Evaluation of nephroprotective and immunomodulatory activities of antioxidants in combination with cisplatin against murine visceral leishmaniasis

Background

Most available drugs against visceral leishmaniasis are toxic, and growing limitations in available chemotherapeutic strategies due to emerging resistant strains and lack of an effective vaccine against visceral leishmaniasis deepens the crisis. Antineoplastic drugs like miltefosine have in the past been effective against the parasitic infections. An antineoplastic drug, cisplatin (cis-diamminedichloroplatinum II; CDDP), is recognized as a DNA-damaging drug which also induces alteration of cell-cycle in both promastigotes and amastigotes leading to cell death. First in vivo reports from our laboratory revealed the leishmanicidal potential of cisplatin. However, high doses of cisplatin produce impairment of kidney, which can be reduced by the administration of antioxidants.

Methodology/Principal Findings

The present study was designed to evaluate the antileishmanial effect of cisplatin at higher doses (5 mg and 2.5 mg/kg body weight) and its combination with different antioxidants (vitamin C, vitamin E and silibinin) so as to eliminate the parasite completely and reduce the toxicity. In addition, various immunological, hematological and biochemical changes induced by it in uninfected and Leishmania donovani infected BALB/c mice were investigated.

Conclusion/Significance

A significant reduction in parasite load, higher IgG2a and lower IgG1 levels, enhanced DTH responses, and greater concentration of Th1 cytokines (IFN-γ, IL-2) with a concomitant down regulation of IL-10 and IL-4 pointed towards the generation of the protective Th1 type of immune response. A combination of cisplatin with antioxidants resulted in successful reduction of nephrotoxicity by normalizing the enzymatic levels of various liver and kidney function tests. Reduction in parasite load, increase in Th1 type of immune responses, and normalization of various biochemical parameters occurred in animals treated with cisplatin in combination with various antioxidants as compared to those treated with the drug only. The above results are promising as antioxidants reduced the potential toxicity of high doses of cisplatin, making the combination a potential anti-leishmanial therapy, especially in resistant cases.     

Radio-attenuated leishmanial parasites as immunoprophylactic agent against experimental murine visceral leishmaniasis

The present study intends to evaluate the role of radio-attenuated leishmania parasites as immunoprophylactic agents for experimental murine visceral leishmaniasis. BALB/c mice were immunized with gamma (γ)-irradiated Leishmania donovani. A second immunization was given after 15 days of first immunization. After two immunizations, mice were infected with virulent L. donovani promastigotes. Protection against Kala-azar (KA) was estimated from spleen and liver parasitic burden along with the measurement of nitrite and superoxide anion generation by isolation of splenocytes and also by T-lymphocyte helper 1(Th1) and T-lymphocyte helper 2(Th2) cytokines release from the experimental groups. It was observed that BALB/c mice having prior immunization with radio-attenuated parasites showed protection against L. donovani infection through higher expression of Th1 cytokines and suppression of Th2 cytokines along with the generation of protective free radicals. The group of mice without prior priming with radio-attenuated parasites surrendered to the disease. Thus it can be concluded that radio-attenuated L. donovani may be used for.     

Antileishmanial IgG and IgE antibodies recognize predominantly carbohydrate epitopes of glycosylated antigens in visceral leishmaniasis

The specificity of human antileishmanial IgG and IgE antibodies to glycosylated antigens of Leishmania chagasi was evaluated. An ELISA was performed with soluble leishmanial antigen (SLA) and a panel of 95 sera including samples from patients with subclinical infection (SC) and visceral leishmaniasis (VL), subjects cured of visceral leishmaniasis (CVL), and from healthy individuals from endemic areas (HIEA). Antileishmanial IgG were verified for 18 (40%) of 45 SC subjects (mean absorbance of 0.49 +/- 0.17). All nine sera from VL patients had such antibody (0.99 +/- 0.21), while 11 (65%) of 17 CVL individuals were seropositive (0.46 +/- 0.05). Only three (12%) of 24 HIEA controls reacted in IgG-ELISA. Antileishmanial IgE was detected in 26 (58%) of 45 SC patients (0.35 +/- 0.14), and in all VL patients (0.65 +/- 0.29). These antibodies were also detected in 13(76%) of 17 CVL subjects (0.42 +/- 0.14) while all HIEA controls were seronegative. There was no correlation between antileishmanial IgG and IgE antibody absorbances. Mild periodate oxidation at acid pH of SLA carbohydrates drastically diminished its antigenicity in both IgG and IgE-ELISA, affecting mainly the antigens of 125, 102, 94, and 63 kDa as demonstrated by western immunoblotting.     

Comparison of BCG,MPL and cationic liposome adjuvant systems in leishmanial antigen vaccine formulations against murine visceral leishmaniasis

Background  

The development of an effective vaccine against visceral leishmaniasis (VL) caused by Leishmania donovani is an essential aim for controlling the disease. Use of the right adjuvant is of fundamental importance in vaccine formulations for generation of effective cell-mediated immune response. Earlier we reported the protective efficacy of cationic liposome-associated L. donovani promastigote antigens (LAg) against experimental VL. The aim of the present study was to compare the effectiveness of two very promising adjuvants, Bacille Calmette-Guerin (BCG) and Monophosphoryl lipid A (MPL) plus trehalose dicorynomycolate (TDM) with cationic liposomes, in combination with LAg, to confer protection against murine VL.     

Dendritic cell-based immunotherapy combined with antimony-based chemotherapy cures established murine visceral leishmaniasis

Dendritic cells (DCs) have been proposed to play a critical role as adjuvants in vaccination and immunotherapy. In this study we evaluated the combined effect of soluble Leishmania donovani Ag (SLDA)-pulsed syngeneic bone marrow-derived DC-based immunotherapy and antimony-based chemotherapy for the treatment of established murine visceral leishmaniasis. Three weekly injections of SLDA-pulsed DCs into L. donovani-infected mice reduced liver and splenic parasite burden significantly, but could not clear parasite load from these organs completely. Strikingly, the conventional antileishmanial chemotherapy (sodium antimony gluconate) along with injections of SLDA-pulsed DCs resulted in complete clearance of parasites from both these organs. Repetitive in vitro stimulation of splenocytes from uninfected or L. donovani-infected mice with SLDA-pulsed DCs led to the emergence of CD4(+) T cells with characteristics of Th1 cells. Our data indicate that DC-based immunotherapy enhances the in vivo antileishmanial potential of antimony or vice versa.     

Acivicin: a highly active potential chemotherapeutic agent against visceral leishmaniasis

Acivicin, a chlorinated amino acid antibiotic, is found to be remarkably effective in killing both the vector and the host form of the parasitic protozoa, Leishmania donovani, the causative agent for visceral leishmaniasis or Kala-azar. The ED50 (50 nM) for the pathogenic amastigote form in in vitro screening system is significantly lower than the reported values for other drugs under trial. The drug irreversibly inactivates both in vitro and in vivo carbamyl phosphate synthetase II, the first enzyme of the pyrimidine biosynthetic pathway. The irreversible inactivation of this sensitive target enzyme and lack of effective reversal by glutamine makes acivicin a preferred candidate for potential chemotherapy against increasing number of Kala-azar cases that are reported to be unresponsive to pentavalent antimonials.     

A long-lasting topical deltamethrin treatment to protect dogs against visceral leishmaniasis

To develop long‐lasting, topical pour‐on insecticides for dogs to control zoonotic visceral leishmaniasis, two deltamethrin‐based formulations (emulsifiable concentrate [EC] and suspension concentrate [SC]) were tested for their efficacy against the phlebotomine sandfly Lutzomyia longipalpis Lutz & Neiva (Diptera: Psychodidae), vector of Leishmania infantum Nicolle (Kinetoplastida: Trypanosomatidae). The entomological outcomes tested were anti‐feeding effect (proportion of female sandflies unfed), lethal effect (24‐h female sandfly mortality) and these two effects combined, and the insecticide persistence time at 50% (residual activity, RA₅₀) and 80% (RA₈₀) efficacy. On initial application, the proportions of female flies that demonstrated anti‐feeding activity or were killed were similar for both formulations, at 0.93 (95% confidence interval [CI] 0.856–0.977) vs. 0.81 (95% CI 0.763–0.858) (anti‐feeding) and 0.86 (95% CI 0.787–0.920) vs. 0.76 (95% CI 0.698–0.817) (24‐h mortality) for EC and SC, respectively. The RA₅₀ rates for anti‐feeding and mortality caused by the EC formulation were 4.7 months (95% CI 4.18–5.84) and 2.5 months (95% CI 2.25–2.90), respectively, compared with 1.1 months (95% CI 0.96–1.15) and 0.6 months (95% CI 0.50–0.61), respectively, for the SC formulation. The RA₅₀ for the combined anti‐feeding and mortality effects of EC was 5.2 months (95% CI 4.73–5.96), compared with only 0.9 months (95% CI 0.85–1.00) for the SC formulation. The four‐ to six‐fold superior residual activity of the EC formulation was attributed to the addition of a solvent‐soluble resin in the formulation which improved fur adhesion and acted as a reservoir for the slow release of the active ingredient. These results identify the potential of such a low‐cost formulation to reduce the inter‐intervention interval to 5–6 months, similar to that recommended for deltamethrin‐impregnated dog collars or for re‐impregnation of conventional bednets, both of which are currently used to combat Leishmania transmission. Finally, a novel bioassay was developed in which sandflies were exposed to fur from treated dogs, revealing no detectable tolerance (24‐h mortality) in wild‐caught sandflies to the insecticide formulations up to 8 months after the initiation of communitywide application of the insecticides to dogs.     

Immunodominant antigens of Leishmania chagasi associated with protection against human visceral leishmaniasis

Background

Protection and recovery from visceral leishmaniasis (VL) have been associated with cell-mediated immune (CMI) responses, whereas no protective role has been attributed to humoral responses against specific parasitic antigens. In this report, we compared carefully selected groups of individuals with distinct responses to Leishmania chagasi to explore antigen-recognizing IgG present in resistant individuals.

Methodology and Principal Findings

VL patients with negative delayed-type hypersensitivity (DTH) were classified into the susceptible group. Individuals who had recovered from VL and converted to a DTH+ response, as well as asymptomatic infected individuals (DTH+), were categorized into the resistant group. Sera from these groups were used to detect antigens from L. chagasi by conventional and 2D Western blot assays. Despite an overall reduction in the reactivity of several proteins after DTH conversion, a specific group of proteins (approximately 110–130 kDa) consistently reacted with sera from DTH converters. Other antigens that specifically reacted with sera from DTH+ individuals were isolated and tandem mass spectrometry followed by database query with the protein search engine MASCO were used to identify antigens. The serological properties of recombinant version of the selected antigens were tested by ELISA. Sera from asymptomatic infected people (DTH+) reacted more strongly with a mixture of selected recombinant antigens than with total soluble Leishmania antigen (SLA), with less cross-reactivity against Chagas disease patients'' sera.

Significance

Our results are the first evidence of leishmania proteins that are specifically recognized by sera from individuals who are putatively resistant to VL. In addition, these data highlight the possibility of using specific proteins in serological tests for the identification of asymptomatic infected individuals.     

Leishmania donovani Aurora kinase: A promising therapeutic target against visceral leishmaniasis

BackgroundAurora kinases are key mitotic kinases executing multiple aspects of eukaryotic cell-division. The apicomplexan homologs being essential for survival, suggest that the Leishmania homolog, annotated LdAIRK, may be equally important.MethodsBioinformatics, stage-specific immunofluorescence microscopy, immunoblotting, RT-PCR, molecular docking, in-vitro kinase assay, anti-leishmanial activity assays, flow cytometry, fluorescence microscopy.ResultsLdairk expression is seen to vary as the cell-cycle progresses from G1 through S and finally G2M and cytokinesis. Kinetic studies demonstrate their enzymatic activity exhibiting a Km and Vmax of 6.12 μM and 82.9pmoles·min^− 1mg^− 1 respectively against ATP using recombinant Leishmania donovani H3, its physiological substrate. Due to the failure of LdAIRK −/+ knock-out parasites to survive, we adopted a chemical knock-down approach. Based on the conservation of key active site residues, three mammalian Aurora kinase inhibitors were investigated to evaluate their potential as inhibitors of LdAIRK activity. Interestingly, the cell-cycle progressed unhindered, despite treatment with GSK-1070916 or Barasertib, inhibitors with greater potencies for the ATP-binding pocket compared to Hesperadin, which at nanomolar concentrations, severely compromised viability at IC50s 105.9 and 36.4 nM for promastigotes and amastigotes, respectively. Cell-cycle and morphological studies implicated their role in both mitosis and cytokinesis.ConclusionWe identified an Aurora kinase homolog in L. donovani implicated in cell-cycle progression, whose inhibition led to aberrant changes in cell-cycle progression and reduced viability.General significanceHuman homologs being actively pursued drug targets and the observations with LdAIRK in both promastigotes and amastigotes suggest their potential as therapeutic-targets. Importantly, our results encourage the exploration of other proteins identified herein as potential novel drug targets.     

Biogenic nanoporous silicon carrier improves the efficacy of buparvaquone against resistant visceral leishmaniasis

Visceral leishmaniasis is a vector-borne protozoan infection that is fatal if untreated. There is no vaccination against the disease, and the current chemotherapeutic agents are ineffective due to increased resistance and severe side effects. Buparvaquone is a potential drug against the leishmaniases, but it is highly hydrophobic resulting in poor bioavailability and low therapeutic efficacy. Herein, we loaded the drug into silicon nanoparticles produced from barley husk, which is an agricultural residue and widely available. The buparvaquone-loaded nanoparticles were several times more selective to kill the intracellular parasites being non-toxic to macrophages compared to the pure buparvaquone and other conventionally used anti-leishmanial agents. Furthermore, the in vivo results revealed that the intraperitoneally injected buparvaquone-loaded nanoparticles suppressed the parasite burden close to 100%. By contrast, pure buparvaquone suppressed the burden only by 50% with corresponding doses. As the conclusion, the biogenic silicon nanoparticles are promising carriers to significantly improve the therapeutic efficacy and selectivity of buparvaquone against resistant visceral leishmaniasis opening a new avenue for low-cost treatment against this neglected tropical disease threatening especially the poor people in developing nations.     

Prophylactic or therapeutic administration of Agaricus blazei Murill is effective in treatment of murine visceral leishmaniasis

The present study aimed to investigate the in vitro antileishmanial activity of five fractions obtained from Agaricus blazei water extract (AbM), namely, Fab1, Fab2, Fab3, Fab4, and Fab5; and use the selected leishmanicidal fraction to treat BALB/c mice infected with Leishmania chagasi. A curve dose-titration was performed to obtain the concentration to be test in infected animals. In this context, Fab5 fraction and AbM were used in the doses of 20 and 100mg/kg/day, respectively, with the product been administered once a day. The effect induced by a chemo-prophylactic regimen, based on the administration Fab5 fraction and AbM 5days before infection, and maintained for an additional 20days post-infection was compared to a therapeutic regimen, in which the compounds were administered from 0 to 20days of infection. Control animals were either treated with amphotericin B deoxycholate (AmpB) or received distilled water. All groups were followed up for 10weeks post-infection, when parasitological and immunological parameters were analyzed. The Fab5 presented the best results of in vitro leishmanicidal activity. In the in vivo experiments, the use of Fab5 or AbM, as compared to control groups, resulted in significant reduced parasite burdens in the liver, spleen, and draining lymph nodes of the infected animals, as compared to control groups. A Type 1 immune response was observed in the Fab5 or AbM treated animals. No significant toxicity was observed. The chemo-prophylactic regimen proved to be more effective to induce theses responses. In this context, the data presented in this study showed the potential of the purified Fab5 fraction of AbM as a therapeutic alternative to treat visceral leishmaniasis. In addition, it can be postulated that this fraction can be also employed in a chemo-prophylactic regimen associated or not with other therapeutic products.     

Experimental visceral leishmaniasis in golden hamsters

As a result of a long passage of L. donovani isolate on golden hamsters (21 passages were observed), in transplanting the agent from animals with a distinct clinical picture there was formed a highly virulent strain "G" of L. donovani for this species of animals. The weight arrest and then body mass losses were the most early signs of the disease. Parasites were regularly accumulated in spleen and liver and to a less extent in bone marrow. The main manifestations of visceral leishmaniasis in hamsters are cachexia, lienal syndrome, polyglandular deficiency on the background of hypoplasia of lymphoid tissue and defects of the system of monocytic phagocytes. Such symptom-complex can be a result of neuroendocrine deficiency during visceral leishmaniasis. Pathohistological picture of experimental visceral leishmaniasis is similar to that of man, so L. donovani infection in hamsters can serve as a model for studies of different medical and biological aspects of leishmaniasis.     

IgG anti-IgE autoantibodies in visceral leishmaniasis

Procedures for IgG depletion in visceral leishmaniasis (VL) and schistosomiasis sera using Sepharose-protein G beads also deplete IgE. In this study, the presence of IgG anti-IgE autoantibodies in sera from patients with VL (n = 10), and hepatic-intestinal schistosomiasis (n = 10) and from healthy individuals (n = 10) was investigated. A sandwich ELISA using goat IgG anti-human IgE to capture serum IgE and goat anti-human IgG peroxidase conjugate to demonstrate the binding of IgG to the IgE captured was performed. VL sera had higher titers (p < 0.05) of IgG anti-IgE autoantibodies (OD = 2.01 +/- 0.43) than sera from healthy individuals (OD = 1.35 +/- 0.16) or persons infected with Schistosoma mansoni (OD = 1.34 +/- 0.18). The immunoblotting carried out with eluates from Sepharose-protein G beads used to deplete IgG from these sera and goat anti-human IgE peroxidase conjugate, showed a similar pattern of bands, predominating the 75 kDa epsilon-heavy chain and also polypeptides resulting from physiological enzymatic digestion of IgE. A frequent additional band immediately above 75 kDa was observed only in VL sera.     

First visceral leishmaniasis focus in Argentina

An eight-year old boy from Posadas (27 masculine 23'S, 55 masculine 54'W) was diagnosed with visceral leishmaniasis (VL) during 2006. Lutzomyia longipalpis was discovered in the backyard of his house, while the spread of canine visceral leishmaniasis was confirmed in Posadas. This is the southernmost report of a VL transmission focus and the first in Argentina.     

Subclinical form of the American visceral leishmaniasis

The subclinical form of visceral leishmaniasis (VL) shows nonspecific clinical manifestations, with difficulties being frequently met in its clinical characterization and diagnostic confirmation. Thus, the objective of the present study was to define the clinical-laboratory profile of this clinical form. A cohort study was conducted in the state of Maranh?o, Brazil, from January/1998 to December/2000, with monthly follow-up of 784 children aged 0-5 years. Based on the clinical-laboratory parameters reported in the literature, four categories were established, with the children being classified (according to their clinical-evolutive behavior) as asymptomatic (N = 144), as having the subclinical form (N = 33) or the acute form (N = 12) or as subjects "without VL" (N = 595). Multiple discriminant analysis demonstrated that the combination of fever, hepatomegaly, hyperglobulinemia, and increased blood sedimentation rate (BSR) can predict the subclinical form of VL as long as it is not associated with splenomegaly or leukopenia. Subjects with the subclinical form did not show prolonged or intermittent evolution or progression to the acute form of VL. Subclinical cases have a profile differing from the remaining clinical forms of VL, being best characterized by the combination of fever, hepatomegaly, hyperglobulinemia, and increased BSR.     

Present situation of visceral leishmaniasis in China

Visceral leishmaniasis or kala azar is a disease that is distributed world-wide from countries around the Mediterranean Sea to Africa, the Middle East, Asia and South America (Fig. 1). Visceral leishmaniasis was highly prevalent in China but since 1958, after a nationwide campaign, it has been brought under control. Only sporadic cases occur in the hilly and newly reclaimed desert areas in NW China.     

Characterization of two proteins from Leishmania donovani and their use for vaccination against visceral leishmaniasis

Two proteins from Leishmania donovani, dp72 and gp70-2, have been previously utilized to specifically serodiagnose patients with visceral leishmaniasis. The proteins were shown by ELISA and Western blotting with monoclonal and polyclonal antibodies to be present in both stages of the parasite. Antibodies to gp70-2 recognize in promastigotes multiple discrete bands of similar m.w. which are common to several isolates of L. donovani. The total amount of Ag and number of bands observed per isolate is not constant. Lectin blots with Con A show gp70-2 to be a glycoprotein. Dp72 shows pronounced microheterogeneity between isolates of L. donovani. The Brazilian isolates examined appear to possess a lower m.w. form (64,000 or 68,000) of this molecule. No reactions were observed with dp72 and lectins in Western blots; and neither tunicamycin, N-glycanase, endoglycosidase H nor F affected the migration of [35S]-methionine-labeled protein on SDS-PAGE. A mAb against dp72 also cross-reacted in Western blots with a 60-kDa protein in Leishmania major, Leishmania aethiopica, and Leishmania tropica. No reaction was observed between the purified promastigote surface protease (gp63) and either monoclonal or polyclonal antibodies produced to dp72 or gp70-2. The ability of the pure proteins to provide protection against a challenge by L. donovani amastigotes was examined. BALB/c mice were immunized with gp70-2 and/or dp72 by using Corynebacterium parvum as an adjuvant. Mice immunized with gp70-2 were not protected; however, mice receiving dp72 showed a 81.1% reduction in the liver parasitemia compared with the adjuvant controls.     

Balancing immunity and pathology in visceral leishmaniasis

Experimental visceral leishmaniasis (VL) caused by infection with Leishmania donovani results in the development of organ-specific immunity in the two main target tissues of infection, the spleen and the liver. The liver is the site of an acute resolving infection associated with the development of inflammatory granulomas around infected Kupffer cells, and resistance to reinfection. Paradoxically, the spleen is an initial site for the generation of cell-mediated immune responses, but ultimately becomes a site of parasite persistence with associated immunopathological changes. These include splenomegaly and a breakdown in tissue architecture that is postulated to contribute to the immunocompromized status of the host. The progressive development of splenic pathology is largely associated with high levels of TNF and interleukin (IL)-10. Follicular dendritic cell (DC) networks are lost, whereas TNF mediates the destruction of marginal zone macrophages and gp38(+) stromal cells, and IL-10 promotes impaired DC migration into T-cell areas with consequent ineffective T-cell priming. Splenic stromal cell function is also altered, promoting the selective development of IL-10-producing DC with immunoregulatory properties. Ultimately, a fine immunological balance determines responses that effectively promote parasite clearance in the liver and those that promote pathology in the spleen, and future investigation aims to separate these responses to offer further means of parasite control in chronically infected VL patients.     

Bacterial infection in patients with visceral leishmaniasis

In an analysis of 63 hospitalized cases with visceral leishmaniasis, the clinical or post-mortem diagnosis of bacterial infection was performed in 33; 13 (39.3%) patients had respiratory infection, 4 (12.1%) had skin infection, 4 had urinary tract infection, 3 (9.0%) showed ear infection and 2 (6.6%) had infection of the oral cavity. It is worth mentioning that in 7 (21%) cases there was infection in multiple sites. Gram positive and/or Gram negative organisms were isolated from 10 patients. In only two (autopsied) cases, infection with less common organisms was recorded, one with disseminated candidiasis and another with disseminated tuberculosis. Death occurred in 9 of the 63 cases, and in 8 of these, concomitant bacterial infection of importance was documented. Patients who had serum globulins lower than 4 g% had significantly more infection (p less than 0.05) than patients with globulin levels higher than 4 g%; there was no significant difference when the number of leucocytes and neutrophils in patients with associated infection was compared with those in patients without bacterial infection. The present study demonstrates that bacterial infection frequently occurs in patients with visceral leishmaniasis, and indicates an unfavourable prognosis. Even though the mechanism of increased susceptibility to infection in this condition was unclear, the widespread range of infections and of infective agents, suggests a multifactorial process.     

Genetics of murine leishmaniasis.

Leishmaniasis encompasses a number of disease syndromes, caused by several species of the digenetic protozoan Leishmania, and is transmitted by sandflies. The mouse model of the disease has been used to identify genes involved in disease susceptibility--for example, the Slc11a1 gene, important in resistance to Leishmania donovani--and to map loci important in Leishmania major infection. The genetics of the host response to L. major has been shown to be complex and to involve much more than the T helper cell response.