Lupeol induces immunity and protective efficacy in a murine model against visceral leishmaniasis

AbstractThe available chemotherapeutics for the cure of visceral leishmaniasis (VL) are linked with many detrimental effects. Moreover, VL is associated with the suppression of protective Th1 immune response of the host and induction of disease exaggerating Th2 immune response. Therefore, there is an urgent requirement of therapeutics which can augment the immune status of the host to cure this disease. In the current investigation, the antileishmanial potential of lupeol was monitored in vitro and in vivo in inbred BALB/c mice against Leishmania donovani. Lupeol showed potent antipromastigote activity via arresting parasites at sub G0/G1 phase in vitro. Lupeol significantly decreased the splenic parasite burden by inducing strong delayed-type hypersensitivity responses in contrary to untreated infected animals. The therapeutic efficacy of lupeol was observed to be similar to the reference drug, AmB. Treatment of infected animals with lupeol depicted enhanced levels of T cells and Th1 cytokines in contrast to only infected controls. Further lupeol treatment upregulated the levels of nuclear factor κ B and nitric oxide synthase genes and elevated the production of reactive oxygen species and nitric oxide. Unlike AmB, lupeol-treated infected animals did not show any toxicity. These findings are promising and indicate that lupeol can serve as a prototype drug for the cure of VL.

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In vitro and in vivo immunomodulatory properties of octyl-β-d-galactofuranoside during Leishmania donovani infection

Parasites & Vectors ◽

10.1186/s13071-019-3858-0 ◽

2019 ◽

Vol 12 (1) ◽

Author(s):

Hélène Guegan ◽

Kevin Ory ◽

Sorya Belaz ◽

Aurélien Jan ◽

Sarah Dion ◽

...

Keyword(s):

Immune Response ◽

Leishmania Donovani ◽

Parasite Burden ◽

Human Monocyte ◽

Fold Increase ◽

Control Group ◽

End Of Treatment ◽

Immunosuppressed Patients

Abstract Background The chemotherapeutic arsenal available to treat visceral leishmaniasis is currently limited, in view of many drawbacks such as high cost, toxicity or emerging resistance. New therapeutic strategies are particularly needed to improve the management and the outcome in immunosuppressed patients. The combination of an immunomodulatory drug to a conventional anti-Leishmania treatment is an emerging concept to reverse the immune bias from Th2 to Th1 response to boost healing and prevent relapses. Methods Here, immunostimulating and leishmanicidal properties of octyl-β-d-galactofuranose (Galf) were assessed in human monocyte-derived macrophages (HM) and in a murine model, after challenge with Leishmania donovani promastigotes. We recorded parasite loads and expression of various cytokines and immune effectors in HM and mouse organs (liver, spleen, bone marrow), following treatment with free (Galf) and liposomal (L-Galf) formulations. Results Both treatments significantly reduced parasite proliferation in HM, as well as liver parasite burden in vivo (Galf, P < 0.05). Consistent with in vitro results, we showed that Galf- and L-Galf-treated mice displayed an enhanced Th1 immune response, particularly in the spleen where pro-inflammatory cytokines TNF-α, IL-1β and IL-12 were significantly overexpressed compared to control group. The hepatic recruitment of myeloid cells was also favored by L-Galf treatment as evidenced by the five-fold increase of myeloperoxidase (MPO) induction, which was associated with a higher number of MPO-positive cells within granulomas. By contrast, the systemic level of various cytokines such as IL-1β, IL-6, IL-17A or IL-27 was drastically reduced at the end of treatment. Conclusions Overall, these results suggest that Galf could be tested as an adjuvant in combination with current anti-parasitic drugs, to restore an efficient immune response against infection in a model of immunosuppressed mice.

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In vitro and in vivo antileishmanial efficacy of a combination therapy of diminazene and artesunate against Leishmania donovani in BALB/c mice

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46652011000300003 ◽

2011 ◽

Vol 53 (3) ◽

pp. 129-132 ◽

Cited By ~ 12

Author(s):

Joshua Muli Mutiso ◽

John Chege Macharia ◽

Mustafa Barasa ◽

Evans Taracha ◽

Alain J. Bourdichon ◽

...

Keyword(s):

Leishmania Donovani ◽

Combined Therapy ◽

Parasite Burden ◽

Mice Model ◽

In Vivo Evaluation ◽

Reference Drug ◽

Drug Treatments ◽

Potential Use

The in vitro and in vivo activity of diminazene (Dim), artesunate (Art) and combination of Dim and Art (Dim-Art) against Leishmania donovani was compared to reference drug; amphotericin B. IC50 of Dim-Art was found to be 2.28 ± 0.24 µg/mL while those of Dim and Art were 9.16 ± 0.3 µg/mL and 4.64 ± 0.48 µg/mL respectively. The IC50 for Amphot B was 0.16 ± 0.32 µg/mL against stationary-phase promastigotes. In vivo evaluation in the L. donovani BALB/c mice model indicated that treatments with the combined drug therapy at doses of 12.5 mg/kg for 28 consecutive days significantly (p < 0.001) reduced parasite burden in the spleen as compared to the single drug treatments given at the same dosages. Although parasite burden was slightly lower (p < 0.05) in the Amphot B group than in the Dim-Art treatment group, the present study demonstrates the positive advantage and the potential use of the combined therapy of Dim-Art over the constituent drugs, Dim or Art when used alone. Further evaluation is recommended to determine the most efficacious combination ratio of the two compounds.

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Use of an Attenuated Leishmanial Parasite as an Immunoprophylactic and Immunotherapeutic Agent against Murine Visceral Leishmaniasis

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.7.2.233-240.2000 ◽

2000 ◽

Vol 7 (2) ◽

pp. 233-240 ◽

Cited By ~ 19

Author(s):

Srirupa Mukhopadhyay ◽

Sandip Bhattacharyya ◽

Ramdhan Majhi ◽

Tripti De ◽

Khudiram Naskar ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Elevated Level ◽

Leishmania Donovani ◽

Peritoneal Macrophages ◽

Delayed Type Hypersensitivity ◽

Self Healing ◽

Immunotherapeutic Agent ◽

Virulent Challenge ◽

Visceral Infection

ABSTRACT The ability of the leishmanial parasite UR6 to act as an immunoprophylactic and immunotherapeutic agent against Leishmania donovani infection in BALB/c mice was investigated. Unlike the virulent L. donovani AG83 (MOHOM/IN/1983/AG83), UR6 given through intracardiac route failed to induce visceral infection, but when it was injected subcutaneously, UR6 induced a short-lived and localized self-healing skin lesion. Priming of peritoneal macrophages with UR6 in vitro induced superoxide (O2 −) generation, whereas similar experiments with virulent AG83 inhibited O2 − generation. It was observed that priming of mice with either live or sonicated UR6 in the absence of any adjuvant provided strong protection against subsequent virulent challenge. Further, UR6-primed infected mice not only displayed a strong antileishmanial delayed-type hypersensitivity (DTH) response but also showed an elevated level of the serum antileishmanial immunoglobulin G2a (IgG2a) isotype, whereas infected mice failed to mount any antileishmanial DTH response and showed an elevated level of IgG1. This indicates that UR6 priming and subsequent L. donovani infection allowed the expansion of Th1 cells. Our studies indicate that UR6 has potential to be used as an immunoprophylactic and immunotherapeutic agent against experimental visceral leishmaniasis.

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IL-10 and TGF-β Induced Arginase Expression Contributes to Deficient Nitric Oxide Response in Human Visceral Leishmaniasis

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2020.614165 ◽

2021 ◽

Vol 10 ◽

Author(s):

Manu Kupani ◽

Smriti Sharma ◽

Rajeev Kumar Pandey ◽

Rajiv Kumar ◽

Shyam Sundar ◽

...

Keyword(s):

Nitric Oxide ◽

Visceral Leishmaniasis ◽

Leishmania Donovani ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

No Production ◽

Peripheral Blood Mononuclear ◽

Before And After ◽

Plasma Nitrite

Nitric oxide (NO) is an anti-microbial effector of the innate immune system which plays major role in non-specific killing of various pathogens including protozoan parasites. However, due to subversion of the host’s immune processes by pathogens, suboptimal production of NO is frequently found in many infection models. Previous studies have shown suppressed NO production during Leishmania donovani infection, the causative agent of visceral leishmaniasis (VL). Availability of L-Arginine, a semi-essential amino acid is required for inducible nitric oxide synthase (iNOS) mediated NO production. However, arginase is another enzyme, which if expressed concomitantly, may strongly compete for L-Arginine, and suppress NO production by iNOS. In the present study, plasma nitrite and arginase levels were measured in VL patients before and after successful drug treatment, endemic and non-endemic healthy donors. We observed significantly lower NO levels in the plasma of VL patients as compared to endemic controls, which improved significantly post-treatment. Significantly elevated arginase activity was also observed in the plasma of VL patients, which may be associated with NO deficiency. VL patients also showed significantly higher levels of IL-10 and TGF-β, which are known to regulate expression of arginase in various immune cells. In vitro studies with human peripheral blood mononuclear cells (PBMCs) further corroborated the role of IL-10 and TGF-β in arginase mediated suppression of NO production.

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Antileishmanial Efficacy and Pharmacokinetics of DB766-Azole Combinations

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01129-17 ◽

2017 ◽

Vol 62 (1) ◽

Cited By ~ 4

Author(s):

April C. Joice ◽

Sihyung Yang ◽

Abdelbasset A. Farahat ◽

Heidi Meeds ◽

Mei Feng ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Leishmania Donovani ◽

Treatment Strategies ◽

Parasite Burden ◽

Pharmacokinetic Analysis ◽

Content Type ◽

New Treatment ◽

Almost All

ABSTRACT Given the limitations of current antileishmanial drugs and the utility of oral combination therapy for other infections, developing an oral combination against visceral leishmaniasis should be a high priority. In vitro combination studies with DB766 and antifungal azoles against intracellular Leishmania donovani showed that posaconazole and ketoconazole, but not fluconazole, enhanced DB766 potency. Pharmacokinetic analysis of DB766-azole combinations in uninfected Swiss Webster mice revealed that DB766 exposure was increased by higher posaconazole and ketoconazole doses, while DB766 decreased ketoconazole exposure. In L. donovani-infected BALB/c mice, DB766-posaconazole combinations given orally for 5 days were more effective than DB766 or posaconazole alone. For example, 81% ± 1% (means ± standard errors) inhibition of liver parasite burden was observed for 37.5 mg/kg of body weight DB766 plus 15 mg/kg posaconazole, while 37.5 mg/kg DB766 and 15 mg/kg posaconazole administered as monotherapy gave 40% ± 5% and 21% ± 3% inhibition, respectively. Combination index (CI) analysis indicated that synergy or moderate synergy was observed in six of nine combined dose groups, while the other three were nearly additive. Liver concentrations of DB766 and posaconazole increased in almost all combination groups compared to monotherapy groups, although many increases were not statistically significant. For DB766-ketoconazole combinations evaluated in this model, two were antagonistic, one displayed synergy, and one was nearly additive. These data indicate that the efficacy of DB766-posaconazole and DB766-ketoconazole combinations in vivo is influenced in part by the pharmacokinetics of the combination, and that the former combination deserves further consideration in developing new treatment strategies against visceral leishmaniasis.

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LmjF.22.0810 from Leishmania major Modulates the Th2-Type Immune Response and Is Involved in Leishmaniasis Outcome

Biomedicines ◽

10.3390/biomedicines8110452 ◽

2020 ◽

Vol 8 (11) ◽

pp. 452

Author(s):

Andrés Vacas ◽

Celia Fernández-Rubio ◽

Esther Larrea ◽

José Peña-Guerrero ◽

Paul A. Nguewa

Keyword(s):

Immune Response ◽

Protein Kinase ◽

Leishmania Major ◽

Parasite Burden ◽

Mrna Levels ◽

Expression Change ◽

Arginase 1 ◽

Th2 Immune Response

A novel serine/threonine protein kinase, LmjF.22.0810, was recently described in Leishmania major. After generating an L. major cell line overexpressing LmjF.22.0810 (named LmJ3OE), the ability of this novel protein to modulate the Th2-type immune response was analyzed. Our results suggest that the protein kinase LmjF.22.0810 might be involved in leishmaniasis outcomes. Indeed, our study outlined the LmJ3OE parasites infectivity in vitro and in vivo. Transgenic parasites displayed lower phagocytosis rates in vitro, and their promastigote forms exhibited lower expression levels of virulence factors compared to their counterparts in control parasites. In addition, LmJ3OE parasites developed significantly smaller footpad swelling in susceptible BALB/c mice. Hematoxylin–eosin staining allowed the observation of a lower inflammatory infiltrate in the footpad from LmJ3OE-infected mice compared to animals inoculated with control parasites. Gene expression of Th2-associated cytokines and effectors revealed a dramatically lower induction in interleukin (IL)-4, IL-10, and arginase 1 (ARG1) mRNA levels at the beginning of the swelling; no expression change was found in Th1-associated cytokines except for IL-12. Accordingly, such results were validated by immunohistochemistry studies, illustrating a weaker expression of ARG1 and a similar induction for inducible NO synthase (iNOS) in footpads from LmJ3OE-infected mice compared to control L. major infected animals. Furthermore, the parasite burden was lower in footpads from LmJ3OE-infected mice. Our analysis indicated that such significant smaller footpad swellings might be due to an impairment of the Th2 immune response that subsequently benefits Th1 prevalence. Altogether, these studies depict LmjF.22.0810 as a potential modulator of host immune responses to Leishmania. Finally, this promising target might be involved in the modulation of infection outcome.

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Effectiveness of the immunomodulatory extract of Kalanchoe pinnata against murine visceral leishmaniasis

Parasitology ◽

10.1017/s0031182009991405 ◽

2009 ◽

Vol 137 (4) ◽

pp. 613-618 ◽

Cited By ~ 21

Author(s):

D. C. O. GOMES ◽

M. F. MUZITANO ◽

S. S. COSTA ◽

B. ROSSI-BERGMANN

Keyword(s):

Visceral Leishmaniasis ◽

Cutaneous Leishmaniasis ◽

Protective Action ◽

Parasite Burden ◽

Serum Levels ◽

Leishmania Chagasi ◽

Reference Drug ◽

Kalanchoe Pinnata ◽

Mouse Model Of Infection

SUMMARYPreviously, we described the protective action of the immunomodulatory extract of Kalanchoe pinnata (Kp) in murine and human cutaneous leishmaniasis. In the present study, we investigated the effectiveness of Kp against visceral leishmaniasis, using the BALB/c mouse model of infection with Leishmania chagasi. Mice receiving oral daily doses of Kp (400 mg/kg) for 30 days displayed significantly reduced hepatic and splenic parasite burden, when compared with untreated animals. Protectiveness was accompanied by a reduction in parasite-specific IgG serum levels, and impaired capacity of spleen cells to produce IL-4, but not IFN-γ and nitric oxide upon antigen recall in vitro. The reference drug Pentostam (72 mg/kg) given by the intra-peritoneal route on alternate days produced an anti-leishmanial effect similar to oral Kp. Our findings show that the oral efficacy of Kp, seen previously in murine cutaneous leishmaniasis, extends also to visceral leishmaniasis caused by L. chagasi, a difficult to treat and lethal disease of man.

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The IL-33/ST2 Axis Is Associated with Human Visceral Leishmaniasis and Suppresses Th1 Responses in the Livers of BALB/c Mice Infected with Leishmania donovani

mBio ◽

10.1128/mbio.00383-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 32

Author(s):

Octavie Rostan ◽

Jean-Pierre Gangneux ◽

Claire Piquet-Pellorce ◽

Christelle Manuel ◽

Andrew N. J. McKenzie ◽

...

Keyword(s):

Immune Response ◽

Visceral Leishmaniasis ◽

Leishmania Donovani ◽

Parasite Burden ◽

World Health ◽

Enzyme Linked Immunosorbent Assay ◽

Cytokine Signaling ◽

Th2 Response ◽

Content Type ◽

Th1 Cytokines

ABSTRACT During visceral leishmaniasis, the control of hepatic parasite burden is mainly due to granuloma assembly in a microenvironment consisting of both Th1 and Th2 components. Using enzyme-linked immunosorbent assay (ELISA) dosages, quantitative PCR (qPCR), immunohistochemistry, and flow cytometry, we studied the role of interleukin-33 (IL-33), a recently described cytokine signaling through the ST2 receptor, during visceral leishmaniasis. We showed that a higher level of IL-33 was detected in the serum of patients with visceral leishmaniasis than in that from healthy donors and demonstrated the presence of IL-33+ cells in a liver biopsy specimen from a patient. Similarly, in BALB/c mice experimentally infected with L. donovani, a higher level of IL-33 was detected in the serum, as well as the presence of IL-33+ cells and ST2+ cells in the mouse liver. In ST2−/− BALB/c mice, better control of the hepatic parasite burden and reduced hepatomegaly were observed. This was associated with strong induction of Th1 cytokines (gamma interferon [IFN-γ] and IL-12) compared to the level in wild-type (WT) mice and better recruitment of myeloid cells associated with strongly induced chemokines (CCL2 and CXCL2) and receptors (CCR2 and CXCR2). Conversely, BALB/c mice treated twice weekly with recombinant IL-33 showed a dramatically reduced induction of Th1 cytokines and delayed inhibition of monocyte and neutrophil recruitment in the liver, which was associated with reduced KC/CXCL1 and CXCR2 expression. Taken together, our results suggest that IL-33 could be a new deleterious regulator of the hepatic immune response against Leishmania donovani, via the repression of the Th1 response and myeloid cell recruitment. IMPORTANCE Visceral leishmaniasis is a life-threatening systemic disease due to the Leishmania protozoa L. infantum and L. donovani and is ranked by the World Health Organization as the second most important protozoan parasitic disease after malaria for its grave morbidity, high mortality, and global distribution. Leishmania parasites subvert the host’s immune response to propagate to target organs, including the spleen, the bone marrow, and the liver. Control of hepatic parasite burdens depends on a delicate and poorly understood Th1/Th2 immune balance. To better understand this complex immune response, new cytokines are interesting targets for research studies. IL-33 is a newly described cytokine usually associated with Th2 response and involved in different diseases, including infectious diseases and hepatitis. Our results suggest that IL-33 could be a new factor of susceptibility and a potential prognostic marker during visceral leishmaniasis.

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Preclinical validation of a second generation leishmanization vaccine against vector transmitted fatal visceral leishmaniasis

10.1101/2020.11.18.388553 ◽

2020 ◽

Author(s):

Subir Karmakar ◽

Nevien Ismail ◽

Fabiano Oliveira ◽

James Oristian ◽

Wen Wei Zhang ◽

...

Keyword(s):

Immune Response ◽

Visceral Leishmaniasis ◽

Leishmania Donovani ◽

Leishmania Major ◽

Protective Efficacy ◽

Sand Fly ◽

Endemic Region ◽

Cytokines And Chemokines ◽

Human Leishmaniasis ◽

Strong Candidate

AbstractVisceral Leishmaniasis (VL) is fatal if untreated. There is no licensed vaccine available against human leishmaniasis. We recently demonstrated protection in mice against L. major infection using a CRISPR genome edited attenuated Leishmania major strain (LmCen−/−). Here, as a pre-clinical step, we evaluated the protective efficacy of LmCen−/− against VL induced by sand fly transmitted Leishmania donovani in hamsters. Intradermal immunization of hamsters with LmCen−/− did not develop any lesion; while still priming a pro-inflammatory immune response. When challenged with L. donovani either by intradermal needle injection or by infected sand flies, LmCen−/−-immunized hamsters were protected, not showing spleen or liver pathology averting VL fatality compared to control animals. Spleen cells from LmCen−/− immunized and infected sand fly challenged hamsters produced significantly higher Th1-associated cytokines and chemokines including IFN-γ and TNF-α, and significantly reduced expression of the anti-inflammatory cytokines IL-10 and IL-21, compared to non-immunized challenged animals. We further developed a GLP-grade LmCen−/− which showed equal protection as laboratory-grade LmCen−/− parasites in hamsters. Importantly, GLP-grade LmCen−/− parasites also induced a proinflammatory immune response in the PBMCs isolated from healthy people living in non-endemic and endemic for VL as well as cured VL people living in endemic region. Together, this study demonstrates that the LmCen−/− parasites are safe and efficacious against VL and it is a strong candidate vaccine to be tested in a human clinical trial.

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A Chimera of Th1 Stimulatory Proteins of Leishmania donovani Offers Moderate Immunotherapeutic Efficacy with a Th1-Inclined Immune Response against Visceral Leishmaniasis

BioMed Research International ◽

10.1155/2021/8845826 ◽

2021 ◽

Vol 2021 ◽

pp. 1-14

Author(s):

Sneha Ratnapriya ◽

Keerti ◽

Narendra Kumar Yadav ◽

Anuradha Dube ◽

Amogh Anant Sahasrabuddhe

Keyword(s):

Immune Response ◽

Visceral Leishmaniasis ◽

Leishmania Donovani ◽

Triosephosphate Isomerase ◽

Chimeric Protein ◽

Delayed Type Hypersensitivity ◽

Effective Vaccine ◽

Host Immune System ◽

Effective Response ◽

Immune System Activation

Immunotherapy, a treatment based on host immune system activation, has been shown to provide a substitute for marginally effective conventional chemotherapy in controlling visceral leishmaniasis (VL), the deadliest form of leishmaniasis. As the majority of endemic inhabitants exhibit either subclinical or asymptomatic infection which often develops into the active disease state, therapeutic intervention seems to be an important avenue for combating infections by stimulating the natural defense system of infected individuals. With this perspective, the present study focuses on two immunodominant Leishmania (L.) donovani antigens (triosephosphate isomerase and enolase) previously proved to be potent prophylactic VL vaccine candidates, for generating a recombinant chimeric antigen. This is based on the premise that in a heterogeneous population, a multivalent antigen vaccine would be required for an effective response against leishmaniasis (a complex parasitic disease). The resulting molecule rLdT-E chimeric protein was evaluated for its immunogenicity and immunotherapeutic efficacy. A Th1 stimulating adjuvant BCG was employed with the protein which showed a remarkable 70% inhibition of splenic parasitic multiplication positively correlated with boosted Th1 dominant immune response against lethal L. donovani challenge in hamsters as evidenced by high IFN-γ and TNF-α and low IL-10. In addition, immunological analysis of antibody subclass presented IgG2-based humoral response besides considerable delayed-type hypersensitivity and lymphocyte proliferative responses in rLdT-E/BCG-treated animals. Our observations indicate the potential of the chimera towards its candidature for an effective vaccine against Leishmania donovani infection.

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