby reducing morbidity and mortality. Consistent with the goals of the VL control and elimination program, this early intervention approach could help interrupt disease transmission.
Previously, through a proteomic analysis, proliferating cell nuclear antigen (PCNA) was found to be overexpressed in the sodium antimony gluconate (SAG)-resistant clinical isolate compared to that in the SAG-sensitive clinical isolate of Leishmania donovani. The present study was designed to explore the potential role of the PCNA protein in SAG resistance in L. donovani. For this purpose, the protein was cloned, overexpressed, purified, and modeled. Western blot (WB) and real-time PCR (RT-PCR) analyses confirmed that PCNA was overexpressed by ≥3-fold in the log phase, stationary phase, and peanut agglutinin isolated procyclic and metacyclic stages of the promastigote form and by ∼5-fold in the amastigote form of the SAG-resistant isolate compared to that in the SAG-sensitive isolate. L. donovani PCNA (LdPCNA) was overexpressed as a green fluorescent protein (GFP) fusion protein in a SAG-sensitive clinical isolate of L. donovani, and modulation of the sensitivities of the transfectants to pentavalent antimonial (SbV) and trivalent antimonial (SbIII) drugs was assessed in vitro against promastigotes and intracellular (J774A.1 cell line) amastigotes, respectively. Overexpression of LdPCNA in the SAG-sensitive isolate resulted in an increase in the 50% inhibitory concentrations (IC50) of SbV (from 41.2 ± 0.6 μg/ml to 66.5 ± 3.9 μg/ml) and SbIII (from 24.0 ± 0.3 μg/ml to 43.4 ± 1.8 μg/ml). Moreover, PCNA-overexpressing promastigote transfectants exhibited less DNA fragmentation compared to that of wild-type SAG-sensitive parasites upon SbIII treatment. In addition, SAG-induced nitric oxide (NO) production was found to be significantly inhibited in the macrophages infected with the transfectants compared with that in wild-type SAG-sensitive parasites. Consequently, we infer that LdPCNA has a significant role in SAG resistance in L. donovani clinical isolates, which warrants detailed investigations regarding its mechanism.
Leishmania donovani is the causative agent of the potentially fatal disease visceral leishmaniasis (VL). Chemotherapeutic options available to treat VL are limited and often face parasite resistance, inconsistent efficacy, and toxic side effects. Paromomycin (PMM) was recently introduced to treat VL as a monotherapy and in combination therapy. It is vital to understand the mechanisms of PMM resistance to safeguard the drug. In the present study, we utilized experimentally generated PMM-resistant L. donovani to elucidate the mechanisms of resistance and parasite biology. We found increased membrane fluidity accompanied by decreased intracellular drug accumulation in the PMM-resistant parasites. There were marked increases in gene expression of ATP-binding cassette (ABC) transporters (MDR1 and MRPA) and protein phosphatase 2A that evince increased drug efflux. Further, evaluation of parasite tolerance toward host leishmanicidal mechanisms revealed PMM-resistant parasites as being more tolerant to nitrosative stress at the promastigote and amastigote stages. The PMM-resistant parasites also predicted a better survival capacity, as indicated by resistance to complement-mediated lysis and increased stimulation of host interleukin-10 (IL-10) expression. The susceptibilities of PMM-resistant isolates to other antileishmanial agents (sodium antimony gluconate and miltefosine) remained unchanged. The data implicated the roles of altered membrane fluidity, decreased drug accumulation, increased expression of ABC transporters, and greater tolerance of parasites to host defense mechanisms in conferring PMM resistance in Leishmania.
Visceral leishmaniasis (VL), caused by protozoa of the Leishmania donovani complex, is a widespread parasitic disease of great public health importance; without effective chemotherapy symptomatic VL is usually fatal. Distinction of asymptomatic carriage from progressive disease and the prediction of relapse following treatment are hampered by the lack of prognostic biomarkers for use at point of care.
All IgG subclass and IgG isotype antibody levels were determined using unpaired serum samples from Indian and Sudanese patients with differing clinical status of VL, which included pre-treatment active VL, post-treatment cured, post-treatment relapsed, and post kala-azar dermal leishmaniasis (PKDL), as well as seropositive (DAT and/or rK39) endemic healthy controls (EHCs) and seronegative EHCs. L. donovani antigen-specific IgG1 levels were significantly elevated in relapsed versus cured VL patients (p<0.0001). Using paired Indian VL sera, consistent with the known IgG1 half-life, IgG1 levels had not decreased significantly at day 30 after the start of treatment (p = 0.8304), but were dramatically decreased by 6 months compared to day 0 (p = 0.0032) or day 15 (p<0.0001) after start of treatment. Similarly, Sudanese sera taken soon after treatment did not show a significant change in the IgG1 levels (p = 0.3939). Two prototype lateral flow immunochromatographic rapid diagnostic tests (RDTs) were developed to detect IgG1 levels following VL treatment: more than 80% of the relapsed VL patients were IgG1 positive; at least 80% of the cured VL patients were IgG1 negative (p<0.0001).
Six months after treatment of active VL, elevated levels of specific IgG1 were associated with treatment failure and relapse, whereas no IgG1 or low levels were detected in cured VL patients. A lateral flow RDT was successfully developed to detect anti-Leishmania IgG1 as a potential biomarker of post-chemotherapeutic relapse.
Visceral leishmaniasis (VL) is a systemic disease with highest prevalence in South Asia, East Africa, and Brazil. VL is caused by protozoan (unicellular) parasites of the Leishmania donovani complex, transmitted to humans when an infected sandfly takes a bloodmeal. Within the human host, the parasites replicate within cells, particularly of bone marrow and spleen. Without effective treatment, symptomatic VL is usually fatal. As outlined in a recent World Health Organisation report, the development of new diagnostic tools to test for successful cure after chemotherapy is a research priority. In this work we investigated the association of clinical status of VL patients (active pre-treatment, and those deemed cured or relapsed post-treatment) with subclasses of the IgG antibody response made to L. donovani infection. We show that high levels of subclass IgG1 are found in pre-treatment and relapsed patients, but are very much lower in patients deemed to be cured. We further show that the decrease in IgG1 is detectable in patients 6 months after successful treatment, and that this detection method can be adapted to a rapid diagnostic test format requiring minimal technical expertise. Thus we believe that IgG1 levels are potentially a biomarker of post-chemotherapeutic monitoring.
Interleukin-18 (IL-18) is a cytokine that mediates Th1 response by inducing interferon-gamma (IFN-γ) production in T cells and natural killer cells. Genetic polymorphisms in the IL-18 gene have been found to be associated with its expression in cancer, tuberculosis, HBV infection, and various other diseases. Lower plasma level of IL-18 in visceral leishmaniasis (VL) patients might be associated with polymorphisms in the regulating or coding region of the gene. Three single nucleotide polymorphisms (SNPs), rs1946519 (−656 G/T) and rs187238 (−137 G/C) in the promoter region and rs549908 (+105 A/C) in the codon region, were genotyped in 204 parasitological confirmed VL patients and 267 controls with no past history of VL. For each locus, polymerase chain reaction (PCR) followed by restriction digestion was performed. IL-18 expression in peripheral blood mononuclear cells (PBMC) collected from VL patients and controls was measured by quantitative real-time RT-PCR. Distribution of G allele at position −656 (P < 0.0001) and double haplotypes GGC/GGA (P = 0.05) were found to be significantly associated with controls while genotypes TT (P < 0.0001) and single haplotypes TGA (P = 0.0002), with cases. The inheritance of G allele at the position −656 might be considered as a protective allele for VL.
Promastigote miltefosine (MIL) susceptibility was performed on Leishmania donovani isolates from Indian patients with visceral leishmaniasis treated with MIL. Isolates that were obtained before the onset of MIL treatment, after completion of treatment (29th day), or at the time of treatment failure, were screened using in vitro promastigote assay. The MIL susceptibility of the pre-treatment isolates (N = 24, mean IC50 ± SEM = 3.74 ± 0.38 μM) was significantly higher than that of the post-treatment group (N = 26, mean IC50 ± SEM = 6.15 ± 0.52 μM; P = 0.0006) but was similar in the cured patients (N = 22, mean IC50 ± SEM = 5.58 ± 0.56 μM) and those who failed treatment (N = 28, mean IC50 ± SEM = 4.53 ± 0.47 μM). The pre/post-treatment results thus showed a 2-fold difference, whereas isolated from cured versus failed patients showed a similar susceptibility, suggesting that this higher tolerance is not responsible for MIL-treatment failure. Our work highlights the need for careful monitoring of MIL susceptibility for implementation in national VL elimination programs.
Visceral leishmaniasis (VL) is associated with increased circulating levels of multiple pro-inflammatory cytokines and chemokines, including IL-12, IFNγ, and TNFα, and elevated expression of IFNγ mRNA in lesional tissue such as the spleen and bone marrow. However, an immunological feature of VL patients is that their peripheral blood mononuclear cells (PBMCs) typically fail to respond to stimulation with leishmanial antigen. Unexpectedly, it was recently shown that Leishmania specific IFNγ, can readily be detected when a whole blood stimulation assay (WBA) is used. We sought to define the conditions that permit whole blood cells to respond to antigen stimulation, and clarify the biological role of the IFNγ found to be released by cells from VL patients. CD4+ T cells were found to be crucial for and the main source of the IFNγ production in Leishmania stimulated whole blood (WB) cultures. Complement, antibodies and red blood cells present in whole blood do not play a significant role in the IFNγ response. The IFNγ production was reduced by blockade of human leukocyte antigen (HLA)-DR, indicating that the response to leishmanial antigens observed in WB of active VL patients is a classical HLA- T cell receptor (TCR) driven reaction. Most importantly, blockade of IFNγ in ex-vivo splenic aspirate cultures demonstrated that despite the progressive nature of their disease, the endogenous IFNγ produced in patients with active VL serves to limit parasite growth.
Our research aims to understand the immune failure underlying progression of human visceral leishmaniasis (VL). A key immunological feature of VL patients is that their peripheral blood mononuclear cells (PBMCs) do not respond to stimulation with leishmanial antigen. Surprisingly, when employing a whole blood assay we discovered significant levels of IFNγ in response to soluble Leishmania donovani antigen (WBA) in VL patients. We were interested to understand the relevance of the IFNγ to the anti-parasitic response. Animal models and in vitro studies have shown that IFNγ is a key effector cytokine required for control of the infection, however, the role of endogenous IFNγ in control of parasites in VL patients, has not been demonstrated. Our results show that CD4 cells were required for and were the source of Leishmania specific IFNγ in WBA of VL patients. Optimal IFNγ response required interaction with HLA-DR, supporting that VL is not due to an intrinsic Th1 response defect per se. The Leishmania driven IFNγ appears to limit parasite growth in patients with active VL, since blockade of IFNγ ex-vivo in splenic aspirate cultures enhanced parasite survival. This suggests that IFNγ may have been prematurely dismissed as an adjunct therapy in treatment of VL.
Background & objectives:
The National AIDS Control Organization (NACO) of India has been providing free ARV (antiretroviral) drugs since 2004. By 2012, 486,173 patients had received treatment through the antiretroviral therapy (ART) centres. The objective of this observational study was to assess the factors determining survival of patients on ART under routine programme conditions in an ART centre in north India five years after its inception.
Treatment naive HIV positive patients who were enrolled in the ART centre between May 2009 and May 2010 and started on ART as per the Revised NACO guidelines 2009, were included in the study and outcome was assessed after two years of follow up.
A total of 1689 patients were included in the analysis, of whom 272 (16.10%) expired, 205 (12.13%) were lost to follow up (LFU), 526 (31.14%) were transferred out to other facilities and 686 (40.63%) were alive at the end of two years. Majority (92%) of the deaths occurred in the first six months of therapy. Age >30 yr, male gender, poor functional status, haemoglobin level <11 g/dl, body weight <45 kg and CD4 count <100/μl at baseline had significantly higher relative hazard of death. Most LFU also occurred in the first six months and these patients had significantly low CD4 count, weight, haemoglobin level and higher number of patients in Stages III and IV as compared to those who survived.
Interpretation & conclusions:
The study findings revealed poor survival in the first six months of therapy especially in those with severe immunosuppression. This emphasizes the need for early enrolment into the programme. The high LFU occurring early after initiation of therapy suggests the urgent need to build an efficient patient retrieval system in the programme.
Antiretroviral therapy; CD4; HIV/AIDS; survival analysis
In visceral leishmaniasis, the recovery from the disease is always associated with the generation of Th1-type of cellular responses. Based on this, we have previously identified several Th1-stimulatory proteins of Leishmania donovani -triose phosphate isomerase (TPI), protein disulfide isomerase (PDI) and elongation factor-2 (EL-2) etc. including heat shock protein 70 (HSP70) which induced Th1-type of cellular responses in both cured Leishmania patients/hamsters. Since, HSPs, being the logical targets for vaccines aimed at augmenting cellular immunity and can be early targets in the immune response against intracellular pathogens; they could be exploited as vaccine/adjuvant to induce long-term immunity more effectively. Therefore, in this study, we checked whether HSP70 can further enhance the immunogenicity and protective responses of the above said Th1-stimulatory proteins. Since, in most of the studies, immunogenicity of HSP70 of L. donovani was assessed in native condition, herein we generated recombinant HSP70 and tested its potential to stimulate immune responses in lymphocytes of cured Leishmania infected hamsters as well as in the peripheral blood mononuclear cells (PBMCs) of cured patients of VL either individually or in combination with above mentioned recombinant proteins. rLdHSP70 alone elicited strong cellular responses along with remarkable up-regulation of IFN-γ and IL-12 cytokines and extremely lower level of IL-4 and IL-10. Among the various combinations, rLdHSP70 + rLdPDI emerged as superior one augmenting improved cellular responses followed by rLdHSP70 + rLdEL-2. These combinations were further evaluated for its protective potential wherein rLdHSP70 + rLdPDI again conferred utmost protection (∼80%) followed by rLdHSP70 + rLdEL-2 (∼75%) and generated a strong cellular immune response with significant increase in the levels of iNOS transcript as well as IFN-γ and IL-12 cytokines which was further supported by the high level of IgG2 antibody in vaccinated animals. These observations indicated that vaccine(s) based on combination of HSP70 with Th1-stimulatory protein(s) may be a viable proposition against intracellular pathogens.
Visceral leishmaniasis (VL) is a deadly parasitic diseases caused by Leishmania donovani; it is a major health problem in many countries. A lack of proper understanding of the disease biology, poor diagnostic methods and increasing drug resistance are the main reasons for the growing burden of VL infection. Comparative plasma proteomics are a relatively useful technique that can be used to investigate disease-associated alterations that can help in understanding host responses against pathogens, and might be useful in disease management and diagnosis.
In this study, a comparative proteomics and glycoproteomics approach using 2DE and 2D-DIGE was employed between early diagnosed VL patients of all age groups and healthy endemic and non-endemic controls in order to aid the recognition of disease-associated alterations in host plasma. Comparative proteomics was performed by the depletion of seven highly abundant plasma proteins. Comparative glycoproteomics was performed by the depletion of albumin and IgG, followed by purification of plasma glycoproteins using a multi lectin affinity column. From these two approaches, 39 differentially expressed protein spots were identified and sequenced using MALDI-TOF/TOF mass spectrometry. This revealed ten distinct proteins that appeared in multiple spots, suggesting micro-heterogeneity. Among these proteins, alpha-1-antitrypsin, alpha-1-B glycoprotein and amyloid-A1 precursor were up-regulated, whereas vitamin-D binding protein, apolipoprotein-A-I and transthyretin were down-regulated in VL. Alterations in the levels of these proteins in VL-infected plasma were further confirmed by western blot and ELISA.
These proteins may be involved in the survival of parasites, resisting neutrophil elastase, and in their multiplication in macrophages, potentially maintaining endogenous anti-inflammatory and immunosuppressive conditions. Consequently, the results of this study may help in understanding the host response against L.donovani, which could help in the discovery of new drugs and disease management. Finally, these alterations on protein levels might be beneficial in improving early diagnosis considering those as biomarkers in Indian VL.
Electronic supplementary material
The online version of this article (doi:10.1186/s12953-014-0048-z) contains supplementary material, which is available to authorized users.
Visceral leishmaniasis; Plasma glycoproteomics; MARS column; M-LAC column; 2D-DIGE; MALDI-TOF/TOF mass spectrometry
India is home to 60% of the total global visceral leishmaniasis (VL) population. Use of long-term oral (e.g. miltefosine) and parenteral drugs, considered the mainstay for treatment of VL, is now faced with increased resistance, decreased efficacy, low compliance and safety issues. The authors evaluated the efficacy and safety of an alternate treatment option, i.e. single infusion of preformed amphotericin B (AmB) lipid emulsion (ABLE) in comparison with that of liposomal formulation (LAmB).
In this multicentric, open-label study, 500 patients with VL were randomly assigned in a 3∶1 ratio to receive 15 mg/kg single infusion of either ABLE (N = 376) or LAmB (N = 124). Initial cure (Day 30/45), clinical improvement (Day 30) and long term definitive cure (Day 180) were assessed.
A total of 326 (86.7%) patients in the ABLE group and 122 (98.4%) patients in the LAmB group completed the study. Initial cure was achieved by 95.9% of patients in the ABLE group compared to 100% in the LAmB group (p = 0.028; 95% CI: −0.0663, −0.0150). Clinical improvement was comparable between treatments (ABLE: 98.9% vs. LAmB: 98.4%). Definitive cure was achieved in 85.9% with ABLE compared to 98.4% with LAmB. Infusion-related pyrexia (37.2% vs. 32.3%) and chills (18.4% vs. 18.5%) were comparable between ABLE and LAmB, respectively. Treatment-related serious adverse events were fewer in ABLE (0.3%) compared to LAmB (1.6%). Two deaths occurred in the ABLE group, of which one was probably related to the study drug. Nephrotoxicity and hepatotoxicity was not observed in either group.
ABLE 15 mg/kg single infusion had favorable efficacy and was well tolerated. Considering the demographic profile of the population in this region, a single dose treatment offers advantages in terms of compliance, cost and applicability.
Visceral leishmaniasis (VL) is highly prevalent in northeastern India, particularly the state of Bihar and its bordering areas with Bangladesh and Nepal. The current standards of treatment, namely, miltefosine (oral) and pentavalent antimonials (parenteral) have long treatment durations and are faced with increasing resistance, decreased efficacy, low compliance and safety issues. In this regard, lipid formulations of amphotericin B (AmB) have become an attractive treatment option due to their high efficacy, shorter treatment regimens and favorable safety profiles. This Phase III study evaluated the efficacy and safety of preformed AmB lipid emulsion (ABLE) versus liposomal AmB (LAmB) (both 15 mg/kg single dose infusions) in the treatment of VL. ABLE showed favorable efficacy measured in terms of initial cure at Day 30/45, and overall clinical improvement. ABLE was well tolerated and its adverse event profile was consistent with previously documented findings. Based on the favorable efficacy and safety profile of ABLE, and considering the demographic profile of the population in the endemic region, a single dose treatment may offer advantages in terms of compliance, cost and applicability.
Nucleostemin is a GTPase residing in the nucleolus that is considered to be an important cancer stem/progenitor cell marker protein due to its high expression levels in breast cancer stem cells and its role in tumor initiation of human mammary tumor cells. It has been proposed that nucleostemin may represent a valuable therapeutic target for breast cancer; however, to date evidence supporting the cellular mechanism has not been elucidated.
Expression of exogenous HER2, a member of the EGF receptor gene family, in the human MCF-10AT preneoplastic mammary epithelial cell line, formed a new breast cancer cell line, 10AT-Her2, which is highly enriched in cells with stem/progenitor cell-like character. 10AT-Her2 cells display a CD44+/CD24-/low phenotype with high levels of the cancer stem/progenitor cell marker proteins nucleostemin, and active aldehyde dehydrogenase-1 (ALDH-1). The overall expression pattern of HER2 protein and the stem/progenitor cell marker proteins in the 10AT-Her2 cell population is similar to that of the luminal HER2+ SKBR3 human breast cancer cell line, whereas both MCF-7 and MDA-MB-231 cells display reduced levels of nucleostemin and no detectable expression of ALDH-1. Importantly, in contrast to the other well-established human breast cancer cell lines, 10AT-Her2 cells efficiently form tumorspheres in suspension cultures and initiate tumor xenograft formation in athymic mice at low cell numbers. Furthermore, 10AT-Her2 cells are highly sensitive to the anti-proliferative apoptotic effects of indole-3-carbinol (I3C), a natural anti-cancer indole carbinol from cruciferous vegetables of the Brassica genus such as broccoli and cabbage. I3C promotes the interaction of nucleostemin with MDM2 (murine double mutant 2), an inhibitor of the p53 tumor suppressor, and disrupts the MDM2 interaction with p53. I3C also induced nucleostemin to sequester MDM2 in a nucleolus compartment, thereby freeing p53 to mediate its apoptotic activity. Small interfering RNA knockdown of nucleostemin functionally documented that nucleostemin is required for I3C to trigger its cellular anti-proliferative responses, inhibit tumorsphere formation, and disrupt MDM2–p53 protein–protein interactions. Furthermore, expression of an I3C-resistant form of elastase, the only known target protein for I3C, prevented I3C anti-proliferative responses in cells and in tumor xenografts in vivo, as well as disrupting the I3C-stimulated nucleostemin–MDM2 interactions.
Our results provide the first evidence that a natural anti-cancer compound mediates its cellular and in vivo tumor anti-proliferative responses by selectively stimulating cellular interactions of the stem/progenitor cell marker nucleostemin with MDM2, which frees p53 to trigger its apoptotic response. Furthermore, our study provides a new mechanistic template that can potentially be exploited for the development of therapeutic strategies targeted at cancer stem/progenitor cells.
nucleostemin; cancer stem/progenitor cell marker; indole-3-carbinol; elastase signaling; nucleostemin–MDM2 interaction; anti-proliferative response in breast cancer cell; tumor xenograft; tumorsphere
Amphotericin B (AmB) has been the first-line treatment for visceral leishmaniasis (VL), a neglected protozoan disease, especially in regions like Bihar, India, where resistance to antimonials is widespread. However, adverse drug reactions are a major limiting factor. We evaluated a novel formulation of AmB conjugated to amine-modified graphene (f-Gr) for safety and efficacy over conventional AmB. The f-Gr was prepared in a gentle one-step process of noncovalent (amine) functionalization with the help of amino acid L-cysteine. This f-Gr was further conjugated to AmB by peptide bond. The conjugate (f-Gr-AmB) was characterized by Raman spectroscopy, Fourier transform infrared spectroscopy, scanning electron microscopy, and transmission electron microscopy. f-Gr-AmB was found to exhibit lesser cytotoxicity toward J774A.1 cells than AmB, and did not induce any hepatic or renal toxicity in Swiss albino mice. In vitro antileishmanial assay in J774A.1 cells showed significantly enhanced efficacy of f-Gr-AmB over AmB. Furthermore, percentage inhibition of amastigote replication in a hamster model of VL was significantly higher in the f-Gr-AmB treated group (87.8%) compared to the AmB group (70.4%). These results suggest that f-Gr-AmB could be a safe and effective alternative to conventional AmB in the treatment of VL.
antileishmanial; efficacy; cytotoxicity; macrophage; Raman spectroscopy; amastigote
Visceral Leishmaniasis (VL) is a vector-borne infectious disease, caused by the protozoan Leishmania donovani, which is transmitted by phlebotomine sand flies. In an earlier study in Bihar, India, we found an association between incidence of VL and housing conditions. In the current study we investigated the influence of housing structure and conditions in and around the house on the indoor abundance of Phlebotomus argentipes, the vector of VL in this area.
In each of 50 study villages in Muzaffarpur district, we randomly selected 10 houses. Light traps were installed in each house for one night during three annual peaks of sand fly density over two successive years. Sand flies captured were morphologically identified and segregated by species, sex and feeding status. Data on housing conditions and socio-economic status were also collected. We fitted a linear mixed-effects regression model with log-transformed P. argentipes counts as outcome variable and village as random effect.
P. argentipes was found in all but four of the 500 households. There was considerable variability between the years and the seasons. On bivariate analysis, housing structure, dampness of the floor, keeping animals inside, presence of animal dung around the house, and socio-economic status were all significantly associated with sand fly density. Highest sand fly densities were observed in thatched houses. In the multivariate model only the housing structure and socio-economic status remained significant.
Better housing conditions are associated with lower sand fly densities, independent of other socio-economic conditions. However, in this area in Bihar even in the better-built houses sand flies are present.
In India the sand fly, Phlebotomus argentipes, transmitted parasitic disease termed kala-azar is caused by Leishmania donovani (LD) in humans. These immune-evading parasites have increasingly developed resistance to the drug sodium antimony gluconate in endemic regions.
Lack of early diagnosis methods for the disease limits the information available regarding the early interactions of this parasite with either human tissues or cell lineages. We reasoned that peripheral blood mononuclear cells (PBMCs) from healthy human beings could help compare some of their immune signatures once they were exposed for up to 8 days, to either pentavalent antimony sensitive (SbS-LD) or resistant (SbR-LD) Leishmania donovani isolates.
At day 2, PBMC cultures exposed to SbS-LD and SbR-LD stationary phase promastigotes had four and seven fold higher frequency of IL-10 secreting monocyte-macrophage respectively, compared to cultures unexposed to parasites. Contrasting with the CD4+CD25−CD127− type-1 T-regulatory (Tr1) cell population that displayed similar features whatever the culture conditions, there was a pronounced increase in the IL-10 producing CD4+CD25+CD127low/− inducible T-regulatory cells (iTregs) in the PBMC cultures sampled at day 8 post addition of SbR-LD.
Sorted iTregs from different cultures on day 8 were added to anti-CD3/CD28 induced naïve PBMCs to assess their suppressive ability. We observed that iTregs from SbR-LD exposed PBMCs had more pronounced suppressive ability compared to SbS-LD counterpart on a per cell basis and is dependent on both IL-10 and TGF-β, whereas IL-10 being the major factor contributing to the suppressive ability of iTregs sorted from PBMC cultures exposed to SbS–LD. Of note, iTreg population frequency value remained at the basal level after addition of genetically modified SbR-LD lacking unique terminal sugar in surface glycan.
Even with limitations of this artificial in vitro model of L. donovani-human PBMC interactions, the present findings suggest that SbR-LD have higher immunomodulatory capacity which may favour aggressive pathology.
The disease Kala-azar is caused by Leishmania donovani (LD). The disease is characterized by the depression of cellular immune response. In the Indian subcontinent LD parasites are mostly resistant to commonly used antileishmanial drug, like sodium antimony gluconate (SAG). It is known that infection with pentavalent antimony (Sb)-resistant parasites induces aggressive pathology- the cause is still not known. Sb-resistant parasites endowed with unique glycan which may also play an important role in the pathogenesis as following removal of terminal sugar of glycan these parasites behave like sensitive parasites. The diagnosis of the disease is possible after the disease sets in and therefore limited information is available on the host-parasite interaction at the onset of disease. As a surrogate of in vivo scenario we studied the interaction between normal human PBMC with Sb-sensitive and Sb-resistant parasites. The Sb-resistant parasites upon interaction with human peripheral blood mononuclear cells (PBMC) in vitro produced two distinct inhibitory cytokines, IL-10 and TGF-β. Similar experiment with Sb-sensitive LD induced much less amount of above cytokines. Thus aggressive pathology induced by Sb-resistant LD, may be, in part attributed to production of dual inhibitory cytokines where surface glycan of the parasite may play a decisive role.
Critical to the search for new anti-leishmanial drugs is the availability of high-throughput screening (HTS) methods to test chemical compounds against the relevant stage for pathogenesis, the intracellular amastigotes. Recent progress in automated microscopy and genetic recombination has produced powerful tools for drug discovery. Nevertheless, a simple and efficient test for measuring drug activity against Leishmania clinical isolates is lacking. Here we describe a quantitative colorimetric assay in which the activity of a Leishmania native enzyme is used to assess parasite viability. Enzymatic reduction of disulfide trypanothione, monitored by a microtiter plate reader, was used to quantify the growth of Leishmania parasites. An excellent correlation was found between the optical density at 412 nm and the number of parasites inoculated. Pharmacological validation of the assay was performed against the conventional alamarBlue method for promastigotes and standard microscopy for intracellular amastigotes. The activity of a selected-compound panel, including several anti-leishmanial reference drugs, demonstrated high consistency between the newly developed assay and the reference method and corroborated previously published data. Quality assessment with standard measures confirmed the robustness and reproducibility of the assay, which performed in compliance with HTS requirements. This simple and rapid assay provides a reliable, accurate method for screening anti-leishmanial agents, with high throughput. The basic equipment and manipulation required to perform the assay make it easy to implement, simplifying the method for scoring inhibitor assays.
High frequency of relapse in miltefosine-treated visceral leishmaniasis (VL) patients in India and Nepal followed up for twelve months.
To identify epidemiological and clinical risk factors for relapse of VL in patients recently treated with standard dosing of miltefosine in India and Nepal.
Prospective observational study in three Primary Health Centers and one reference center in Muzaffarpur district, Bihar, India; and two zonal hospitals and a university hospital in South-east Nepal; records of all consenting patients diagnosed with VL and treated with miltefosine according to the current treatment guidelines of the Kala azar elimination program between 2009 and 2011.
We compared the clinical records of 78 cases of relapse with those of 775 patients who had no record of subsequent relapse. Relapse was 2 times more common amongst male patients (IRR 2.14, 95% CI 1.27–3.61), and 2 to 3 times more frequent in the age groups below 15 compared to the over 25 year olds (age 10 to 14: IRR 2.53; 95% CI 1.37–4.65 and Age 2 to 9: IRR 3.19; 95% CI 1.77–5.77). History of earlier VL episodes, or specific clinical features at time of diagnosis such as duration of symptoms or spleen size were no predictors of relapse.
Young age and male gender were associated with increased risk of VL relapse after miltefosine, suggesting that the mechanism of relapse is mainly host-related i.e. immunological factors and/or drug exposure (pharmacokinetics). The observed decrease in efficacy of miltefosine may be explained by the inclusion of younger patients compared to the earlier clinical trials, rather than by a decreased susceptibility of the parasite to miltefosine. Our findings highlight the importance of proper clinical trials in children, including pharmacokinetics, to determine the safety, efficacy, drug exposure and therapeutic response of new drugs in this age group.
The present study includes cloning and expression of recombinant Leishmania donovani histone proteins (rLdH2B, rLdH3, rLdH2A and rLdH4), assessment of their immunogenicity in Leishmania infected cured patients/endemic contacts as well as in cured hamsters and finally evaluation of their prophylactic efficacy in hamsters against L. donovani challenge. All recombinant proteins were expressed and purified from the heterologous bacterial host system. Leishmania infected cured patients/endemic contacts as well as cured hamsters exhibited significantly higher proliferative responses to individual recombinant histones and their pooled combination (rLdH2B+rLdH3+rLdH2A+rLdH4) than those of L.donovani infected hosts. The L.donovani soluble antigens (SLD) stimulated PBMCs of cured/exposed and Leishmania patients to produce a mixed Thl/Th2-type cytokine profile, whereas rLdH2B, rLdH3, rLdH2A, rLdH4 and pooled combination (rLdH2-4) stimulated the production of Th1 cytokines IFN-γ, IL-12 and TNF-α but not Th2 cytokines IL-4 or IL-10. The immunogenicity of these histone proteins along with their combination was also checked in cured hamsters where they stimulated higher lymphoproliferation and Nitric oxide production in lymphocytes of cured hamsters than that of infected controls. Moreover, significantly increased IgG2 response, an indicative of cell mediated immunity, was observed in cured hamsters against these individual proteins and their combination as compared to infected hamsters. Further, it was demonstrated that rLdH2B, rLdH3, rLdH2A and rLdH4 and pooled combination were able to provide considerable protection for hamsters against L. donovani challenge. The efficacy was supported by the increased inducible Nitric Oxide Synthase (iNOS) mRNA transcripts and Th1-type cytokines - IFN-γ, IL-12 and TNF-α and down-regulation of IL-4, IL-10 and TGF-β. Hence, it is inferred that pooled rLdH2-4 elicits Thl-type of immune responses exclusively and confer considerable protection against experimental Visceral Leishmaniasis.
Rapid diagnostic tests (RDTs) based on the detection of specific antibodies in serum are commonly used for the diagnosis of visceral leishmaniasis (VL). Several commercial kits are available, and some of them allow the use of whole-blood samples instead of serum. An RDT is much more user-friendly for blood samples than for serum samples. In this study, we examined the sensitivities and specificities of six different commercially available immunochromatographic tests for their accuracy in detecting Leishmania infection in whole blood and serum of parasitologically confirmed VL cases. This study was performed in areas of India and Nepal where VL is endemic. A total of 177 confirmed VL cases, 208 healthy controls from areas of endemicity (EHCs), 26 malaria patients (MP), and 37 tuberculosis (TB) patients were enrolled. The reproducibilities of the blood and serum results and between-reader and between-laboratory results were tested. In India, the sensitivities of all the RDTs ranged between 94.7 and 100.0%, with no significant differences between whole blood and serum. The specificities ranged between 92.4 and 100.0%, except for the specificity of the Onsite Leishmania Ab RevB kit, which was lower (33.6 to 42.0%). No differences in specificities were observed for blood and serum. In Nepal, the sensitivities of all the test kits, for whole-blood as well as serum samples, ranged between 96.3 and 100.0%, and the specificities ranged between 90.1 and 96.1%, again with the exception of that of the Onsite Leishmania Ab RevB test, which was markedly lower (48.7 to 49.3%). The diagnostic accuracies of all the tests, except for one brand, were excellent for the whole-blood and serum samples. We conclude that whole blood is an adequate alternative for serum in RDTs for VL, with sensitivities and specificities comparable to those obtained in serum samples, provided that the test kit is of overall good quality.
Majority of HIV/AIDS patients who are on Highly Active Anti Retroviral Therapy (HAART), are not aware about drug adherence and its importance which is the most important factor for drug adherence.
To study the level of drug adherence in patients accessing antiretroviral therapy (ART) through the National program and factor influencing drug adherence.
Materials and Methods:
In present study, we enrolled 102 newly diagnosed patients, among them in 79 patients, ART was started. To study the drug adherence a pretested, semistructured questionnaire was formed and patients were followed up for 6 months of the study. Pretest and posttest counseling was done to all such patients.
A total of 28 patients missed the dose in 1st follow-up, nine patients missed in 2nd follow-up, eight patients missed in 3rd follow-up. Three patients lost follow-up in 2nd follow-up, three patients further lost follow-up in 3rd follow-up. Running out of pills (40.0%), side effect (15.5%), and family problem (13.3%), poor transport facility for taking drug (8.9%) and forgetfulness (11.1%) are five major causes related to miss dose. In females patients, drug adherence (69%) was initially less than male patients (76%) but latter on female patients (96.3%) had better adherence than males (95.2%).
This study suggest that drug adherence can be increased by proper counseling and close monitoring of the patients which may have a great role in preventing the drug resistance and ART response.
Adherence; HIV/AIDS; antiretroviral therapy adherence; counseling
Rapid diagnostic test using rk39 antigen is widely used for visceral leishmaniasis. However it detects anti-rk39 antibodies in 20-32% of endemic healthy individuals. In search for a better biomarker of infection, we identified a protein of molecular weight 70 kDa (BHUP1), specifically recognized by sera of visceral leishmaniasis (VL) patients.
The protein was cloned as His-tagged fusion protein and purified. We evaluated the sensitivity and specificity of this protein in an enzyme linked immunosorbant assay (ELISA) format in comparison to the rk39 antigen using sera collected from various groups of individuals.
The sensitivity of rBHUP1 was 96.5% compared to 98.8% with rk39. For healthy controls from non endemic and endemic regions, the specificity of rBHUP1 was 100% and 95.6% compared to 100% and 84.9% for rk39, respectively. For other infectious diseases such as malaria, tuberculosis, viral fever, etc., specificity of rBHUP1 was as low as 74.5% when compared to 94% of rk39. At six month and one year follow-up, 74% and 22.5% patients tested positive with rBHUP1, respectively, compared to 97% and 77.4% with rk39 antigen.
Though the high sensitivity and specificity of rBHUP1 antigen for VL and healthy controls would have made it a good diagnostic biomarkers, however, its non-specific reaction with other infectious diseases limit its utility.
ELISA; rBHUP1; Visceral leishmaniasis; rk39
In the absence of effective vector control measures and vaccines against leishmaniasis, effective chemotherapy remains the mainstay of treatment. As the armoury of antileishmanial drugs is limited, strategies should be made to target the emergence of drug resistance. The loss of efficacy of antimonials such as sodium stibogluconate in the Indian subcontinent which has been the mainstay of treatment for more than six decades has raised concern to save the other drugs. In the current review, we highlight various steps which could be implemented to halt the increasing unresponsiveness of drugs such as monitoring of therapy in the form of rational dosing and duration of treatment, understanding the mechanism of action of the drugs and drug resistance, identification of markers of resistance, distribution of drugs free of cost, evolution of effective combination therapy and immunotherapy, and proper management of HIV/VL coinfection and post-kala-azar dermal leishmaniasis (PKDL). Strong support from governmental agencies and local communities in the form of education and orientation programmes for feasibility of implementing these strategies and affordability within the context of their health systems is needed in controlling and preventing leishmaniasis.