scholarly journals Evaluation of modulators of cAMP-response in terms of their impact on cell cycle and mitochondrial activity of Leishmania donovani

2020 ◽  
Author(s):  
Amrita Saha ◽  
Anindita Bhattacharjee ◽  
Amit Vij ◽  
Pijush K. Das ◽  
Arijit Bhattacharya ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Leishmania Donovani ◽  
Parasite Burden ◽  
Careful Examination ◽  
Mammalian Host ◽  
Infection Model ◽  
Mitochondrial Activity ◽  
Macrophage Infection ◽  
Pde Inhibitors

AbstractWith the identification of novel cAMP binding effecter molecules in Trypanosoma, role of cAMP in kinetopalstida parasites gained an intriguing break through. Despite earlier demonstrations of role of cAMP in survival of Leishmania during macrophage infection, there is essential need to specifically clarify involvement of cAMP in various cellular processes in the parasite. In this context, we sought to gain a comprehensive understanding of the effect of cAMPanalogs and cAMP-phosphodiesterase (PDE) inhibitors on proliferation of log phase parasites. Administration of both hydrolysable (8-pCPT-cAMP) and non-hydrolysable analogs (Sp-8-pCPT-cAMPS) of cAMP resulted in significant decrease of Leishmania proliferation. Amongst the various PDE inhibitors, etazolate was found to be potently anti-proliferative. BrdU cell proliferation and K/N/F-enumeration microscopic study revealed that both cAMP analogues and selective PDE inhibitors resulted in significant cell cycle arrest at G1 phase with reduced S-phase population. Furthermore, careful examination of the flagellar motility patterns revealed significantly reduced coordinated forward flagellar movement of the promastigotes with a concomitant decrease in cellular ATP levels. Alongside, 8-pCPT-cAMP and PDE inhibitors etazolate and trequinsin showed marked reduction in mitochondrial membrane potential. Treatment of etazolate at subcytotoxic concentration to infected macrophages significantly reduced parasite burden and administration of etazolate to Leishmania-infected BALB/c mice showed reduced liver and spleen parasite burden. Collectively, these results imply involvement of cAMP in various crucial processes paving the avenue for developing potent anti-leishmanial agent.Author SummaryLeishmania donovani is the causative agent of fatal Visceral Leishmaniasis. The current available medications are toxic, expensive and require long term daily administrations. With an aim to develop improved therapeutic, components of cAMP homeostasis, particularly cAMP-phosphodiesteares, has been targeted for Leishmania and other kinetoplastid pathogens. cAMP plays diverse roles in functional processes involved in cell division, transition into different stages of the life cycle of Leishmania and motility. In this study, the authors found administration of both hydrolysable and non-hydrolysable analogs of cAMP and certain PDE inhibitors resulted in remarkable decrease proliferation with considerable cytopathic impact on promastigotes. The mammalian phosphodiestearse inhibitor etazolate caused significant reduction in parasite load in L. donovani infected macrophages and demonstrated considerable reduction of liver and spleen parasite burden in in vivo mouse infection model. The study suggested that etazolate, with its slightest impact on mammalian host, can be repurposed for developing effective anti-leishmanials.

scholarly journals The role of Rad51 in safeguarding mitochondrial activity during the meiotic cell cycle in mammalian oocytes

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Kyeoung-Hwa Kim ◽  
Ji-Hoon Park ◽  
Eun-Young Kim ◽  
Jung-Jae Ko ◽  
Kyung-Soon Park ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mitochondrial Activity ◽  
Meiotic Cell

scholarly journals Evaluation of Modulators of cAMP-Response in Terms of Their Impact on Cell Cycle and Mitochondrial Activity of Leishmania donovani

2020 ◽  
Vol 11 ◽  
Author(s):  
Amrita Saha ◽  
Anindita Bhattacharjee ◽  
Amit Vij ◽  
Pijush K. Das ◽  
Arijit Bhattacharya ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Leishmania Donovani ◽  
Mitochondrial Activity

scholarly journals PREPARATION AND CHARACTERIZATION OF ANDROGRAPHOLIDE NANOPARTICLES FOR VISCERAL LEISHMANIASIS CHEMOTHERAPY: IN VITRO AND IN VIVO EVALUATIONS

2016 ◽  
Vol 8 (12) ◽  
pp. 102
Author(s):  
Pallab Ghosh ◽  
Subhasish Mondal ◽  
Tanmoy Bera
Keyword(s):  
Visceral Leishmaniasis ◽  
Leishmania Donovani ◽  
Parasite Burden ◽  
Plga Nanoparticles ◽  
Antileishmanial Activity ◽  
Infection Model ◽  
Vitamin E Tpgs ◽  
Polyethylene Glycol 1000

<p><strong>Objective: </strong>To overcome low physiological solubility, poor bioavailability, the short plasma half-life of andrographolide (AG), a delivery system based on poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) were developed to increase the efficiency of AG against visceral leishmaniasis (VL).<strong> </strong></p><p><strong>Methods: </strong>Andrographolide-PLGA nanoparticles (AGnp) were prepared with Pgp efflux inhibitor vitamin E TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate) by emulsion solvent evaporation method and characterized. Antileishmanial activity was evaluated using<em> in vitro</em> and<em> in vivo</em> VL infection model. <strong></strong></p><p><strong>Results: </strong>The particle size of AGnp was found to be171.4±11.5 nm with an encapsulation efficiency of 81%. The AGnp reduced AG cellular toxicity, retained it's<em> in vitro</em> antileishmanial activity and lead to a reduction (99.9%) of parasite burden in the <em>Leishmania donovani</em> infected spleen and liver. AGnp was more active in infected mice liver at low dose than in spleen. Therapeutic indexes (TI) were 6.9-fold greater in AG and 68-fold in AGnp compared to amphotericin B (AmB) when evaluated in <em>L. donovani</em> infected spleen.<strong> </strong></p><p><strong>Conclusion: </strong>Incorporation of AG in PLGA nanoparticles, provided controlled and improved <em>in vivo</em> performance against VL</p>

scholarly journals Hematological consequences of malaria infection in mice previously treated for visceral leishmaniasis

2021 ◽  
Vol 6 ◽  
pp. 83
Author(s):  
Gulab Fatima Rani ◽  
Helen Ashwin ◽  
Najmeeyah Brown ◽  
Ian S. Hitchcock ◽  
Paul M. Kaye
Keyword(s):  
Leishmania Donovani ◽  
Severe Disease ◽  
Parasite Burden ◽  
Subsequent Development ◽  
Blood Parameters ◽  
Inos Expression ◽  
Infection Model ◽  
Clinical Responses ◽  
Previously Treated ◽  
The Impact

Background: Polyparasitism is commonplace in countries where endemicity for multiple parasites exists, and studies in animal models of coinfection have made significant inroads into understanding the impact of often competing demands on the immune system. However, few studies have addressed how previous exposure to and treatment for one infection impacts a subsequent heterologous infection.   Methods: We used a C57BL/6 mouse model of drug-treated Leishmania donovani infection followed by experimental Plasmodium chabaudi AS malaria, focusing on hematological dysfunction as a common attribute of both infections. We measured parasite burden, blood parameters associated with anemia and thrombocytopenia, and serum thrombopoietin. In addition, we quantified macrophage iNOS expression through immunohistological analysis of the liver and spleen.   Results: We found that the thrombocytopenia and anemia that accompanies primary L. donovani infection was rapidly reversed following single dose AmBisome® treatment, along with multiple other markers associated with immune activation (including restoration of tissue microarchitecture and reduced macrophage iNOS expression). Compared to naive mice, mice cured of previous VL showed comparable albeit delayed clinical responses (including peak parasitemia and anemia) to P. chabaudi AS infection. Thrombocytopenia was also evident in these sequentially infected mice, consistent with a decrease in circulating levels of thrombopoietin. Architectural changes to the spleen were also comparable in sequentially infected mice compared to those with malaria alone. Conclusions: Our data suggest that in this sequential infection model, previously-treated VL has limited impact on the subsequent development of malaria, but this issue deserves further attention in models of more severe disease or through longitudinal population studies in humans.

scholarly journals Super enhancer-mediated transcription of miR146a-5p drives M2 polarization during Leishmania donovani infection

2021 ◽  
Vol 17 (2) ◽  
pp. e1009343
Author(s):  
Sonali Das ◽  
Sohitri Mukherjee ◽  
Nahid Ali
Keyword(s):  
Transcription Factors ◽  
Leishmania Donovani ◽  
Nuclear Translocation ◽  
Differential Expression Analysis ◽  
Parasite Burden ◽  
Arginase 1 ◽  
M2 Polarization ◽  
Super Enhancer ◽  
Immune Precipitation

The outcome of Leishmania donovani infection depends upon the dynamic interchanges between M1 and M2 macrophages. Information of the involvement of microRNAs (miRNAs) and epigenetic modifiers in regulating macrophage plasticity during L. donovani infection is still elusive. Differential expression analysis of polarization-regulating miRNAs, revealed significant enrichment of miR146a-5p during Leishmania donovani infection. A sustained enrichment of miR146a-5p was observed in both infected bone marrow derived macrophages (BMDMs) and BALB/c mice organs. We found involvement of miR146a-5p in phagocytosis and survivability of parasites. Moreover, miR146a-5pgot enriched in interleukin 4- stimulated BMDMs, indicating its possible involvement in M2 polarization. Upon transfecting BMDMs with miRVANA anti-146a oligos, M2 markers (CCR7, YM-1, FIZZ-1, arginase-1, IL10 and IL4) and transcription factors (p-STAT6 and c/EBPβ) got depleted with concomitant augmentation of M1-polarizing transcription factors (p-STAT1, AP1 and IRF-1), miR146a target genes (TRAF6 and IRAK1), M1 cytokines (IL12 and TNFα), iNOS, nitric oxide, and nuclear translocation of phospho p-65 subunit. Neutralization of intracellular mature miR146a-5p pool in infected BALB/c mice lower organ parasite burden and expressions of M2 markers and IL10 with enrichment of M1 markers like iNOS and IL12. Additionally, we explored the novel role of super enhancer (SE), a cis-acting regulatory component, to enrich miR146a-5p expression during infection. Enhanced expression and nuclear retention of SE components like BET bromodomain 4 (BRD4) and p300 were found in infected BMDMs. Upon silencing BRD4, expressions of miR146a-5p and M2 markers were down regulated and TRAF6, IRAK1 and iNOS levels increased. STRING V.11 based predication and immune precipitation confirmed the strong interaction amongst BRD4, p300 and RNA pol II (RpbI). Chromatin immune precipitation studies suggested the recruitment of BRD4 at the enhancer loci of miR146a-5p gene during infection. Altogether, our findings revealed a novel role of BRD4/p300-depdendent super-enhancer in regulating miR146a expression during L. donovani infection which in turn mediates M2 polarization and immune-suppression.

scholarly journals Leishmania parasite arginine deprivation response pathway influences the host macrophage lysosomal arginine sensing machinery

2021 ◽  
Author(s):  
Evanka Madan ◽  
Madhu Puri ◽  
Rohini Muthuswami ◽  
Dan Zilberstein ◽  
Rentala Madhubala
Keyword(s):  
Amino Acid ◽  
Leishmania Donovani ◽  
Amino Acid Transporter ◽  
Host Cells ◽  
Mammalian Host ◽  
Host Macrophage ◽  
Arginine Deprivation ◽  
Acid Transporter ◽  
Null Mutants

AbstractExtensive interaction between the host and pathogen metabolic networks decidedly shapes the outcome of infection. Infection with Leishmania donovani, an intracellular protozoan parasite, leads to a competition for arginine between the host and the parasite. L. donovani transports arginine via a high-affinity transporter LdAAP3, encoded by the two genes LdAAP3.1 and LdAAP3.2. Earlier reports show that upon arginine starvation, cultured Leishmania parasites promptly activate an Arginine Deprivation Response (ADR) pathway, resulting in the stoichiometric up-regulation of LdAAP3.2 mRNA, protein and activity. Lysosomes, on the other hand, are known to employ a specific sensor and an arginine-activated amino acid transporter, solute carrier family 38 member 9 (SLC38A9) that monitors intra-lysosome arginine sufficiency and subsequently up-regulates cellular mTORkinase activity. The present study investigates the interaction between Leishmania and macrophage-lysosome arginine sensing machinery. We show that infection with L. donovani activates SLC38A9 arginine sensing in the human monocyte like-macrophage cell line (THP-1) when grown under physiological concentrations of arginine (0.1 mM). However, supplementing the macrophage growth medium with excess arginine (1.5 mM) followed by infection led to the down-regulation of SLC38A9. Similarly, THP-1 cells infected with LdAAP3.2 null mutants grown in 0.1 mM arginine resulted in reduced expression of SLC38A9 and mTOR. These results indicate that inside the host macrophage, Leishmania overcome low arginine levels by up-regulating the transport of arginine via LdAAP3 and SLC38A9 signalling. Furthermore, while LdAAP3.2 null mutants were impaired in their ability to develop inside THP-1 macrophages, their infectivity and intracellular growth were restored in SLC38A9 silenced macrophages. This study provides the first identification of regulatory role of SLC38A9 in the expression and role of LdAAP3.Author SummaryLeishmania donovani, the causative agent of kala-azar, exhibits a digenetic life cycle. Following infection of the mammalian host, promastigotes differentiate into intracellular amastigotes within the phagolysosome of macrophages. Arginine is a central point of competition between the host and the pathogen. L. donovani senses lack of arginine in the surrounding micro-environment and activates a unique ADR pathway, thus upregulating the expression of the arginine transporter (LdAAP3). The arginine-activated amino acid transporter SLC38A9 localizes to the lysosome surface of mammalian cells and acts as a sensor that transmits information about arginine levels in the lysosome lumen to the mechanistic target of rapamycin (mTOR) kinase. In the present study, we identified the functional interaction of host SLC38A9 and parasite LdAAP3 in macrophages infected with L. donovani. We report that host SLC38A9 upregulation is critical for enhancing and maintaining high LdAAP3 levels in intracellular L. donovani. Our results decode crucial information regarding the molecular mechanism involved in the arginine sensing response in L. donovani-infected host cells. These findings increase our understanding of the interaction of signalling intermediates during Leishmania infection which may lead to the discovery of novel therapeutic interventions.

scholarly journals Interferon-driven alterations of the host’s amino acid metabolism in the pathogenesis of typhoid fever

2016 ◽  
Vol 213 (6) ◽  
pp. 1061-1077 ◽  
Author(s):  
Christoph J. Blohmke ◽  
Thomas C. Darton ◽  
Claire Jones ◽  
Nicolas M. Suarez ◽  
Claire S. Waddington ◽  
...  
Keyword(s):  
Typhoid Fever ◽  
Enteric Fever ◽  
Public Health Problem ◽  
Tryptophan Metabolism ◽  
Infection Model ◽  
Type I ◽  
Macrophage Infection ◽  
Dominant Type ◽  
Cytokine Responses

Enteric fever, caused by Salmonella enterica serovar Typhi, is an important public health problem in resource-limited settings and, despite decades of research, human responses to the infection are poorly understood. In 41 healthy adults experimentally infected with wild-type S. Typhi, we detected significant cytokine responses within 12 h of bacterial ingestion. These early responses did not correlate with subsequent clinical disease outcomes and likely indicate initial host–pathogen interactions in the gut mucosa. In participants developing enteric fever after oral infection, marked transcriptional and cytokine responses during acute disease reflected dominant type I/II interferon signatures, which were significantly associated with bacteremia. Using a murine and macrophage infection model, we validated the pivotal role of this response in the expression of proteins of the host tryptophan metabolism during Salmonella infection. Corresponding alterations in tryptophan catabolites with immunomodulatory properties in serum of participants with typhoid fever confirmed the activity of this pathway, and implicate a central role of host tryptophan metabolism in the pathogenesis of typhoid fever.

scholarly journals Hematological consequences of malaria in mice previously treated for visceral leishmaniasis

2021 ◽  
Vol 6 ◽  
pp. 83
Author(s):  
Gulab Fatima Rani ◽  
Helen Ashwin ◽  
Najmeeyah Brown ◽  
Ian S. Hitchcock ◽  
Paul M. Kaye
Keyword(s):  
Leishmania Donovani ◽  
Severe Disease ◽  
Parasite Burden ◽  
Subsequent Development ◽  
Blood Parameters ◽  
Inos Expression ◽  
Infection Model ◽  
Plasmodium Infection ◽  
Previously Treated ◽  
The Impact

Background: Polyparasitism is commonplace in countries where endemicity for multiple parasites exists, and studies in animal models of coinfection have made significant inroads into understanding the impact of often competing demands on the immune system. However, few studies have addressed how previous exposure to and treatment for one infection impacts a subsequent heterologous infection.   Methods: We used a C57BL/6 mouse model of drug-treated Leishmania donovani infection followed by experimental Plasmodium chabaudi AS malaria, focusing on hematological dysfunction as a common attribute of both infections. We measured parasite burden, blood parameters associated with anemia and thrombocytopenia, and serum thrombopoietin. In addition, we quantified macrophage iNOS expression through immunohistological analysis of the liver and spleen.   Results: We found that the thrombocytopenia and anemia that accompanies primary L. donovani infection was rapidly reversed following single dose AmBisome® treatment, along with multiple other markers associated with immune activation (including restoration of tissue microarchitecture and reduced macrophage iNOS expression). Compared to naive mice, mice cured of previous L. donovani infection showed comparable albeit delayed clinical responses (including peak parasitemia and anemia) to P. chabaudi AS infection. Thrombocytopenia was also evident in these sequentially infected mice, consistent with a decrease in circulating levels of thrombopoietin. Architectural changes to the spleen were also comparable in sequentially infected mice compared to those with Plasmodium infection alone. Conclusions: Our data suggest that in this sequential infection model, previously-treated L. donovani infection has limited impact on the subsequent development of Plasmodium infection, but this issue deserves further attention in models of more severe disease or through longitudinal population studies in humans.

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