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

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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Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model

Parasites & Vectors ◽

10.1186/s13071-021-04618-4 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Hai-Jun Gao ◽

Xu-Dong Sun ◽

Yan-Ping Luo ◽

Hua-Sheng Pang ◽

Xing-Ming Ma ◽

...

Keyword(s):

Protein Kinase ◽

Mrna Expression ◽

Echinococcus Granulosus ◽

Calcium Content ◽

Mrna Levels ◽

Dependent Protein Kinase ◽

Calcium Calmodulin Dependent ◽

Dependent Protein

Abstract Background Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice. Methods The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction. Results In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 μg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm. Conclusions Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.

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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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Zinc(II)-Dipicolylamine Coordination Complexes as Targeting and Chemotherapeutic Agents for Leishmania major

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00410-16 ◽

2016 ◽

Vol 60 (5) ◽

pp. 2932-2940 ◽

Cited By ~ 14

Author(s):

Douglas R. Rice ◽

Paola Vacchina ◽

Brianna Norris-Mullins ◽

Miguel A. Morales ◽

Bradley D. Smith

Keyword(s):

Cutaneous Leishmaniasis ◽

Mammalian Cells ◽

Leishmania Major ◽

Parasite Burden ◽

Coordination Complexes ◽

Chemotherapeutic Agents ◽

Selective Toxicity ◽

Content Type

ABSTRACTCutaneous leishmaniasis is a neglected tropical disease that causes painful lesions and severe disfigurement. Modern treatment relies on a few chemotherapeutics with serious limitations, and there is a need for more effective alternatives. This study describes the selective targeting of zinc(II)-dipicolylamine (ZnDPA) coordination complexes towardLeishmania major, one of the species responsible for cutaneous leishmaniasis. Fluorescence microscopy ofL. majorpromastigotes treated with a fluorescently labeled ZnDPA probe indicated rapid accumulation of the probe within the axenic promastigote cytosol. The antileishmanial activities of eight ZnDPA complexes were measured using anin vitroassay. All tested complexes exhibited selective toxicity againstL. majoraxenic promastigotes, with 50% effective concentration values in the range of 12.7 to 0.3 μM. Similar toxicity was observed against intracellular amastigotes, but there was almost no effect on the viability of mammalian cells, including mouse peritoneal macrophages.In vivotreatment efficacy studies used fluorescence imaging to noninvasively monitor changes in the red fluorescence produced by an infection of mCherry-L. majorin a mouse model. A ZnDPA treatment regimen reduced the parasite burden nearly as well as the reference care agent, potassium antimony(III) tartrate, and with less necrosis in the local host tissue. The results demonstrate that ZnDPA coordination complexes are a promising new class of antileishmanial agents with potential for clinical translation.

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Obesity impairs resistance to Leishmania major infection in C57BL/6 mice

10.1101/342642 ◽

2018 ◽

Author(s):

Franciele Carolina Silva ◽

Vinicius Dantas Martins ◽

Felipe Caixeta ◽

Matheus Batista Carneiro ◽

Graziele Ribeiro Goes ◽

...

Keyword(s):

Immune Response ◽

Lymph Node ◽

Leishmania Major ◽

Parasite Burden ◽

Public Health Problem ◽

Obese Mice ◽

Obese People ◽

Diet Induced Obesity ◽

Draining Lymph Node

AbstractAn association between increased susceptibility to infectious diseases and obesity has been described as a result of impaired immunity in obese individuals. It is not clear whether a similar linkage can be drawn between obesity and parasitic diseases. To evaluate the effect of obesity in the immune response to cutaneous L. major infection, we studied the ability of C57BL/6 mice submitted to a high fat and sugar diet to control leishmaniasis. Mice with diet-induced obesity presented thicker lesions with higher parasite burden and more inflammatory infiltrate in the infected ear when infected with L. major. We observe no difference in IFN-γ or IL-4 production by draining lymph node cells between control and obese mice, but obese mice presented higher production of IgG1 and IL-17. A higher percentage of in vitro-infected peritoneal macrophages was found when these cells were obtained from obese mice when compared to lean mice. In vitro stimulation of macrophages with IL-17 decreased the capacity of cells from control mice to kill the parasite. Moreover, macrophages from obese mice presented higher arginase activity. Together our results indicate that diet-induced obesity impairs resistance to L. major in C57BL/6 mice without affecting the development of Th1 response.Author SummaryThe obesity is a public health problem and it is reaching extraordinary numbers in the world and others diseases are being involved and aggravated as consequence of obesity. What we know is that some diseases are more severe in obese people than in normal people. We did not know how obesity changes the profile of immune response to infectious agents, leading to the more severe diseases. That‘s why we decided to investigate how obese mice lead with Leishmania major infection. Leishmaniasis is a protozoa parasite infection considered a neglected disease. To try our hypothesis we gave a hipercaloric diet to induce obesity in C57BL/6 mice. After that, we injected L. major in the mice ear and followed the lesion for 8 weeks. We observed a ticker lesion and the cells from draining lymph node from obese mice produced more IL-17 than cells from normal mice. We also infected in vitro, macrophages from obese mice and stimulated the cells with IL-17, and we observed that the macrophages from obese mice are more infected by the L. major and it is worst in the presence of IL-17. Our results suggest that diet induced obesity decrease the resistance to infection.

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BRF1 Protein Turnover and mRNA Decay Activity Are Regulated by Protein Kinase B at the Same Phosphorylation Sites

Molecular and Cellular Biology ◽

10.1128/mcb.01099-06 ◽

2006 ◽

Vol 26 (24) ◽

pp. 9497-9507 ◽

Cited By ~ 62

Author(s):

Don Benjamin ◽

Martin Schmidlin ◽

Lu Min ◽

Brigitte Gross ◽

Christoph Moroni

Keyword(s):

Protein Kinase ◽

Mrna Decay ◽

Protein Kinase B ◽

Proteasomal Degradation ◽

Mrna Levels ◽

Cell Compartment ◽

Protein Levels ◽

Dependent Protein

ABSTRACT BRF1 posttranscriptionally regulates mRNA levels by targeting ARE-bearing transcripts to the decay machinery. We previously showed that protein kinase B (PKB) phosphorylates BRF1 at Ser92, resulting in binding to 14-3-3 and impairment of mRNA decay activity. Here we identify an additional regulatory site at Ser203 that cooperates in vivo with Ser92. In vitro kinase labeling and wortmannin sensitivity indicate that Ser203 phosphorylation is also performed by PKB. Mutation of both serines to alanine uncouples BRF1 from PKB regulation, leading to constitutive mRNA decay even in the presence of stabilizing signals. BRF1 protein is labile because of proteasomal degradation (half-life, <3 h) but becomes stabilized upon phosphorylation and is less stable in PKBα−/− cells. Surprisingly, phosphorylation-dependent protein stability is also regulated by Ser92 and Ser203, with parallel phosphorylation required at these sites. Phosphorylation-dependent binding to 14-3-3 is abolished only when both sites are mutated. Cell compartment fractionation experiments support a model in which binding to 14-3-3 sequesters BRF1 through relocalization and prevents it from executing its mRNA decay activity, as well as from proteasomal degradation, thereby maintaining high BRF1 protein levels that are required to reinstate decay upon dissipation of the stabilizing signal.

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Stanniocalcin 1 and 2 are secreted as phosphoproteins from human fibrosarcoma cells

Biochemical Journal ◽

10.1042/bj3500453 ◽

2000 ◽

Vol 350 (2) ◽

pp. 453-461 ◽

Cited By ~ 34

Author(s):

Derek A. JELLINEK ◽

Andy C. CHANG ◽

Martin R. LARSEN ◽

Xin WANG ◽

Phillip J. ROBINSON ◽

...

Keyword(s):

Protein Kinase ◽

Mammalian Cells ◽

Phosphorylation Site ◽

Mrna Levels ◽

Intact Cells ◽

Fibrosarcoma Cells ◽

Ht1080 Cells ◽

Human Fibrosarcoma Cells

Stanniocalcin 1 (STC1) and stanniocalcin 2 (STC2) are two recently identified mammalian peptide hormones. STC1 plays a role in calcium and phosphate homoeostasis, while the role of STC2 is unknown. We examined a human fibrosarcoma cell line, HT1080, that has high steady-state STC1 and STC2 mRNA levels, to determine whether these proteins are secreted. Following incubation of HT1080 cells with 32P, labelled STC1 and STC2 were found to be secreted into the medium. STC1 was phosphorylated in vitro by protein kinase C (PKC). In vitro and in vivo phosphorylation both occurred exclusively on serine and the phosphopeptide maps were similar, suggesting that PKC might be the in vivo kinase. STC2 was phosphorylated in vitro by casein kinase II (CK2), in vitro and in vivo phosphorylation were exclusively on serine and the phosphopeptide maps were indistinguishable. Phosphorylation of STC2 in intact cells resulted from the action of an ecto-protein kinase, since exogenous STC2 was phosphorylated by HT1080 cells and no phosphorylated STC2 was detectable inside the cells. The ectokinase activity was abolished by heparin and GTP could substitute for ATP as the phosphate donor, indicative of an ecto-CK2-like activity. The in vitro CK2 phosphorylation site was shown by matrix-assisted laser-desorption ionization–time-of-flight MS to be a single serine located between Ser-285 and Ser-298 in the C-terminal region of STC2. This is the first report of the secretion of STC1 or STC2 from mammalian cells. We conclude that these human fibrosarcoma cells express both STC1 and STC2 as secreted phosphoproteins in vivo, with STC2 being phosphorylated by an ecto-CK2-like enzyme.

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In Vitro and In Vivo Anti-Parasitic Activity of Artemisinin Combined With Glucantime and Shark Cartilage Extract on Iranian Strain of Leishmania major (MRHO/IR/75/ER)

Jundishapur Journal of Microbiology ◽

10.5812/jjm.113313 ◽

2021 ◽

Vol 14 (3) ◽

Author(s):

Fatemeh Ghaffarifar ◽

Soheila Molaei ◽

Zuhair Mohammad Hassan ◽

Mohammad Saaid Dayer ◽

Abdolhossein Dalimi ◽

...

Keyword(s):

Leishmania Major ◽

Human Subjects ◽

Parasite Burden ◽

Lesion Size ◽

In Vivo Experiments ◽

Parasitic Activity ◽

Shark Cartilage ◽

Immune System Modulation

Background: The adverse effects and increased resistance of drugs necessities the discovery of novel combination therapy. Objectives: This study aimed to examine the effects of Artemisinin plus glucantime or shark cartilage extract on the Iranian strain of Leishmania major (MRHO/IR/75/ER) in vitro and in vivo. Methods: In in vitro experiments, the effects of drugs and their combination in different concentrations (3.12 - 400 µg/mL) on the promastigotes, amastigotes, and un-infected macrophage cells were evaluated. In in vivo experiments, infected BALB/c mice were used as a cutaneous leishmaniasis model to evaluate the effects of the drugs and their combinations with different routes of administrations (namely Artemisinin: oral, ointment, and intraperitoneal; glucantime: intraperitoneal, intramuscular, intralesional, and subcutaneous; shark cartilage extract: oral) on parasite burden, lesion size, and immune system modulation. Results: The results revealed that Artemisinin and glucantime in combination with shark cartilage extract had greater effects on promastigotes than either Artemisinin or glucantime (P < 0.05), and that the combinations also had high cytotoxic effects on promastigotes and uninfected macrophages (P = 0.001). These combinations had more inhibitory effects on amastigotes and infected macrophages than promastigotes. The lesion sizes and parasite burden in the spleen decreased against the combinations of the drugs in different administrations. It was also noticed that the best combination administration route of Artemisinin and glucantime, as strong inducers of INF-γ and Th1 immune response, were ointment and IM, respectively (P < 0.05). Conclusions: The findings indicate that Artemisinin- glucantime or Artemisinin- Shark cartilage combinations are effective inhibitors of L. major. However, further clinical trials are recommended to evaluate the effects of these combinations in human subjects.

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Evaluation of Skin Permeation and Retention of Topical Dapsone in Murine Cutaneous Leishmaniasis Lesions

Pharmaceutics ◽

10.3390/pharmaceutics11110607 ◽

2019 ◽

Vol 11 (11) ◽

pp. 607

Author(s):

Moreno ◽

Calvo ◽

Schwartz ◽

Navarro-Blasco ◽

González-Peñas ◽

...

Keyword(s):

Cutaneous Leishmaniasis ◽

Leishmania Major ◽

Ex Vivo ◽

Skin Permeation ◽

Topical Therapy ◽

Parasite Burden ◽

Skin Penetration ◽

High Plasma

The oral administration of dapsone (DAP) for the treatment of cutaneous leishmaniasis (CL) is effective, although serious hematological side effects limit its use. In this study, we evaluated this drug for the topical treatment of CL. As efficacy depends on potency and skin penetration, we first determined its antileishmanial activity (IC50 = 100 μM) and selectivity index in vitro against Leishmania major-infected macrophages. In order to evaluate the skin penetration ex vivo, we compared an O/W cream containing DAP that had been micronized with a pluronic lecithin emulgel, in which the drug was solubilized with diethylene glycol monoethyl ether. For both formulations we obtained similar low flux values that increased when the stratum corneum and the epidermis were removed. In vivo efficacy studies performed on L. major-infected BALB/c mice revealed that treatment not only failed to cure the lesions but made their evolution and appearance worse. High plasma drug levels were detected and were concomitant with anemia and iron accumulation in the spleen. This side effect was correlated with a reduction of parasite burden in this organ. Our results evidenced that DAP in these formulations does not have an adequate safety index for use in the topical therapy of CL.

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Leishmania major Phosphoglycans Influence the Host Early Immune Response by Modulating Dendritic Cell Functions

Infection and Immunity ◽

10.1128/iai.01447-08 ◽

2009 ◽

Vol 77 (8) ◽

pp. 3272-3283 ◽

Cited By ~ 26

Author(s):

Dong Liu ◽

Chahnaz Kebaier ◽

Nazzy Pakpour ◽

Althea A. Capul ◽

Stephen M. Beverley ◽

...

Keyword(s):

Immune Response ◽

Antigen Presentation ◽

Leishmania Major ◽

Mhc Ii ◽

Cell Functions ◽

Early Immune Response ◽

Ifn Γ ◽

Th1 And Th2 Responses

ABSTRACT The precise role of Leishmania glycoconjugate molecules including phosphoglycans (PGs) and lipophosphoglycan (LPG) on host cellular responses is still poorly defined. Here, we investigated the interaction of Leishmania major LPG2 null mutant (lpg2 − ), which lacks both PGs and LPG, with dendritic cells (DCs) and the subsequent early immune response in infected mice. Surprisingly, the absence of phosphoglycans did not influence expression pattern of major histocompatibility complex class II (MHC II), CD40, CD80, and CD86 on DCs in vitro and in vivo. However, lpg2 − L. major induced significantly higher production of interleukin-12p40 (IL-12p40) by infected bone marrow-derived DCs (BMDCs) than wild-type (WT) parasites in vitro. Furthermore, the production of IL-12p40 by draining lymph node cells from lpg2 − mutant-infected mice was higher than those from WT L. major-infected mice. In model antigen presentation experiments, DCs from lpg2 − mutant-infected mice induced more gamma interferon (IFN-γ) and IL-2 production by Leishmania-specific T cells than those from WT-infected mice. Lymphocytes isolated from mice infected for 3 days with lpg2 − parasites produce similar levels of IFN-γ, but significantly less IL-4 and IL-10 than WT controls. Decreased IL-4 production was also seen in another general PG-deficient mutant lacking the Golgi UDP-galactose transporters (lpg5A − lpg5B − ), but not with the lpg1 − mutant lacking only LPG, thereby implicating PGs generally in the reduction of IL-4 production. Thus, Leishmania PGs influence host early immune response by modulating DC functions in a way that inhibits antigen presentation and promotes early IL-4 response, and their absence may impact the balance between Th1 and Th2 responses.

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In vivo regulation of Na/Ca exchanger expression by adrenergic effectors

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.280.3.h1376 ◽

2001 ◽

Vol 280 (3) ◽

pp. H1376-H1382 ◽

Cited By ~ 22

Author(s):

Kish L. Golden ◽

Jun Ren ◽

Jessica O'Connor ◽

Andrea Dean ◽

Stephen E. DiCarlo ◽

...

Keyword(s):

Protein Kinase ◽

Rat Heart ◽

Day Treatment ◽

Functional Expression ◽

Fluorescence Decay ◽

Mrna Levels ◽

Adult Rat ◽

Intracellular Ca

The Na/Ca exchanger encoded by the NCX1gene plays an important role in calcium homeostasis in cardiac muscle. We previously identified three in vitro signaling pathways that are of major importance in the regulation of Na/Ca exchanger gene expression in neonatal cardiac myocytes, the protein kinase A (PKA) and protein kinase C (PKC) pathways, and intracellular Ca2+. To determine whether these pathways are important in vivo, we stimulated the PKA and PKC pathways and examined functional expression of the Na/Ca exchanger in adult rat heart. After a 3- and 7-day treatment, norepinephrine (200 μg · kg−1 · h−1), isoproterenol (150 μg · kg−1 · h−1), and phenylephrine (200 μg · kg−1 · h−1) each stimulated a significant increase in NCX1 mRNA levels (35–85%, P < 0.05). Norepinephrine also stimulated a 35% increase in protein abundance ( P < 0.05), a 20% decrease in relaxation duration ( P < 0.05), and a 25% reduction in the fluorescence decay constant ( P < 0.05) after a 7-day treatment. We conclude that a 7-day treatment of α- and β-adrenergic agonists increases the expression of functional Na/Ca exchangers in adult rat heart.

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