The site of the bite: Leishmania interaction with macrophages, neutrophils and the extracellular matrix in the dermis.

PubMed

de Menezes, Juliana Perrone; Saraiva, Elvira M; da Rocha-Azevedo, Bruno

2016-05-04

Leishmania spp., the causative agents of leishmaniasis, are intracellular parasites, transmitted to humans via the bite of their sand fly vectors. Once inoculated, the promastigotes are exposed to the dermis, which is composed of extracellular matrix (ECM), growth factors and its resident cells. Promastigote forms are phagocytosed by macrophages recruited to the site of the sand fly bite, either directly or after interaction with neutrophils. Since Leishmania is an intracellular parasite, its interaction with the host ECM has been neglected as well as the immediate steps after the sand fly bite. However, promastigotes must overcome the obstacles presented by the dermis ECM in order to establish the infection. Thus, the study of the interaction between Leishmania promastigotes and ECM components as well as the earliest stages of infection are important steps to understand the establishment of the disease, and could contribute in the future to new drug developments towards leishmaniasis.

Phloroglucinol derivatives from Hypericum species trigger mitochondrial dysfunction in Leishmania amazonensis.

PubMed

Dagnino, Ana Paula Aquistapase; Mesquita, Camila Saporiti; Dorneles, Gilson Pires; Teixeira, Vivian de Oliveira Nunes; de Barros, Francisco Maikon Corrêa; Vidal Ccana-Ccapatinta, Gari; Fonseca, Simone Gonçalves; Monteiro, Marta Chagas; Júnior, Luiz Carlos Rodrigues; Peres, Alessandra; von Poser, Gilsane Lino; Romão, Pedro Roosevelt Torres

2018-02-27

Bioactive molecules isolated from plants are promising sources for the development of new therapies against leishmaniasis. We investigated the leishmanicidal activity of cariphenone A (1), isouliginosin B (2) and uliginosin B (3) isolated from Hypericum species. Promastigotes and amastigotes of Leishmania amazonensis were incubated with compounds 1-3 at concentrations 1-100 µ m for 48 h. The anti-promastigote effect of compounds was also tested in combinations. The cytotoxicity against macrophages and human erythrocytes were determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method and hemolysis assay, respectively. The compounds 1-3 showed high leishmanicidal activity against promastigotes, IC50 values of 10.5, 17.5 and 11.3 µ m, respectively. Synergistic interactions were found to the associations of compounds 1 and 2 [Σ fractional inhibitory concentration (FIC) = 0.41], and 2 and 3 (ΣFIC = 0.28) on promastigotes. All Hypericum compounds induced mitochondrial hyperpolarization and reactive oxygen species production in promastigotes. The compounds showed low cytotoxicity toward mammalian cells, high selectivity index and killed intracellular amastigotes probably mediated by oxidative stress. These results indicate that these compounds are promising candidates for the development of drugs against leishmaniasis.

Susceptibility of promastigotes and intracellular amastigotes from distinct Leishmania species to the calpain inhibitor MDL28170.

PubMed

de Sousa Araújo, Pedro Soares; de Oliveira, Simone Santiago Carvalho; d'Avila-Levy, Claudia Masini; Dos Santos, André Luis Souza; Branquinha, Marta Helena

2018-05-04

Despite the available drug options, leishmaniasis treatment remains unsatisfactory. The repurposing of calpain inhibitors originally developed for human diseases became an interesting alternative, since Leishmania cells express calpain-related proteins. The susceptibility of six Leishmania species (L. amazonensis, L. braziliensis, L. major, L. mexicana, L. chagasi, and L. donovani) to the calpain inhibitor MDL28170 was determined. Promastigote and intracellular amastigote viability in the presence of MDL28170 was evaluated. MDL28170 was able to reduce promastigote proliferation in a dose-dependent manner for all the parasites. A significant reduction on the general parasite metabolism was detected, as judged by resazurin assay, as well as induced important morphological alterations, including rounding promastigotes and loss of the flagellum. MDL28170 was also able to reduce the number of intracellular amastigotes in RAW macrophages. The susceptibility of both parasite stages (promastigotes and amastigotes) to MDL28170 was similar for all Leishmania species tested. MDL28170 showed a much higher toxicity to Leishmania amastigotes when compared with mammalian macrophages, displaying selectivity index values varying from 13.1 to 39.8. These results suggest that the development of calpain inhibitors may represent an interesting alternative in the treatment of leishmaniasis.

In vitro activity of Tridax procumbens against promastigotes of Leishmania mexicana.

PubMed

Martín-Quintal, Zhelmy; Moo-Puc, Rosa; González-Salazar, Francisco; Chan-Bacab, Manuel J; Torres-Tapia, Luis W; Peraza-Sánchez, Sergio R

2009-04-21

Tridax procumbens is an active herb against leishmaniasis. Leishmaniasis is a group of diseases caused by Leishmania protozoa. We investigated the antileishmanial activity of Tridax procumbens extracts and a pure compound against promastigotes of Leishmania mexicana, the causative agent of cutaneous leishmaniasis in the New World. Extracts and (3S)-16,17-didehydrofalcarinol (1) were obtained by chromatographic methods from Tridax procumbens, and the latter identified by spectroscopic analysis. The effect of these extracts and 1 on the growth inhibition of promastigotes of Leishmania mexicana was evaluated. In order to test the safety of extracts and 1, mammalian cells were treated with them, and cell viability was assessed using trypan blue and MTT. We demonstrated that extracts of Tridax procumbens and 1 showed a pronounced activity against Leishmania mexicana. The methanol extract inhibited promastigotes growth of Leishmania mexicana with a 50% inhibitory concentration (IC(50)) of 3 microg/ml, while oxylipin 1 exhibited the highest inhibition at IC(50)=0.478 microg/ml. In this study we report the biological activity of extracts and (3S)-16,17-didehydrofalcarinol (1), obtained from Tridax procumbens, on the promastigote form of Leishmania mexicana, with no effect upon mammalian cells.

Leishmanicidal Activities of Novel Synthetic Furoxan and Benzofuroxan Derivatives

PubMed Central

Dutra, Luiz Antônio; de Almeida, Letícia; Passalacqua, Thais G.; Reis, Juliana Santana; Torres, Fabio A. E.; Martinez, Isabel; Peccinini, Rosangela Gonçalves; Chin, Chung Man; Chegaev, Konstantin; Guglielmo, Stefano; Fruttero, Roberta

2014-01-01

A novel series of furoxan (1,2,5-oxadiazole 2-oxide) (compounds 3, 4a and -b, 13a and -b, and 14a to -f) and benzofuroxan (benzo[c][1,2,5]oxadiazole 1-oxide) (compounds 7 and 8a to -c) derivatives were synthesized, characterized, and evaluated for in vitro activity against promastigote and intracellular amastigote forms of Leishmania amazonensis. The furoxan derivatives exhibited the ability to generate nitric oxide at different levels (7.8% to 27.4%). The benzofuroxan derivative 8a was able to increase nitrite production in medium supernatant from murine macrophages infected with L. amazonensis at 0.75 mM after 48 h. Furoxan and benzofuroxan derivatives showed remarkable leishmanicidal activity against both promastigote and intracellular amastigote forms. Compounds 8a, 14a and -b, and 14d exerted selective leishmanicidal activities superior to those of amphotericin B and pentamidine. In vitro studies at pH 5.4 reveal that compound 8a is stable until 8 h and that compound 14a behaves as a prodrug, releasing the active aldehyde 13a. These compounds have emerged as promising novel drug candidates for the treatment of leishmaniasis. PMID:24913171

In vitro selection of miltefosine resistance in promastigotes of Leishmania donovani from Nepal: genomic and metabolomic characterization.

PubMed

Shaw, C D; Lonchamp, J; Downing, T; Imamura, H; Freeman, T M; Cotton, J A; Sanders, M; Blackburn, G; Dujardin, J C; Rijal, S; Khanal, B; Illingworth, C J R; Coombs, G H; Carter, K C

2016-03-01

In this study, we followed the genomic, lipidomic and metabolomic changes associated with the selection of miltefosine (MIL) resistance in two clinically derived Leishmania donovani strains with different inherent resistance to antimonial drugs (antimony sensitive strain Sb-S; and antimony resistant Sb-R). MIL-R was easily induced in both strains using the promastigote-stage, but a significant increase in MIL-R in the intracellular amastigote compared to the corresponding wild-type did not occur until promastigotes had adapted to 12.2 μM MIL. A variety of common and strain-specific genetic changes were discovered in MIL-adapted parasites, including deletions at the LdMT transporter gene, single-base mutations and changes in somy. The most obvious lipid changes in MIL-R promastigotes occurred to phosphatidylcholines and lysophosphatidylcholines and results indicate that the Kennedy pathway is involved in MIL resistance. The inherent Sb resistance of the parasite had an impact on the changes that occurred in MIL-R parasites, with more genetic changes occurring in Sb-R compared with Sb-S parasites. Initial interpretation of the changes identified in this study does not support synergies with Sb-R in the mechanisms of MIL resistance, though this requires an enhanced understanding of the parasite's biochemical pathways and how they are genetically regulated to be verified fully. © 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

In vivo and in vitro phagocytosis of Leishmania (Leishmania) amazonensis promastigotes by B-1 cells.

PubMed

Geraldo, M M; Costa, C R; Barbosa, F M C; Vivanco, B C; Gonzaga, W F K M; Novaes E Brito, R R; Popi, A F; Lopes, J D; Xander, P

2016-06-01

Leishmaniasis is caused by Leishmania parasites that infect several cell types. The promastigote stage of Leishmania is internalized by phagocytic cells and transformed into the obligate intracellular amastigote form. B-1 cells are a subpopulation of B cells that are able to differentiate in vitro and in vivo into mononuclear phagocyte-like cells with phagocytic properties. B-1 cells use several receptors for phagocytosis, such as the mannose receptor and third complement receptor. Leishmania binds to the same receptors on macrophages. In this study, we demonstrated that phagocytes derived from B-1 cells (B-1 CDP) were able to internalize promastigotes of L. (L.) amazonensis in vitro. The internalized promastigotes differentiated into amastigotes. Our results showed that the phagocytic index was higher in B-1 CDP compared to peritoneal macrophages and bone marrow-derived macrophages. The in vivo phagocytic ability of B-1 cells was also demonstrated. Parasites were detected inside purified B-1 cells after intraperitoneal infection with L. (L.) amazonensis promastigotes. Intraperitoneal stimulation with the parasites led to an increase in both IL-10 and TNF-α. These results highlight the importance of studying B-1 CDP cells as phagocytic cells that can participate and contribute to immunity to parasites. © 2016 John Wiley & Sons Ltd.

In vitro anti-leishmanial activity of methanolic extracts of Calendula officinalis flowers, Datura stramonium seeds, and Salvia officinalis leaves.

PubMed

Nikmehr, Banafsheh; Ghaznavi, Habib; Rahbar, Amir; Sadr, Samira; Mehrzadi, Saeed

2014-06-01

The anti-leishmanial activity of methanolic extracts of Calendula officinalis flowers, Datura stramonium seeds, and Salvia officinalis leaves against extracellular (promastigote) and intracellular (amastigote) forms of Leishmania major were evaluated in this study. In the first stage, promastigote forms of L. major, were treated with different doses of the plant extracts in a 96-well tissue-culture microplate and IC50 values for each extract were measured with colorimetric MTT assay. In the second stage, macrophage cells were infected with L. major promastigotes. Infected macrophages were treated with plant extracts. Then the macrophages were stained with Gimsa and the number of infected macrophages and amastigotes were counted with a light microscope. The results indicated that the plant extracts inhibited the growth of promastigotes and amastigotes of L. major. Inhibitory concentrations (IC50) for promastigote assay were 108.19, 155.15, and 184.32 μgmL(-1) for C. officinalis flowers, D. stramonium seeds and S. officinalis, respectively. The extracts also reduced the number of amastigotes in macrophage cells from 264 for control group to 88, 97, and 102 for test groups. Although the anti-leishmanial activity of the extracts were not comparable with the standard drug, miltefosine; but they showed significant efficiency in reducing the number of amastigotes in macrophages, in comparison with the control group (P < 0.001). These plant extracts had lower toxicity compared with miltefosine. This study demonstrates the potential efficacy of the methanolic extracts of C. officinalis flowers, D. stramonium seeds, and S. officinalis leaves to control of cutaneous leishmaniasis. Copyright © 2014 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Stachytarpheta cayennensis extract inhibits promastigote and amastigote growth in Leishmania amazonensis via parasite arginase inhibition.

PubMed

Maquiaveli, Claudia do Carmo; Oliveira E Sá, Amanda Maria; Vieira, Paulo Cezar; da Silva, Edson Roberto

2016-11-04

Stachytarpheta cayennensis is a plant that is traditionally used to treat tegumentary leishmaniasis and as an anti-inflammatory agent. This study aimed to evaluate the action of S. cayennensis extracts on the Leishmania (Leishmania) amazonensis arginase enzyme. S. cayennensis was collected from the Brazilian Amazon region. Aqueous extracts were fractionated with n-butanol. The leishmanicidal effects of the n-butanolic fraction (BUF) were evaluated in L. (L.) amazonensis promastigotes and amastigotes. BUF was tested against recombinant arginase from both L. (L.) amazonensis and macrophage arginase. Promastigote cultures and infected macrophage cultures were supplemented with L-ornithine to verify arginase inhibition. NMR analysis was used to identify the major components of BUF. BUF showed an EC 50 of 51 and 32µg/mL against promastigotes and amastigotes of L. (L.) amazonensis, respectively. BUF contains a mixture of verbascoside and isoverbascoside (7:3 ratio) and is a potent L. (L.) amazonensis arginase inhibitor (IC 50 =1.2µg/mL), while macrophage arginase was weakly inhibited (IC 50 >1000µg/mL). The inhibition of arginase by BUF in promastigotes and amastigotes could be demonstrated by culture media supplementation with L-ornithine, a product of the hydrolysis of L-arginine by arginase. Leishmanicidal effects of the S. cayennensis BUF fraction on L. (L.) amazonensis are associated with selective parasite arginase inhibition. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Iron uptake controls the generation of Leishmania infective forms through regulation of ROS levels

PubMed Central

Mittra, Bidyottam; Cortez, Mauro; Haydock, Andrew; Ramasamy, Gowthaman; Myler, Peter J.

2013-01-01

During its life cycle, Leishmania undergoes extreme environmental changes, alternating between insect vectors and vertebrate hosts. Elevated temperature and decreased pH, conditions encountered after macrophage invasion, can induce axenic differentiation of avirulent promastigotes into virulent amastigotes. Here we show that iron uptake is a major trigger for the differentiation of Leishmania amazonensis amastigotes, independently of temperature and pH changes. We found that iron depletion from the culture medium triggered expression of the ferrous iron transporter LIT1 (Leishmania iron transporter 1), an increase in iron content of the parasites, growth arrest, and differentiation of wild-type (WT) promastigotes into infective amastigotes. In contrast, LIT1-null promastigotes showed reduced intracellular iron content and sustained growth in iron-poor media, followed by cell death. LIT1 up-regulation also increased iron superoxide dismutase (FeSOD) activity in WT but not in LIT1-null parasites. Notably, the superoxide-generating drug menadione or H2O2 was sufficient to trigger differentiation of WT promastigotes into fully infective amastigotes. LIT1-null promastigotes accumulated superoxide radicals and initiated amastigote differentiation after exposure to H2O2 but not to menadione. Our results reveal a novel role for FeSOD activity and reactive oxygen species in orchestrating the differentiation of virulent Leishmania amastigotes in a process regulated by iron availability. PMID:23382545

In Vitro Susceptibility of Leishmania infantum to Artemisinin Derivatives and Selected Trioxolanes

PubMed Central

Albuquerque, Andreia; Cabral, Lília I. L.; Lopes, Liliana; Campino, Lenea

2015-01-01

Leishmaniasis is among the world's most neglected diseases. Currently available drugs for treatment present drawbacks, urging the need for more effective, safer, and cheaper drugs. A small library of artemisinin-derived trioxanes and synthetic trioxolanes was tested against promastigote and intramacrophage amastigote forms of Leishmania infantum. The trioxolanes LC50 and LC95 presented the best activity and safety profiles, showing potential for further studies in the context of leishmanial therapy. Our results indicate that the compounds tested exhibit peroxide-dependent activity. PMID:26014947

Apoptosis-like cell death in Leishmania donovani treated with KalsomeTM10, a new liposomal amphotericin B

PubMed Central

Shadab, Md.; Jha, Baijayanti; Asad, Mohammad; Deepthi, Makaraju; Kamran, Mohd.; Ali, Nahid

2017-01-01

Objective The present study aimed to elucidate the cell death mechanism in Leishmania donovani upon treatment with KalsomeTM10, a new liposomal amphotericin B. Methodology/Principal findings We studied morphological alterations in promastigotes through phase contrast and scanning electron microscopy. Phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and disruption of mitochondrial integrity was determined by flow cytometry using annexinV-FITC, JC-1 and mitotraker, respectively. For analysing oxidative stress, generation of H2O2 (bioluminescence kit) and mitochondrial superoxide O2− (mitosox) were measured. DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase mediated dUTP nick-end labelling (TUNEL) and DNA laddering assay. We found that KalsomeTM10 is more effective then Ambisome against the promastigote as well as intracellular amastigote forms. The mechanistic study showed that KalsomeTM10 induced several morphological alterations in promastigotes typical of apoptosis. KalsomeTM10 treatment showed a dose- and time-dependent exposure of PS in promastigotes. Further, study on mitochondrial pathway revealed loss of mitochondrial membrane potential as well as disruption in mitochondrial integrity with depletion of intracellular pool of ATP. KalsomeTM10 treated promastigotes showed increased ROS production, diminished GSH levels and increased caspase-like activity. DNA fragmentation and cell cycle arrest was observed in KalsomeTM10 treated promastigotes. Apoptotic DNA fragmentation was also observed in KalsomeTM10 treated intracellular amastigotes. KalsomeTM10 induced generation of ROS and nitric oxide leads to the killing of the intracellular parasites. Moreover, endocytosis is indispensable for KalsomeTM10 mediated anti-leishmanial effect in host macrophage. Conclusions KalsomeTM10 induces apoptotic-like cell death in L. donovani parasites to exhibit its anti-leishmanial function. PMID:28170432

Leishmanicidal activity of Nystatin (mycostatin): a potent polyene compound.

PubMed

Ali, S A; Iqbal, J; Nabeel; Khalil, Y; Manzoor, A; Bukhari, I; Ahmad, B; Yasinzai, M M

1997-10-01

The susceptibility of promastigote of Leishmania major to Nystatin in vitro was examined. L. major (MHOM/PK/88/DESTO) promastigote were cultured in medium 199 supplemented with 10% heat inactivated foetal bovine serum and 2% urine. The growth of the promastigote was monitored in the absence and presence of the experimental compound (Nystatin) for upto 5 days post-inoculation. The EC50 value (the concentration of drug necessary to inhibit the growth rate of cells to 50% of the control value) obtained for Nystatin against the promastigote of L. major was less than 9.76 iu ml. Certain polyene compounds like Amphotericin-B and Nystatin (mycostatin) are familiar for their fungicidal activity. Amphotericin-B is used since long as antileishmanial drug as well. Results obtained suggest that Nystatin has a very good anti leishmanial activity in vitro. The mode of action proposed for this drug is same as for Amphotericin-B as both of these polyene compounds interact with the various sterols present on the surface of the parasite, thus unusual gaps and pores are formed on the surface that results in the leakage of the ions. This leakage finally leads to the destruction of the parasite.

Lulo cell line derived from Lutzomyia longipalpis (Diptera: Psychodidae): a novel model to assay Leishmania spp. and vector interaction.

PubMed

Côrtes, Luzia Mc; Silva, Roger Mm; Pereira, Bernardo As; Guerra, Camila; Zapata, Angela C; Bello, Felio J; Finkelstein, Léa C; Madeira, Maria F; Brazil, Reginaldo P; Côrte-Real, Suzana; Alves, Carlos R

2011-11-14

Leishmania (Vianna) braziliensis, Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) chagasi are important parasites in the scenario of leishmaniasis in Brazil. During the life cycle of these parasites, the promastigote forms adhere to the midgut epithelial microvillii of phlebotomine insects to avoid being secreted along with digestive products. Lulo cells are a potential model that will help to understand the features of this adhesion phenomenon. Here, we analyze the interaction between Leishmania spp. promastigotes and Lulo cells in vitro, specifically focusing on adhesion events occurring between three Leishmania species and this cell line. Confluent monolayers of Lulo cells were incubated with promastigotes and adhesion was assessed using both light microscopy and scanning electron microscopy. The results indicate that species from the subgenera Leishmania and Viannia have great potential to adhere to Lulo cells. The highest adherence rate was observed for L. (L.) chagasi after 24 h of incubation with Lulo cells (27.3 ± 1.8% of cells with adhered promastigotes), followed by L. (L.) amazonensis (16.0 ± 0.7%) and L. (V.) braziliensis (3.0 ± 0.7%), both after 48 h. In the ultrastructural analysis, promastigote adherence was also assessed by scanning electron microscopy, showing that, for parasites from both subgenera, adhesion occurs by both the body and the flagellum. The interaction of Lulo cells with Leishmania (L.) chagasi showed the participation of cytoplasmic projections from the former closely associating the parasites with the cells. We present evidence that Lulo cells can be useful in studies of insect-parasite interactions for Leishmania species.

A unique, highly conserved secretory invertase is differentially expressed by promastigote developmental forms of all species of the human pathogen, Leishmania

PubMed Central

Lyda, Todd A.; Joshi, Manju B.; Andersen, John F.; Kelada, Andrew Y.; Owings, Joshua P.; Bates, Paul A.; Dwyer, Dennis M.

2015-01-01

Leishmania are protozoan pathogens of humans that exist as extracellular promastigotes in the gut of their sand fly vectors and as obligate intracellular amastigotes within phagolysosomes of infected macrophages. Between infectious blood meal feeds, sand flies take plant juice meals that contain sucrose and store these sugars in their crop. Such sugars are regurgitated into the sand fly anterior midgut where they impact the developing promastigote parasite population. In this report we showed that promastigotes of all Leishmania species secreted an invertase/sucrase enzyme during their growth in vitro. In contrast, neither L. donovani nor L. mexicana amastigotes possessed any detectable invertase activity. Importantly, no released/secreted invertase activity was detected in culture supernatants from either Trypanosoma brucei or Trypanosoma cruzi. Using HPLC, the L. donovani secretory invertase was isolated and subjected to amino acid sequencing. Subsequently, we used a molecular approach to identify the LdINV and LmexINV genes encoding the ~72 kDa invertases produced by these organisms. Interestingly, we identified high fidelity LdINV-like homologs in the genomes of all Leishmania sp. but none were present in either T. brucei or T. cruzi. Northern blot and RT-PCR analyses showed that these genes were developmentally/differentially expressed in promastigotes but not amastigotes of these parasites. Homologous transfection studies demonstrated that these genes in fact encoded the functional secretory invertases produced by these parasites. Cumulatively, our results suggest that these secretory enzymes play critical roles in the survival/growth/development and transmission of all Leishmania parasites within their sand fly vector hosts. PMID:25763714

A unique, highly conserved secretory invertase is differentially expressed by promastigote developmental forms of all species of the human pathogen, Leishmania.

PubMed

Lyda, Todd A; Joshi, Manju B; Andersen, John F; Kelada, Andrew Y; Owings, Joshua P; Bates, Paul A; Dwyer, Dennis M

2015-06-01

Leishmania are protozoan pathogens of humans that exist as extracellular promastigotes in the gut of their sand fly vectors and as obligate intracellular amastigotes within phagolysosomes of infected macrophages. Between infectious blood meal feeds, sand flies take plant juice meals that contain sucrose and store these sugars in their crop. Such sugars are regurgitated into the sand fly anterior midgut where they impact the developing promastigote parasite population. In this report we showed that promastigotes of all Leishmania species secreted an invertase/sucrase enzyme during their growth in vitro. In contrast, neither L. donovani nor L. mexicana amastigotes possessed any detectable invertase activity. Importantly, no released/secreted invertase activity was detected in culture supernatants from either Trypanosoma brucei or Trypanosoma cruzi. Using HPLC, the L. donovani secretory invertase was isolated and subjected to amino acid sequencing. Subsequently, we used a molecular approach to identify the LdINV and LmexINV genes encoding the ~72 kDa invertases produced by these organisms. Interestingly, we identified high fidelity LdINV-like homologs in the genomes of all Leishmania sp. but none were present in either T. brucei or T. cruzi. Northern blot and RT-PCR analyses showed that these genes were developmentally/differentially expressed in promastigotes but not amastigotes of these parasites. Homologous transfection studies demonstrated that these genes in fact encoded the functional secretory invertases produced by these parasites. Cumulatively, our results suggest that these secretory enzymes play critical roles in the survival/growth/development and transmission of all Leishmania parasites within their sand fly vector hosts.

Identification, biochemical characterization, and in-vivo expression of the intracellular invertase BfrA from the pathogenic parasite Leishmania major.

PubMed

Belaz, Sorya; Rattier, Thibault; Lafite, Pierre; Moreau, Philippe; Routier, Françoise H; Robert-Gangneux, Florence; Gangneux, Jean-Pierre; Daniellou, Richard

2015-10-13

The parasitic life cycle of Leishmania includes an extracellular promastigote stage that occurs in the gut of the insect vector. During that period, the sucrose metabolism and more specifically the first glycosidase of this pathway are essential for growth and survival of the parasite. We investigated the expression of the invertase BfrA in the promastigote and amastigote stages of three parasite species representative of the three various clinical forms and of various geographical areas, namely Leishmania major, L. donovani and L. braziliensis. Thereafter, we cloned, overexpressed and biochemically characterized this invertase BfrA from L. major, heterologously expressed in both Escherichia coli and L. tarentolae. For all species, expression levels of BfrA mRNA were correlated to the time of the culture and the parasitic stage (promastigotes > amastigotes). BfrA exhibited no activity when expressed as a glycoprotein in L. tarentolae but proved to be an invertase when not glycosylated, yet owing low sequence homology with other invertases from the same family. Our data suggest that BfrA is an original invertase that is located inside the parasite. It is expressed in both parasitic stages, though to a higher extent in promastigotes. This work provides new insight into the parasite sucrose metabolism. Copyright © 2015 Elsevier Ltd. All rights reserved.

Leishmanicidal activity of nine novel flavonoids from Delphinium staphisagria.

PubMed

Ramírez-Macías, Inmaculada; Marín, Clotilde; Díaz, Jesús G; Rosales, María José; Gutiérrez-Sánchez, Ramón; Sánchez-Moreno, Manuel

2012-01-01

To evaluate the in vitro leishmanicidal activity of nine flavonoid derivatives from Delphinium staphisagria against L. infantum and L. braziliensis. The in vitro activity of compounds 1-9 was assayed on extracellular promastigote and axenic amastigote forms and on intracellular amastigote forms of the parasites. Infectivity and cytotoxicity tests were carried on J774.2 macrophage cells using Glucantime as the reference drug. The mechanisms of action were analysed performing metabolite excretion and transmission electronic microscope ultrastructural alteration studies. Nine flavonoids showed leishmanicidal activity against promastigote as well as amastigote forms of Leishmania infantum and L. braziliensis. These compounds were nontoxic to mammalian cells and were effective at similar concentrations up to or lower than that of the reference drug (Glucantime). The results showed that 2(″)-acetylpetiolaroside (compound 8) was clearly the most active. This study has demonstrated that flavonoid derivatives are active against L. infantum and L. braziliensis.

Leishmania amazonensis promastigotes in 3D Collagen I culture: an in vitro physiological environment for the study of extracellular matrix and host cell interactions.

PubMed

Petropolis, Debora B; Rodrigues, Juliany C F; Viana, Nathan B; Pontes, Bruno; Pereira, Camila F A; Silva-Filho, Fernando C

2014-01-01

Leishmania amazonensis is the causative agent of American cutaneous leishmaniasis, an important neglected tropical disease. Once Leishmania amazonensis is inoculated into the human host, promastigotes are exposed to the extracellular matrix (ECM) of the dermis. However, little is known about the interaction between the ECM and Leishmania promastigotes. In this study we established L. amazonensis promastigote culture in a three-dimensional (3D) environment mainly composed of Collagen I (COL I). This 3D culture recreates in vitro some aspects of the human host infection site, enabling the study of the interaction mechanisms of L. amazonensis with the host ECM. Promastigotes exhibited "freeze and run" migration in the 3D COL I matrix, which is completely different from the conventional in vitro swimming mode of migration. Moreover, L. amazonensis promastigotes were able to invade, migrate inside, and remodel the 3D COL I matrix. Promastigote trans-matrix invasion and the freeze and run migration mode were also observed when macrophages were present in the matrix. At least two classes of proteases, metallo- and cysteine proteases, are involved in the 3D COL I matrix degradation caused by Leishmania. Treatment with a mixture of protease inhibitors significantly reduced promastigote invasion and migration through this matrix. Together our results demonstrate that L. amazonensis promastigotes release proteases and actively remodel their 3D environment, facilitating their migration. This raises the possibility that promastigotes actively interact with their 3D environment during the search for their cellular "home"-macrophages. Supporting this hypothesis, promastigotes migrated faster than macrophages in a novel 3D co-culture model.

Leishmania amazonensis promastigotes in 3D Collagen I culture: an in vitro physiological environment for the study of extracellular matrix and host cell interactions

PubMed Central

Rodrigues, Juliany C.F.; Viana, Nathan B.; Pontes, Bruno; Pereira, Camila F.A.; Silva-Filho, Fernando C.

2014-01-01

Leishmania amazonensis is the causative agent of American cutaneous leishmaniasis, an important neglected tropical disease. Once Leishmania amazonensis is inoculated into the human host, promastigotes are exposed to the extracellular matrix (ECM) of the dermis. However, little is known about the interaction between the ECM and Leishmania promastigotes. In this study we established L. amazonensis promastigote culture in a three-dimensional (3D) environment mainly composed of Collagen I (COL I). This 3D culture recreates in vitro some aspects of the human host infection site, enabling the study of the interaction mechanisms of L. amazonensis with the host ECM. Promastigotes exhibited “freeze and run” migration in the 3D COL I matrix, which is completely different from the conventional in vitro swimming mode of migration. Moreover, L. amazonensis promastigotes were able to invade, migrate inside, and remodel the 3D COL I matrix. Promastigote trans-matrix invasion and the freeze and run migration mode were also observed when macrophages were present in the matrix. At least two classes of proteases, metallo- and cysteine proteases, are involved in the 3D COL I matrix degradation caused by Leishmania. Treatment with a mixture of protease inhibitors significantly reduced promastigote invasion and migration through this matrix. Together our results demonstrate that L. amazonensis promastigotes release proteases and actively remodel their 3D environment, facilitating their migration. This raises the possibility that promastigotes actively interact with their 3D environment during the search for their cellular “home”—macrophages. Supporting this hypothesis, promastigotes migrated faster than macrophages in a novel 3D co-culture model. PMID:24765565

Molecular differentiation of Leishmania protozoarium using CdS quantum dots as biolabels

NASA Astrophysics Data System (ADS)

Santos, Beate S.; de Farias, Patrícia M. A.; de Menezes, Frederico D.; Mariano, Erick L.; de C. Ferreira, Ricardo; Giorgio, Selma; Bosetto, Maira C.; Ayres, Diana C.; Lima, Paulo M.; Fontes, Adriana; de Thomas, André A.; Cesar, Carlos L.

2006-02-01

In this work we applied core-shell CdS/Cd(OH)2 quantum dots (QDs) as fluorescent labels in the Leishmania amazonensis protozoarium. The nanocrystals (8-9 nm) are obtained via colloidal synthesis in aqueous medium, with final pH=7 using sodium polyphosphate as the stabilizing agent. The surface of the particles is passivated with a cadmium hydroxide shell and the particle surface is functionalized with glutaraldehyde. The functionalized and non-functionalized particles were conjugated to Leishmania organisms in the promastigote form. The marked live organisms were visualized using confocal microscopy. The systems exhibit a differentiation of the emission color for the functionalized and non-functionalized particles suggesting different chemical interactions with the promastigote moieties. Two photon emision spectra (λ exc=795nm) were obtained for the promastigotes labeled with the functionalized QDs showing a significant spectral change compared to the original QDs suspension. These spectral changes are discussed in terms of the possible energy deactivation processes.

Antiproliferative and ultrastructural effects of phenethylamine derivatives on promastigotes and amastigotes of Leishmania (Leishmania) infantum chagasi.

PubMed

Brasil, Paula Ferreira; de Freitas, Júlia Araújo; Barreto, Anna Léa Silva; Adade, Camila Marques; Reis de Sá, Leandro Figueira; Constantino-Teles, Pamella; Toledo, Fabiano Travanca; de Sousa, Bruno A; Gonçalves, Augusto Cesar; Romanos, Maria Teresa Villela; Comasseto, João V; Dos Santos, Alcindo A; Tessis, Ana Claudia; Souto-Padrón, Thais; Soares, Rosangela Maria A; Ferreira-Pereira, Antonio

2017-04-01

Leishmania (Leishmania) infantum chagasi is one of the agents that cause visceral leishmaniasis. This disease occurs more frequently in third world countries, such as Brazil. The treatment is arduous, and is dependent on just a few drugs like the antimonial derivatives and amphotericin B. Moreover, these drugs are not only expensive, but they can also cause severe side effects and require long-term treatment. Therefore, it is very important to find new compounds that are effective against leishmaniasis. In the present work we evaluated a new group of synthetic amides against the promastigote and amastigote forms of L. infantum chagasi. The results showed that one of these amides in particular, presented very effective activity against the promastigotes and amastigotes of L. infantum chagasi at low concentrations and it also presented low toxicity for mammal cells, which makes this synthetic amide a promising drug for combating leishmaniasis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Liposomal formulation of turmerone-rich hexane fractions from Curcuma longa enhances their antileishmanial activity.

PubMed

Amaral, Ana Claudia F; Gomes, Luciana A; Silva, Jefferson Rocha de A; Ferreira, José Luiz P; Ramos, Aline de S; Rosa, Maria do Socorro S; Vermelho, Alane B; Rodrigues, Igor A

2014-01-01

Promastigote forms of Leishmania amazonensis were treated with different concentrations of two fractions of Curcuma longa cortex rich in turmerones and their respective liposomal formulations in order to evaluate growth inhibition and the minimal inhibitory concentration (MIC). In addition, cellular alterations of treated promastigotes were investigated under transmission and scanning electron microscopies. LipoRHIC and LipoRHIWC presented lower MIC, 5.5 and 12.5 μg/mL, when compared to nonencapsulated fractions (125 and 250 μg/mL), respectively, and to ar-turmerone (50 μg/mL). Parasite growth inhibition was demonstrated to be dose-dependent. Important morphological changes as rounded body and presence of several roles on plasmatic membrane could be seen on L. amazonensis promastigotes after treatment with subinhibitory concentration (2.75 μg/mL) of the most active LipoRHIC. In that sense, the hexane fraction from the turmeric cortex of Curcuma longa incorporated in liposomal formulation (LipoRHIC) could represent good strategy for the development of new antileishmanial agent.

Chemotherapy and Drug Targeting in the Treatment of Leishmaniasis

DTIC Science & Technology

1991-05-29

flagellated promastigote forms reside in the alimentary tract of their sandfly vector hosts, and the obligate intracellular amastigote form exists within the...LKB Biotechnology (Piscataway, NJ, USA). Centricon-10 microconcentrator was from .-\\micon (Danvers, \\V1A, USA). Enzyme grade ammonium sulfate and G4

Inhibitory activity of pentacyano(isoniazid)ferrate(II), IQG-607, against promastigotes and amastigotes forms of Leishmania braziliensis

PubMed Central

Amorim, Camila F.; Galina, Luiza; Carvalho, Natália B.; Sperotto, Nathalia D. M.; Pissinate, Kenia; Machado, Pablo; Campos, Maria M.; Basso, Luiz A.; Carvalho, Edgar M.; Santos, Diógenes Santiago

2017-01-01

M. tuberculosis and parasites of the genus Leishmania present the type II fatty acid biosynthesis system (FASII). The pentacyano(isoniazid)ferrate(II) compound, named IQG-607, inhibits the enzyme 2-trans-enoyl-ACP(CoA) reductase from M. tuberculosis, a key component in the FASII system. Here, we aimed to evaluate the inhibitory activity of IQG-607 against promastigote and amastigote forms of Leishmania (Viannia) braziliensis isolated from patients with different clinical forms of L. braziliensis infection, including cutaneous, mucosal and disseminated leishmaniasis. Importantly, IQG-607 inhibited the proliferation of three different isolates of L. braziliensis promastigotes associated with cutaneous, mucosal and disseminated leishmaniasis. The IC50 values for IQG-607 ranged from 32 to 75 μM, for these forms. Additionally, IQG-607 treatment decreased the proliferation of intracellular amastigotes in infected macrophages, after an analysis of the percentage of infected cells and the number of intracellular parasites/100 cells. IQG-607 reduced from 58% to 98% the proliferation of L. braziliensis from cutaneous, mucosal and disseminated strains. Moreover, IQG-607 was also evaluated regarding its potential toxic profile, by using different cell lines. Cell viability of the lineages Vero, HaCat and HepG2 was significantly reduced after incubation with concentrations of IQG-607 higher than 2 mM. Importantly, IQG-607, in a concentration of 1 mM, did not induce DNA damage in HepG2 cells, when compared to the untreated control group. Future studies will confirm the mechanism of action of IQG-607 against L. braziliensis. PMID:29281707

Lulo cell line derived from Lutzomyia longipalpis (Diptera: Psychodidae): a novel model to assay Leishmania spp. and vector interaction

PubMed Central

2011-01-01

Background Leishmania (Vianna) braziliensis, Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) chagasi are important parasites in the scenario of leishmaniasis in Brazil. During the life cycle of these parasites, the promastigote forms adhere to the midgut epithelial microvillii of phlebotomine insects to avoid being secreted along with digestive products. Lulo cells are a potential model that will help to understand the features of this adhesion phenomenon. Here, we analyze the interaction between Leishmania spp. promastigotes and Lulo cells in vitro, specifically focusing on adhesion events occurring between three Leishmania species and this cell line. Methods Confluent monolayers of Lulo cells were incubated with promastigotes and adhesion was assessed using both light microscopy and scanning electron microscopy. Findings The results indicate that species from the subgenera Leishmania and Viannia have great potential to adhere to Lulo cells. The highest adherence rate was observed for L. (L.) chagasi after 24 h of incubation with Lulo cells (27.3 ± 1.8% of cells with adhered promastigotes), followed by L. (L.) amazonensis (16.0 ± 0.7%) and L. (V.) braziliensis (3.0 ± 0.7%), both after 48 h. In the ultrastructural analysis, promastigote adherence was also assessed by scanning electron microscopy, showing that, for parasites from both subgenera, adhesion occurs by both the body and the flagellum. The interaction of Lulo cells with Leishmania (L.) chagasi showed the participation of cytoplasmic projections from the former closely associating the parasites with the cells. Conclusions We present evidence that Lulo cells can be useful in studies of insect-parasite interactions for Leishmania species. PMID:22082050

In vitro antileishmanial activity of fisetin flavonoid via inhibition of glutathione biosynthesis and arginase activity in Leishmania infantum.

PubMed

Adinehbeigi, Keivan; Razi Jalali, Mohammad Hossein; Shahriari, Ali; Bahrami, Somayeh

2017-06-01

With the increasing emergence of drug resistant Leishmania sp. in recent years, combination therapy has been considered as a useful way to treat and control of Leishmaniasis. The present study was designed to evaluate the antileishmanial effects of the fisetin alone and combination of fisetin plus Meglumine antimoniate (Fi-MA) against Leishmania infantum. The IC50 values for fisetin were obtained 0.283 and 0.102 μM against promastigotes and amastigote forms, respectively. Meglumine antimoniate (MA, Glucantime) as control drug also revealed IC50 values of 0.247 and 0.105 μM for promastigotes and amastigotes of L. infantum, respectively. In order to determine the mode of action of fisetin and Meglumine antimoniate (MA, Glucantime), the activities of arginase (ARG), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were measured. Moreover, intracellular glutathione (GSH) and nitric oxide (NO) levels in L. infantum-infected macrophages and L. infantum promastigotes which were treated with IC50 concentrations of fisetin, MA and Fi-MA were investigated. Our results showed that MA decreased CAT and SOD activity and increased NO levels in L. infantum-infected macrophages. In promastigotes, MA inhibited parasite SOD activity and reduced parasite NO production. The decreased levels of most of the antioxidant enzymes, accompanying by the raised level of NO in treated macrophages with MA, were observed to regain their normal profiles due to Fi-MA treatment. Furthermore, fisetin could prevent the growth of promastigotes by inhibition of ARG activity and reduction of GSH levels and NO production. In conclusion, these findings showed that fisetin improves MA side effects.

In vitro antileishmanial activity of fisetin flavonoid via inhibition of glutathione biosynthesis and arginase activity in Leishmania infantum

PubMed Central

Razi Jalali, Mohammad Hossein; Shahriari, Ali; Bahrami, Somayeh

2017-01-01

With the increasing emergence of drug resistant Leishmania sp. in recent years, combination therapy has been considered as a useful way to treat and control of Leishmaniasis. The present study was designed to evaluate the antileishmanial effects of the fisetin alone and combination of fisetin plus Meglumine antimoniate (Fi-MA) against Leishmania infantum. The IC50 values for fisetin were obtained 0.283 and 0.102 μM against promastigotes and amastigote forms, respectively. Meglumine antimoniate (MA, Glucantime) as control drug also revealed IC50 values of 0.247 and 0.105 μM for promastigotes and amastigotes of L. infantum, respectively. In order to determine the mode of action of fisetin and Meglumine antimoniate (MA, Glucantime), the activities of arginase (ARG), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were measured. Moreover, intracellular glutathione (GSH) and nitric oxide (NO) levels in L. infantum-infected macrophages and L. infantum promastigotes which were treated with IC50 concentrations of fisetin, MA and Fi-MA were investigated. Our results showed that MA decreased CAT and SOD activity and increased NO levels in L. infantum-infected macrophages. In promastigotes, MA inhibited parasite SOD activity and reduced parasite NO production. The decreased levels of most of the antioxidant enzymes, accompanying by the raised level of NO in treated macrophages with MA, were observed to regain their normal profiles due to Fi-MA treatment. Furthermore, fisetin could prevent the growth of promastigotes by inhibition of ARG activity and reduction of GSH levels and NO production. In conclusion, these findings showed that fisetin improves MA side effects. PMID:28385129

Influence of the Microenvironment in the Transcriptome of Leishmania infantum Promastigotes: Sand Fly versus Culture

PubMed Central

Alcolea, Pedro J.; Alonso, Ana; Domínguez, Mercedes; Parro, Víctor; Jiménez, Maribel; Molina, Ricardo; Larraga, Vicente

2016-01-01

Zoonotic visceral leishmaniasis is a vector-borne disease caused by Leishmania infantum in the Mediterranean Basin, where domestic dogs and wild canids are the main reservoirs. The promastigote stage replicates and develops within the gut of blood-sucking phlebotomine sand flies. Mature promastigotes are injected in the dermis of the mammalian host and differentiate into the amastigote stage within parasitophorous vacuoles of phagocytic cells. The major vector of L. infantum in Spain is Phlebotomus perniciosus. Promastigotes are routinely axenized and cultured to mimic in vitro the conditions inside the insect gut, which allows for most molecular, cellular, immunological and therapeutical studies otherwise inviable. Culture passages are known to decrease infectivity, which is restored by passage through laboratory animals. The most appropriate source of promastigotes is the gut of the vector host but isolation of the parasite is technically challenging. In fact, this option is not viable unless small samples are sufficient for downstream applications like promastigote cultures and nucleic acid amplification. In this study, in vitro infectivity and differential gene expression have been studied in cultured promastigotes at the stationary phase and in promastigotes isolated from the stomodeal valve of the sand fly P. perniciosus. About 20 ng RNA per sample could be isolated. Each sample contained L. infantum promastigotes from 20 sand flies. RNA was successfully amplified and processed for shotgun genome microarray hybridization analysis. Most differentially regulated genes are involved in regulation of gene expression, intracellular signaling, amino acid metabolism and biosynthesis of surface molecules. Interestingly, meta-analysis by hierarchical clustering supports that up-regulation of 22.4% of the differentially regulated genes is specifically enhanced by the microenvironment (i.e. sand fly gut or culture). The correlation between cultured and naturally developed promastigotes is strong but not very high (Pearson coefficient R2 = 0.727). Therefore, the influence of promastigote culturing should be evaluated case-by-case in experimentation. PMID:27163123

Influence of the Microenvironment in the Transcriptome of Leishmania infantum Promastigotes: Sand Fly versus Culture.

PubMed

Alcolea, Pedro J; Alonso, Ana; Domínguez, Mercedes; Parro, Víctor; Jiménez, Maribel; Molina, Ricardo; Larraga, Vicente

2016-05-01

Zoonotic visceral leishmaniasis is a vector-borne disease caused by Leishmania infantum in the Mediterranean Basin, where domestic dogs and wild canids are the main reservoirs. The promastigote stage replicates and develops within the gut of blood-sucking phlebotomine sand flies. Mature promastigotes are injected in the dermis of the mammalian host and differentiate into the amastigote stage within parasitophorous vacuoles of phagocytic cells. The major vector of L. infantum in Spain is Phlebotomus perniciosus. Promastigotes are routinely axenized and cultured to mimic in vitro the conditions inside the insect gut, which allows for most molecular, cellular, immunological and therapeutical studies otherwise inviable. Culture passages are known to decrease infectivity, which is restored by passage through laboratory animals. The most appropriate source of promastigotes is the gut of the vector host but isolation of the parasite is technically challenging. In fact, this option is not viable unless small samples are sufficient for downstream applications like promastigote cultures and nucleic acid amplification. In this study, in vitro infectivity and differential gene expression have been studied in cultured promastigotes at the stationary phase and in promastigotes isolated from the stomodeal valve of the sand fly P. perniciosus. About 20 ng RNA per sample could be isolated. Each sample contained L. infantum promastigotes from 20 sand flies. RNA was successfully amplified and processed for shotgun genome microarray hybridization analysis. Most differentially regulated genes are involved in regulation of gene expression, intracellular signaling, amino acid metabolism and biosynthesis of surface molecules. Interestingly, meta-analysis by hierarchical clustering supports that up-regulation of 22.4% of the differentially regulated genes is specifically enhanced by the microenvironment (i.e. sand fly gut or culture). The correlation between cultured and naturally developed promastigotes is strong but not very high (Pearson coefficient R2 = 0.727). Therefore, the influence of promastigote culturing should be evaluated case-by-case in experimentation.

Differential protein abundance in promastigotes of nitric oxide-sensitive and resistant Leishmania chagasi strains.

PubMed

Alcolea, Pedro J; Tuñón, Gabriel I L; Alonso, Ana; García-Tabares, Francisco; Ciordia, Sergio; Mena, María C; Campos, Roseane N S; Almeida, Roque P; Larraga, Vicente

2016-11-01

Leishmania chagasi is the causative agent of zoonotic visceral leishmaniasis in Brazil. Domestic and stray dogs are the main reservoirs. The life cycle of the parasite involves two stages. Promastigotes are extracellular and develop within the sand fly gut. Amastigotes survive inside the harsh environment of the phagolysosome of mammalian host phagocytes, which display the nitric oxide defense mechanism. Surprisingly, we were able to isolate promastigotes that are also resistant to NO. This finding may be explained by the preadaptative hypothesis. An insight into the proteome of NO-sensitive and resistant promastigotes is presented herein. Total protein extracts were prepared from promastigote cultures of an NO-sensitive and a resistant strain at early-logarithmic, mid-logarithmic and stationary phase. A population enriched in metacyclic promastigotes was also isolated by Percoll gradient centrifugation. In vitro infectivity of both strains was compared. Differential protein abundance was analyzed by 2DE-MALDI-TOF/TOF. The most striking results were tested at the mRNA level by qRT-PCR. Three biological replicates were performed in all cases. NO-resistant L. chagasi promastigotes are more infective than NO-sensitive ones. Among the differentially abundant spots, 40 proteins could be successfully identified in the sensitive strain and 38 in resistant promastigotes. The increase of G6PD and the decrease of ARG and GPX transcripts and proteins contribute to NO resistance in L. chagasi promastigotes. These proteins may be studied as potential drug targets and/or vaccine candidates in the future. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Liposomal Formulation of Turmerone-Rich Hexane Fractions from Curcuma longa Enhances Their Antileishmanial Activity

PubMed Central

Amaral, Ana Claudia F.; Gomes, Luciana A.; Silva, Jefferson Rocha de A.; Ferreira, José Luiz P.; Ramos, Aline de S.; Rosa, Maria do Socorro S.; Vermelho, Alane B.; Rodrigues, Igor A.

2014-01-01

Promastigote forms of Leishmania amazonensis were treated with different concentrations of two fractions of Curcuma longa cortex rich in turmerones and their respective liposomal formulations in order to evaluate growth inhibition and the minimal inhibitory concentration (MIC). In addition, cellular alterations of treated promastigotes were investigated under transmission and scanning electron microscopies. LipoRHIC and LipoRHIWC presented lower MIC, 5.5 and 12.5 μg/mL, when compared to nonencapsulated fractions (125 and 250 μg/mL), respectively, and to ar-turmerone (50 μg/mL). Parasite growth inhibition was demonstrated to be dose-dependent. Important morphological changes as rounded body and presence of several roles on plasmatic membrane could be seen on L. amazonensis promastigotes after treatment with subinhibitory concentration (2.75 μg/mL) of the most active LipoRHIC. In that sense, the hexane fraction from the turmeric cortex of Curcuma longa incorporated in liposomal formulation (LipoRHIC) could represent good strategy for the development of new antileishmanial agent. PMID:25045693

Antileishmanial activity evaluation of adunchalcone, a new prenylated dihydrochalcone from Piper aduncum L.

PubMed

Dal Picolo, Camilla R; Bezerra, Mariana P; Gomes, Kaio S; Passero, Luiz Felipe D; Laurenti, Marcia D; Martins, Euder Glendes A; Sartorelli, Patricia; Lago, João Henrique G

2014-09-01

Bioactivity-guided fractionation of EtOH extract from the leaves of Piper aduncum L. (Piperaceae) afforded a new dihydrochalcone, named adunchalcone. Its structure was elucidated on the basis of their spectroscopic data, primarily NMR and MS. Adunchalcone was evaluated against promastigote forms of Leishmania (L.) amazonensis, L. (V.) braziliensis, L. (V.) shawi, and L. (L.) chagasi and displayed 50% effective concentrations (EC50) of 11.03, 26.70, and 11.26 μM, as well as selective indexes of 4.86, 2.01, 4.76 and 0.50, respectively. This compound was also tested against intracellular forms of L. (L.) amazonensis, displaying weak activity, in comparison to reference drug amphotericin B. However, despite reduced effect of adunchalcone against amastigotes of L. (L.) amazonensis, this work opens the perspective to use this particular molecule as a scaffold for the design of novel and selective drug candidates for neglected diseases, mainly leishmaniasis. Copyright © 2014 Elsevier B.V. All rights reserved.

Anti-Trypanosoma, anti-Leishmania and cytotoxic activities of natural products from Psidium brownianum Mart. ex DC. and Psidium guajava var. Pomifera analysed by LC-MS.

PubMed

de Souza, Celestina Elba Sobral; da Silva, Ana Raquel Pereira; Gomez, Maria Celeste Vega; Rolóm, Míriam; Coronel, Cathia; da Costa, José Galberto Martins; Sousa, Amanda K; Rolim, Larissa A; de Souza, Francisco Hugo Sobral; Coutinho, Henrique Douglas Melo

2017-12-01

Neglected diseases are those that are prevalent in developing countries, even with a rich biodiversity. These diseases still persist because of the lack of scientific studies, government negligence or failures of the public health system. This study aims to identify the composition of extracts and fractions from Psidium brownianum and Psidium guajava through LC-MS, to evaluate its in vitro anti-parasitic and cytotoxic activity against Trypanosoma cruzi, Leishmania brasiliensis and L. infantum epismastigote and promastigote forms, as well as mammalian cells. The results showed the presence of chemical constituents in the two Psidium species as quercetin, myricetin and gallic acid derivatives. The P. brownianum extract and fractions showed low toxicity at all tested concentrations and all samples were effective at the concentration of 1000μg/mL against the parasites, with the extract being the most efficient against the L. infantum promastigote form. The ethanolic extract, and the flavonoid and tannic fractions, from P. guajava showed low toxicity for the fibroblasts. All samples showed effectiveness at the highest concentration tested and the extract was more effective against the promastigote forms tested. The results showed that the species Psidium brownianum and Psidium guajava demonstrated an anti-parasitic activity against the T. cruzi, L. brasiliensis and L. infantum parasite cell lines indicating these species as an alternative therapy given their efficacy in the in vitro assays performed, opening the possibility for new biological studies to further this knowledge through in vivo assays. Copyright © 2017 Elsevier B.V. All rights reserved.

Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs

PubMed Central

Petitdidier, Elodie; Pagniez, Julie; Papierok, Gérard; Vincendeau, Philippe; Lemesre, Jean-Loup; Bras-Gonçalves, Rachel

2016-01-01

Preventive vaccination is a highly promising strategy for interrupting leishmaniasis transmission that can, additionally, contribute to elimination. A vaccine formulation based on naturally excreted secreted (ES) antigens was prepared from L. infantum promastigote culture supernatant. This vaccine achieved successful results in Phase III trials and was licensed and marketed as CaniLeish. We recently showed that newly identified ES promastigote surface antigen (PSA), from both viable promastigotes and axenically-grown amastigotes, represented the major constituent and the highly immunogenic antigen of L. infantum and L. amazonensis ES products. We report here that three immunizations with either the recombinant ES LaPSA-38S (rPSA) or its carboxy terminal part LaPSA-12S (Cter-rPSA), combined with QA-21 as adjuvant, confer high levels of protection in naive L. infantum-infected Beagle dogs, as checked by bone marrow parasite absence in respectively 78.8% and 80% of vaccinated dogs at 6 months post-challenge. The parasite burden in infected vaccinated dogs was significantly reduced compared to placebo group, as measured by q-PCR. Moreover, our results reveal humoral and cellular immune response clear-cut differences between vaccinated and control dogs. An early increase in specific IgG2 antibodies was observed in rPSA/QA-21- and Cter-rPSA/QA-21-immunized dogs only. They were found functionally active in vitro and were highly correlated with vaccine protection. In vaccinated protected dogs, IFN-γ and NO productions, as well as anti-leishmanial macrophage activity, were increased. These data strongly suggest that ES PSA or its carboxy-terminal part, in recombinant forms, induce protection in a canine model of zoonotic visceral leishmaniasis by inducing a Th1-dominant immune response and an appropriate specific antibody response. These data suggest that they could be considered as important active components in vaccine candidates. PMID:27223609

Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs.

PubMed

Petitdidier, Elodie; Pagniez, Julie; Papierok, Gérard; Vincendeau, Philippe; Lemesre, Jean-Loup; Bras-Gonçalves, Rachel

2016-05-01

Preventive vaccination is a highly promising strategy for interrupting leishmaniasis transmission that can, additionally, contribute to elimination. A vaccine formulation based on naturally excreted secreted (ES) antigens was prepared from L. infantum promastigote culture supernatant. This vaccine achieved successful results in Phase III trials and was licensed and marketed as CaniLeish. We recently showed that newly identified ES promastigote surface antigen (PSA), from both viable promastigotes and axenically-grown amastigotes, represented the major constituent and the highly immunogenic antigen of L. infantum and L. amazonensis ES products. We report here that three immunizations with either the recombinant ES LaPSA-38S (rPSA) or its carboxy terminal part LaPSA-12S (Cter-rPSA), combined with QA-21 as adjuvant, confer high levels of protection in naive L. infantum-infected Beagle dogs, as checked by bone marrow parasite absence in respectively 78.8% and 80% of vaccinated dogs at 6 months post-challenge. The parasite burden in infected vaccinated dogs was significantly reduced compared to placebo group, as measured by q-PCR. Moreover, our results reveal humoral and cellular immune response clear-cut differences between vaccinated and control dogs. An early increase in specific IgG2 antibodies was observed in rPSA/QA-21- and Cter-rPSA/QA-21-immunized dogs only. They were found functionally active in vitro and were highly correlated with vaccine protection. In vaccinated protected dogs, IFN-γ and NO productions, as well as anti-leishmanial macrophage activity, were increased. These data strongly suggest that ES PSA or its carboxy-terminal part, in recombinant forms, induce protection in a canine model of zoonotic visceral leishmaniasis by inducing a Th1-dominant immune response and an appropriate specific antibody response. These data suggest that they could be considered as important active components in vaccine candidates.

VOSalophen: a vanadium complex with a stilbene derivative-induction of apoptosis, autophagy, and efficiency in experimental cutaneous leishmaniasis.

PubMed

Machado, Patrícia de A; Morais, Jessica O F; Carvalho, Gustavo S G; Lima, Wallace P; Macedo, Gilson C; Britta, Elizandra A; Nakamura, Celso V; da Silva, Adilson D; Cuin, Alexandre; Coimbra, Elaine S

2017-08-01

In our previous work, we demonstrated the promising in vitro effect of VOSalophen, a vanadium complex with a stilbene derivative, against Leishmania amazonensis. Its antileishmanial activity has been associated with oxidative stress in L. amazonensis promastigotes and L. amazonensis-infected macrophages. In the present study, the mechanism involved in the death of parasites after treatment with VOSalophen, as well as in vivo effect in the murine model cutaneous leishmaniasis, has been investigated. Promastigotes of L. amazonensis treated with VOSalophen presented apoptotic cells features, such as cell volume decrease, phosphatidylserine externalization, and DNA fragmentation. An increase in autophagic vacuoles formation in treated promastigotes was also observed, showing that autophagy also may be involved in the death of these parasites. In intracellular amastigotes, DNA fragmentation was observed after treatment with VOSalophen, but this effect was not observed in host cells, highlighting the selective effect of this vanadium complex. In addition, VOSalophen showed activity in the murine model of cutaneous leishmaniasis, without hepatic and renal damages. The outcome described here points out that VOSalophen had promising antileishmanial properties and these data also contribute to the understanding of the mechanisms involved in the death of protozoa induced by metal complexes.

Leishmania sand fly interaction: progress and challenges.

PubMed

Bates, Paul A

2008-08-01

Complex interactions occurs between Leishmania parasites and their sand fly vectors. Promastigotes of Leishmania live exclusively within the gut, possess flagella and are motile, and kinesins, kinases and G proteins have been described that play a role in regulating flagellar assembly. Movement within the gut is not random: promastigotes can detect gradients of solutes via chemotaxis and osmotaxis. Further they use their flagella to attach to the fly midgut using surface glyconconjugates, a key step in establishment of the infection. Differentiation of mammal-infective stages is characterised by significant biochemical and cellular remodelling. Further, the parasites can manipulate the behaviour of the vector to maximise their transmission, and flies may even deliver altruistic apoptotic forms to aid transmission of infective stages.

Regulated Degradation of an Endoplasmic Reticulum Membrane Protein in a Tubular Lysosome in Leishmania mexicana

PubMed Central

Mullin, Kylie A.; Foth, Bernardo J.; Ilgoutz, Steven C.; Callaghan, Judy M.; Zawadzki, Jody L.; McFadden, Geoffrey I.; McConville, Malcolm J.

2001-01-01

The cell surface of the human parasite Leishmania mexicana is coated with glycosylphosphatidylinositol (GPI)-anchored macromolecules and free GPI glycolipids. We have investigated the intracellular trafficking of green fluorescent protein- and hemagglutinin-tagged forms of dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis in L. mexicana promastigotes. These functionally active chimeras are found in the same subcompartment of the endoplasmic reticulum (ER) as endogenous DPMS but are degraded as logarithmically growing promastigotes reach stationary phase, coincident with the down-regulation of endogenous DPMS activity and GPI biosynthesis in these cells. We provide evidence that these chimeras are constitutively transported to and degraded in a novel multivesicular tubule (MVT) lysosome. This organelle is a terminal lysosome, which is labeled with the endocytic marker FM 4-64, contains lysosomal cysteine and serine proteases and is disrupted by lysomorphotropic agents. Electron microscopy and subcellular fractionation studies suggest that the DPMS chimeras are transported from the ER to the lumen of the MVT via the Golgi apparatus and a population of 200-nm multivesicular bodies. In contrast, soluble ER proteins are not detectably transported to the MVT lysosome in either log or stationary phase promastigotes. Finally, the increased degradation of the DPMS chimeras in stationary phase promastigotes coincides with an increase in the lytic capacity of the MVT lysosome and changes in the morphology of this organelle. We conclude that lysosomal degradation of DPMS may be important in regulating the cellular levels of this enzyme and the stage-dependent biosynthesis of the major surface glycolipids of these parasites. PMID:11514622

Evaluation of Leishmania species reactivity in human serologic diagnosis of leishmaniasis.

PubMed

Silvestre, Ricardo; Santarém, Nuno; Teixeira, Lúcia; Cunha, Joana; Schallig, Henk; Cordeiro-da-Silva, Anabela

2009-08-01

The sensitivities and specificities of IgG-ELISA and IgG flow cytometry based techniques using different Leishmania species were determined using sera collected from 40 cutaneous or visceral leishmaniasis patients. The flow cytometry technique, using promastigote parasite forms, performed better than total soluble extract IgG-ELISA. At the species level, the use of Leishmania amazonensis and Leishmania major as antigens in enzyme linked immunosorbent assay (ELISA) decreased the overall sensitivity. To assess the specificity of these tests, sera from malaria, toxoplasmosis, amoebiasis, schistosomiasis, and leprosy patients were used. We also included sera from Leishmania non-infected endemic individuals. The cutaneous species displayed a decreased specificity in both assays. Although more sensitive, flow cytometry using promastigote parasite forms generally presented lower levels of specificity when compared with total extract of IgG-ELISA. Overall, the results of the study show the potential of IgG flow cytometry for the diagnosis of leishmaniasis. Although highly sensitive, a refinement of the flow cytometry method should be performed to improve the overall specificity.

Sinergism between alkaloids piperine and capsaicin with meglumine antimoniate against Leishmania infantum.

PubMed

Vieira-Araújo, Francisco Marcelo; Macedo Rondon, Fernanda Cristina; Pinto Vieira, Ícaro Gusmão; Pereira Mendes, Francisca Noelia; Carneiro de Freitas, José Claudio; Maia de Morais, Selene

2018-05-01

The primary choice of drugs to treat Leishmaniasis are the pentavalent antimony-based compounds, nevertheless these drugs presented undesirable side effects. However, safe natural compounds could be used in combination with these drugs to enhance their activity. The aim of this study was to evaluate the sinergism of capsaicin and piperine, isolated from Capsicum frutescens and Piper nigrum, respectively, in combination with meglumine antimoniate against Leishmania infantum promastigote and amastigote forms. Each compound was mixed with the standard drug in several percentage mixtures and tested at various concentrations. Capsaicin and piperine in combination with meglumine antimoniate (25% + 75%) showed better anti-leishmanial activity with EC 50  = 4.31 ± 0.44 e 7.25 ± 4.84 μg/mL against promastigote and amastigote forms, respectively. The results point that these spice alkaloids are suitable compounds to be administered in combinations with antileishmanial drugs to improve their action. Copyright © 2018 Elsevier Inc. All rights reserved.

The potent cell permeable calpain inhibitor MDL28170 affects the interaction of Leishmania amazonensis with macrophages and shows anti-amastigote activity.

PubMed

Marinho, Fernanda A; Sangenito, Leandro S; Oliveira, Simone S C; De Arruda, Luciana B; D'Ávila-Levy, Claudia M; Santos, André L S; Branquinha, Marta H

2017-10-01

Since the discovery of the28 first drugs used in leishmaniasis treatment up to now, the search for compounds with anti-Leishmania activity without toxic effects and able to overcome the emergency of resistant strains remains a major goal to combat this neglected disease. With this in mind, in the present work, we evaluated the effects of the calpain inhibitor MDL28170 on the interaction process of Leishmania amazonensis promastigote forms with murine peritoneal macrophages and on the intracellular amastigotes. Our results showed that the calpain inhibitor MDL28170 at 15 and 30μM significantly reduced the interaction process of promastigotes with macrophages by 16% and 41%, respectively. The inhibitor was also able to drastically reduce the number of infected macrophages in a time- and dose-dependent manner: after only 24h, MDL28170 was able to significantly diminish the infection rate, presenting an IC 50 value of 18.2μM for amastigotes. The treatment with MDL28170 did not alter the nitric oxide production, but the production of TNF-α was significantly raised. Altogether, the results presented here contribute to the search of new proteolytic inhibitors able to act in a selective and effective manner against the diseases caused by trypanosomatids. Copyright © 2017 Elsevier B.V. All rights reserved.

Increased Glycolytic ATP Synthesis Is Associated with Tafenoquine Resistance in Leishmania major▿

PubMed Central

Manzano, José Ignacio; Carvalho, Luis; Pérez-Victoria, José M.; Castanys, Santiago; Gamarro, Francisco

2011-01-01

Tafenoquine (TFQ), an 8-aminoquinoline used to treat and prevent Plasmodium infections, could represent an alternative therapy for leishmaniasis. Indeed, TFQ has shown significant leishmanicidal activity both in vitro and in vivo, where it targets Leishmania mitochondria and activates a final apoptosis-like process. In order not to jeopardize the life span of this potential antileishmania drug, it is important to determine the likelihood that Leishmania will develop resistance to TFQ and the mechanisms of resistance induced. To address this issue, a TFQ-resistant Leishmania major promastigote line (R4) was selected. This resistance, which is unstable in a drug-free medium (revertant line), was maintained in intramacrophage amastigote forms, and R4 promastigotes were found to be cross-resistant to other 8-aminoquinolines. A decreased TFQ uptake, which is probably associated with an alkalinization of the intracellular pH rather than drug efflux, was observed for both the R4 and revertant lines. TFQ induces a decrease in ATP synthesis in all Leishmania lines, although total ATP levels were maintained at higher values in R4 parasites. In contrast, ATP synthesis by glycolysis was significantly increased in R4 parasites, whereas mitochondrial ATP synthesis was similar to that in wild-type parasites. We therefore conclude that increased glycolytic ATP synthesis is the main mechanism underlying TFQ resistance in Leishmania. PMID:21199921

Increased glycolytic ATP synthesis is associated with tafenoquine resistance in Leishmania major.

PubMed

Manzano, José Ignacio; Carvalho, Luis; Pérez-Victoria, José M; Castanys, Santiago; Gamarro, Francisco

2011-03-01

Tafenoquine (TFQ), an 8-aminoquinoline used to treat and prevent Plasmodium infections, could represent an alternative therapy for leishmaniasis. Indeed, TFQ has shown significant leishmanicidal activity both in vitro and in vivo, where it targets Leishmania mitochondria and activates a final apoptosis-like process. In order not to jeopardize the life span of this potential antileishmania drug, it is important to determine the likelihood that Leishmania will develop resistance to TFQ and the mechanisms of resistance induced. To address this issue, a TFQ-resistant Leishmania major promastigote line (R4) was selected. This resistance, which is unstable in a drug-free medium (revertant line), was maintained in intramacrophage amastigote forms, and R4 promastigotes were found to be cross-resistant to other 8-aminoquinolines. A decreased TFQ uptake, which is probably associated with an alkalinization of the intracellular pH rather than drug efflux, was observed for both the R4 and revertant lines. TFQ induces a decrease in ATP synthesis in all Leishmania lines, although total ATP levels were maintained at higher values in R4 parasites. In contrast, ATP synthesis by glycolysis was significantly increased in R4 parasites, whereas mitochondrial ATP synthesis was similar to that in wild-type parasites. We therefore conclude that increased glycolytic ATP synthesis is the main mechanism underlying TFQ resistance in Leishmania.

Attenuation of Leishmania infantum chagasi Metacyclic Promastigotes by Sterol Depletion

PubMed Central

Gaur Dixit, Upasna; Barker, Jason H.; Teesch, Lynn M.; Love-Homan, Laurie; Donelson, John E.; Wilson, Mary E.

2013-01-01

The infectious metacyclic promastigotes of Leishmania protozoa establish infection in a mammalian host after they are deposited into the dermis by a sand fly vector. Several Leishmania virulence factors promote infection, including the glycosylphosphatidylinositol membrane-anchored major surface protease (MSP). Metacyclic Leishmania infantum chagasi promastigotes were treated with methyl-beta-cyclodextrin (MβCD), a sterol-chelating reagent, causing a 3-fold reduction in total cellular sterols as well as enhancing MSP release without affecting parasite viability in vitro. MβCD-treated promastigotes were more susceptible to complement-mediated lysis than untreated controls and reduced the parasite load 3-fold when inoculated into BALB/c mice. Paradoxically, MβCD-treated promastigotes caused a higher initial in vitro infection rate in human or murine macrophages than untreated controls, although their intracellular multiplication was hindered upon infection establishment. There was a corresponding larger amount of covalently bound C3b than iC3b on the parasite surfaces of MβCD-treated promastigotes exposed to healthy human serum in vitro, as well as loss of MSP, a protease that enhances C3b cleavage to iC3b. Mass spectrometry showed that MβCD promotes the release of proteins into the extracellular medium, including both MSP and MSP-like protein (MLP), from virulent metacyclic promastigotes. These data support the hypothesis that plasma membrane sterols are important for the virulence of Leishmania protozoa at least in part through retention of membrane virulence proteins. PMID:23630964

Leishmanicidal activity in vitro of Musa paradisiaca L. and Spondias mombin L. fractions.

PubMed

Accioly, Marina Parissi; Bevilaqua, Claudia Maria Leal; Rondon, Fernanda C M; de Morais, Selene Maia; Machado, Lyeghyna K A; Almeida, Camila A; de Andrade, Heitor Franco; Cardoso, Roselaine P A

2012-06-08

Visceral leishmaniasis (VL) is a zoonotic disease characterized by infection of mononuclear phagocytes by Leishmania chagasi. The primary vector is Lutzomyia longipalpis and the dog is the main domestic reservoir. The control and current treatment of dogs using synthetic drugs have not shown effectiveness in reducing the incidence of disease in man. In attempt to find new compounds with leishmanicidal action, plant secondary metabolites have been studied in search of treatments of VL. This study aimed to evaluate the leishmanicidal activity of Musa paradisiaca (banana tree) and Spondias mombin (cajazeira) chemical constituents on promastigotes and amastigotes of L. chagasi. Phytochemical analysis by column chromatography was performed on ethanol extracts of two plants and fractions were isolated. Thin layer chromatography was used to compare the fractions and for isolation the substances to be used in vitro tests. The in vitro tests on promastigotes of L. chagasi used the MTT colorimetric method and the method of ELISA in situ was used against amastigotes besides the cytotoxicity in RAW 264.7 cells. Of the eight fractions tested, Sm1 and Sm2 from S. mombin had no action against promastigotes, but had good activity against amastigotes. The fractions Mp1 e Mp4 of M. paradisiaca were very cytotoxic to RAW 264.7 cells. The best result was obtained with the fraction Sm3 from S. mombin with IC(50) of 11.26 μg/ml against promastigotes and amastigotes of 0.27 μg/ml. The fraction Sm3 characterized as tannic acid showed the best results against both forms of Leishmania being a good candidate for evaluation in in vivo tests. Copyright © 2012. Published by Elsevier B.V.

Cytotoxic Activity of Holothuria leucospilota Extract against Leishmania infantum In Vitro

PubMed Central

Khademvatan, Shahram; Eskandari, Alborz; Saki, Jasem; Foroutan-Rad, Masoud

2016-01-01

Leishmaniasis is a tropical parasitic infection. The resistance and toxicity issues are the major complications and remain significant consequences related to the treatment of leishmaniasis with the recent and classical drugs. Thus there is an immediate requirement to develop new compounds for the treatment of this protozoan disease. Sea cucumbers or holothurians are potentially presented as the marine sources of antimicrobial and cytotoxic compounds. The aim of this study was investigation of in vitro antileishmanial activity of methanol extract of body wall, coelomic fluid, and cuvierian organs of Holothuria leucospilota obtained from coastal parts of Persian Gulf against Leishmania infantum promastigotes and axenic amastigotes. The colorimetric MTT assay was used to determine L. infantum promastigotes and axenic amastigotes viability at different concentrations of the extracts and drug control (Glucantime®) at time dependent manner and the results are represented as IC50 (50% of inhibitory concentration). Coelomic fluid was the most active extract among the three different extracts of H. leucospilota against L. infantum promastigotes and axenic amastigotes with IC50s of 62.33 μg/mL and 22.4 μg/mL and 73 μg/mL and 46 μg/mL at 48 and 72 hours after treatment, respectively. Cuvierian organs extract showed less toxicity with IC50s more than 1000 μg/mL for both Leishmania infantum axenic amastigotes and promastigotes forms after 48 and 72 hours of exposure. Results acquired from the present study propose that the sea cucumber H. leucospilota may be a provoking source of antileishmanial compounds and could be a lead source in the development of the potent antileishmanial and cytotoxic drugs. PMID:27022392

Photoacoustic monitoring of life cycles of Leishmania Mexicana

NASA Astrophysics Data System (ADS)

Arguello, C.; Acosta-Avalos, D.; Alvarado-Gil, J. J.; Vargas, H.

1999-03-01

Photoacoustic spectroscopy is used to monitor in situ, the difference between the two forms of the protozoan Leishmania Mexicana. Differences are the result of changes in the respiratory chain and could be attributed, according to our results, to the presence of cytochrome b in promastigotes and cytochrome c in amastigotes.

In vitro antileishmanial activity of Mexican medicinal plants.

PubMed

Delgado-Altamirano, Ronna; Monzote, Lianet; Piñón-Tápanes, Abel; Vibrans, Heike; Rivero-Cruz, J Fausto; Ibarra-Alvarado, César; Rojas-Molina, Alejandra

2017-09-01

To evaluate the anti-leishmanial activity and cytotoxicity of aqueous and organic extracts of ten plants used in Mexican traditional medicine as anti-parasitics. For the organic extracts, plant material was macerated in dichloromethane (CH 2 Cl 2 ) and dichloromethane/methanol (CH 2 Cl 2 /MeOH) (1:1) during two weeks; the aqueous extracts were prepared by infusion. The extracts were tested against promastigotes and intracellular amastigotes of Leishmania amazonensis . The cytotoxicity was assayed in parallel on peritoneal macrophages of BALB/c mice. Four of the thirty extracts tested were active and selective against L. amazonensis promastigotes: Schinus molle (CH 2 Cl 2 and CH 2 Cl 2 /MeOH), Lantana camara (CH 2 Cl 2 ) and Prosopis laevigata (aqueous). These extracts had a median inhibitory concentration (IC 50 ) against intracellular amastigotes under 50 μg/mL and a selectivity index (SI) higher than 5, which indicates that they constitute valuable candidates to obtain secondary metabolites with leishmanicidal activity. The results derived from this study indicate that L. camara, P. laevigata, and S. molle might provide interesting new leads for the development of antileishmanial drugs.

Spectroscopic study of antileishmanial drug incubated in the promastigotes of Leishmania mexicana

NASA Astrophysics Data System (ADS)

Hung, J.; Castillo, J.; Jiménez, G.; Hasegawa, M.; Rodriguez, M.

2003-11-01

In this work we present spectroscopic study of Boldine (aporphine alkaloid) that possesses important biological activities, in particular, in interaction with the promastigotes of Leishmania mexicana. The results show the applicability of autofluorescence of this drug to determinate the possible mechanism of its biological action. The blue shift and hyperchromic effect in the emission spectrum of the drug in interaction with the parasite cells indicate an energy transference process between them. The morphological change of cell shape of the promastigotes treated with the drug is observed using confocal microscopy. This morphological cell-shape transformation evidences an important interaction between the drug studied and some protein of the parasite cell. Here we describe for the first time the fluorescence properties of the Boldine in the promastigotes of L. mexicana.

In vitro permissiveness of bovine neutrophils and monocyte derived macrophages to Leishmania donovani of Ethiopian isolate.

PubMed

Tasew, Geremew; Gadisa, Endalamaw; Abera, Adugna; Zewude, Aboma; Chanyalew, Menberework; Aseffa, Abraham; Abebe, Markos; Ritter, Uwe; van Zandbergen, Ger; Laskay, Tamás; Tafess, Ketema

2016-04-18

Epidemiological studies in Ethiopia have documented that the risk of visceral leishmaniasis (VL, Kala-azar) is higher among people living with domestic animals. The recent report on isolation of Leishmania donovani complex DNA and the detected high prevalence of anti-leishmanial antibodies in the blood of domestic animals further strengthen the potential role of domestic animals in the epidemiology of VL in Ethiopia. In mammalian hosts polymorphonuclear cells (PMN) and macrophages are the key immune cells influencing susceptibility or control of Leishmania infection. Thus to substantiate the possible role of cattle in VL transmission we investigate the permissiveness of bovine PMN and monocyte derived macrophages (MDM) for Leishmania (L.) donovani infection. Whole blood was collected from pure Zebu (Boss indicus) and their cross with Holstein Friesian cattle. L. donovani (MHOM/ET/67/HU3) wild and episomal green fluorescent protein (eGFP) labelled stationary stage promastigotes were co-incubated with whole blood and MDM to determine infection of these cells. Engulfment of promastigotes by the cells and their transformation to amastigote forms in MDM was studied with direct microscopy. Microscopy and flow cytometry were used to measure the infection rate while PCR-RLFP was used to confirm the infecting parasite. L. donovani infected bovine whole blood PMN in the presence of plasma factors and all cellular elements. Morphological examinations of stained cytospin smears revealed that PMN engulfed promastigotes. Similarly, we were able to show that bovine MDM can be infected by L. donovani, which transformed to amastigote forms in the cells. The in vitro infection of bovine PMN and MDM by L. donovani further strengthens the possibility that cattle might serve as source of L. donovani infection for humans.

Kinetics and molecular characteristics of arginine transport by Leishmania donovani promastigotes.

PubMed

Kandpal, M; Fouce, R B; Pal, A; Guru, P Y; Tekwani, B L

1995-05-01

Characteristics of transport of L-arginine were studied in Leishmania donovani promastigotes grown in vitro in a defined medium. The promastigotes exhibited a time-dependent, temperature-sensitive, pH-dependent and saturable uptake of arginine. Metabolic inhibitors caused 81-92% inhibition, indicating that arginine influx in promastigotes is an energy requiring process. The presence of Na+ ions was necessary for full activity. Considerable inhibition was also noticed with valinomycin, gramicidin and amiloride. The transporter seems to involve an -SH group at the active site. The most distinctive feature of the leishmanial transporter was that lysine and ornithine did not show significant competition with arginine transport. Other neutral and acidic amino acids, as well as polyamines were also ineffective. The arginine analogues, viz., nitro-L-arginine methyl ester, N-nitro-L-arginine, aminoguanidine, agmatine and D-arginine were not recognised by the transporter, while N-methyl-L-arginine acetate and phospho-L-arginine showed competition, indicating stereo-specificity of the transporter and recognition of both the guanidino group, as well as the arginine side chain by the transporter. No exchange of intracellular [14C]arginine taken up by the promastigotes was noticed during incubation with 2 or 5 mM arginine in the extracellular medium. Eighty percent of the arginine taken up remained in the trichloroacetic acid-soluble fraction. Pentamidine caused competitive inhibition of arginine transport, exhibiting an IC50 value of 40 microM. Results indicate the presence of a novel distinct arginine transporter in Leishmania promastigotes.

Curcumin overcomes the inhibitory effect of nitric oxide on Leishmania.

PubMed

Chan, Marion Man-Ying; Adapala, Naga Suresh; Fong, Dunne

2005-04-01

Upon Leishmania infection, macrophages are activated to produce nitrogen and oxygen radicals simultaneously. It is well established that the infected host cells rely on nitric oxide (NO) as the major weapon against the intracellular parasite. In India where leishmaniasis is endemic, the spice turmeric is used prolifically in food and for insect bites. Curcumin, the active principle of turmeric, is a scavenger of NO. This report shows that curcumin protects promastigotes and amastigotes of the visceral species, Leishmania donovani, and promastigotes of the cutaneous species, L. major, against the actions of S-nitroso-N-acetyl-D,L-penicillamine (SNAP) and DETANONOate, which release NO, 3-morpholino-sydnonimine hydrochloride (SIN-1), which releases NO and superoxide, and peroxynitrite, which is formed from the reaction of NO with superoxide. Thus, curcumin, as an antioxidant, is capable of blocking the action of both NO and NO congeners on the Leishmania parasite.

P-type proton ATPases are involved in intracellular calcium and proton uptake in the plant parasite Phytomonas francai.

PubMed

Miranda, Kildare; Vercesi, Anibal E; Catisti, Rosana; De Souza, Wanderley; Rodrigues, Claudia O; Docampo, Roberto

2005-01-01

The use of digitonin to permeabilize the plasma membrane of promastigotes of Phytomonas francai allowed the identification of two non-mitochondrial Ca(2+) compartments; one sensitive to ionomycin and vanadate (neutral or alkaline), possibly the endoplasmic reticulum, and another sensitive to the combination of nigericin plus ionomycin (acidic), possibly the acidocalcisomes. A P-type (phospho-intermediate form) Ca(2+)-ATPase activity was found to be responsible for intracellular Ca(2+) transport in these cells, with no evidence of a mitochondrial Ca(2+) transport activity. ATP-driven acidification of internal compartments in cell lysates and cells mechanically permeabilized was assayed spectrophotometrically with acridine orange. This activity was inhibited by low concentrations of vanadate and digitonin, was insensitive to bafilomycin A(1), and stimulated by Na(+) ions. Taken together, our results indicate that P-type ATPases are involved in intracellular Ca(2+) and H(+) transport in promastigotes of P. francai.

The proliferation potential of promastigotes of the main Leishmania species of the old world in NNN culture medium prepared using blood of four different mammals.

PubMed

Ladopoulos, Theodoros; Ntais, Pantelis; Tsirigotakis, Nikolaos; Dokianakis, Emmanouil; Antoniou, Maria

2015-10-01

The efficacy of the in vitro cultivation of promastigotes of four Leishmania spp. was tested in the biphasic Novy-MacNeal-Nicolle (NNN) medium prepared using blood from different animals (horse, donkey, goat and sheep). The aim was to test which NNN preparation gave the best yield in the shortest time for different parasite species, in order to obtain a large crop of promastigotes for experimental work and for antigen preparation. Promastigotes of Leishmania infantum, Leishmania donovani, Leishmania tropica and Leishmania major, the four main parasite species occurring in the old world, were defrosted from -80 °C and placed, at equal numbers, in the 4 different NNN preparations. At the end of the 7th day, the NNN medium using horse blood produced the greatest number of promastigotes for all Leishmania spp. tested, whilst goat blood proved the poorest medium, providing culture results only for L. infantum. This finding may be explained by the fact that Leishmania is a nicotinamide adenine dinucleotide (NAD) auxotroph and horse erythrocytes support NAD-dependent microorganisms. Copyright © 2015 Elsevier Inc. All rights reserved.

In Vitro and In Vivo Activities of 2,3-Diarylsubstituted Quinoxaline Derivatives against Leishmania amazonensis

PubMed Central

Kaplum, Vanessa; Cogo, Juliana; Sangi, Diego Pereira; Ueda-Nakamura, Tânia; Corrêa, Arlene Gonçalves

2016-01-01

Leishmaniasis is endemic in 98 countries and territories worldwide. The therapies available for leishmaniasis have serious side effects, thus prompting the search for new therapies. The present study investigated the antileishmanial activities of 2,3-diarylsubstituted quinoxaline derivatives against Leishmania amazonensis. The antiproliferative activities of 6,7-dichloro-2,3-diphenylquinoxaline (LSPN329) and 2,3-di-(4-methoxyphenyl)-quinoxaline (LSPN331) against promastigotes and intracellular amastigotes were assessed, and the cytotoxicities of LSPN329 and LSPN331 were determined. Morphological and ultrastructural alterations were examined by electron microscopy, and biochemical alterations, reflected by the mitochondrial membrane potential (ΔΨm), mitochondrial superoxide anion (O2·−) concentration, the intracellular ATP concentration, cell volume, the level of phosphatidylserine exposure on the cell membrane, cell membrane integrity, and lipid inclusions, were evaluated. In vivo antileishmanial activity was evaluated in a murine cutaneous leishmaniasis model. Compounds LSPN329 and LSPN331 showed significant selectivity for promastigotes and intracellular amastigotes and low cytotoxicity. In promastigotes, ultrastructural alterations were observed, including an increase in lipid inclusions, concentric membranes, and intense mitochondrial swelling, which were associated with hyperpolarization of ΔΨm, an increase in the O2·− concentration, decreased intracellular ATP levels, and a decrease in cell volume. Phosphatidylserine exposure and DNA fragmentation were not observed. The cellular membrane remained intact after treatment. Thus, the multifactorial response that was responsible for the cellular collapse of promastigotes was based on intense mitochondrial alterations. BALB/c mice treated with LSPN329 or LSPN331 showed a significant decrease in lesion thickness in the infected footpad. Therefore, the antileishmanial activity and mitochondrial mechanism of action of LSPN329 and LSPN331 and the decrease in lesion thickness in vivo brought about by LSPN329 and LSPN331 make them potential candidates for new drug development for the treatment of leishmaniasis. PMID:27001812

A defined medium for Leishmania culture allows definition of essential amino acids.

PubMed

Nayak, Archana; Akpunarlieva, Snezhana; Barrett, Michael; Burchmore, Richard

2018-02-01

Axenic culture of Leishmania is generally performed in rich, serum-supplemented media which sustain robust growth over multiple passages. The use of such undefined media, however, obscures proteomic analyses and confounds the study of metabolism. We have established a simple, defined culture medium that supports the sustained growth of promastigotes over multiple passages and which yields parasites that have similar infectivity to macrophages to parasites grown in a conventional semi-defined medium. We have exploited this medium to investigate the amino acid requirements of promastigotes in culture and have found that phenylalanine, tryptophan, arginine, leucine, lysine and valine are essential for viability in culture. Most of the 20 proteogenic amino acids promote growth of Leishmania promastigotes, with the exception of alanine, asparagine, and glycine. This defined medium will be useful for further studies of promastigote substrate requirements, and will facilitate future proteomic and metabolomic analyses. Copyright © 2018 Elsevier Inc. All rights reserved.

Selective effect of 2',6'-dihydroxy-4'-methoxychalcone isolated from Piper aduncum on Leishmania amazonensis.

PubMed

Torres-Santos, E C; Moreira, D L; Kaplan, M A; Meirelles, M N; Rossi-Bergmann, B

1999-05-01

2',6'-Dihydroxy-4'-methoxychalcone (DMC) was purified from the dichloromethane extract of Piper aduncum inflorescences. DMC showed significant activity in vitro against promastigotes and intracellular amastigotes of Leishmania amazonensis, with 50% effective doses of 0.5 and 24 micrograms/ml, respectively. Its inhibitory effect on amastigotes is apparently a direct effect on the parasites and is not due to activation of the nitrogen oxidative metabolism of macrophages, since the production of nitric oxide by both unstimulated and recombinant gamma interferon-stimulated macrophages was decreased rather than increased with DMC. The phagocytic activity of macrophages was functioning normally even with DMC concentrations as high as 80 micrograms/ml, as seen by electron microscopy and by the uptake of fluorescein isothiocyanate-labeled beads. Ultrastructural studies also showed that in the presence of DMC the mitochondria of promastigotes were enlarged and disorganized. Despite destruction of intracellular amastigotes, no disarrangement of macrophage organelles were observed, even at 80 micrograms of DMC/ml. These observations suggest that DMC is selectively toxic to the parasites. Its simple structure may well enable it to serve as a new lead compound for the synthesis of novel antileishmanial drugs.

Decanethiol functionalized silver nanoparticles are new powerful leishmanicidals in vitro.

PubMed

Isaac-Márquez, A P; Talamás-Rohana, P; Galindo-Sevilla, N; Gaitan-Puch, S E; Díaz-Díaz, N A; Hernández-Ballina, G A; Lezama-Dávila, C M

2018-02-19

We evaluated, for the first time, the leishmanicidal potential of decanethiol functionalized silver nanoparticles (AgNps-SCH) on promastigotes and amastigotes of different strains and species of Leishmania: L. mexicana and L. major isolated from different patients suffering from localized cutaneous leishmaniasis (CL) and L. mexicana isolated from a patient suffering from diffuse cutaneous leishmaniasis (DCL). We recorded the kinetics of promastigote growth by daily parasite counting for 5 days, promastigote mobility, parasite reproduction by CFSE staining's protocol and promastigote killing using the propidium iodide assay. We also recorded IC 50 's of promastigotes and amastigotes, therapeutic index, and cytotoxicity by co-culturing macrophages with AgNps-SCH or sodium stibogluconate (Sb) used as reference drug. We used Sb as a reference drug since it is used as the first line treatment for all different types of leishmaniasis. At concentrations 10,000 times lower than those used with Sb, AgNps-SCH had a remarkable leishmanicidal effect in all tested strains of parasites and there was no toxicity to J774A.1 macrophages since > 85% were viable at the concentrations used. Therapeutic index was about 20,000 fold greater than the corresponding one for Sb treated cells. AgNps-SCH inhibited > 80% promastigote proliferation in all tested parasites. These results demonstrate there is a high leishmanicidal potential of AgNps-SCH at concentrations of 0.04 µM. Although more studies are needed, including in vivo testing of AgNps-SCH against different types of leishmaniasis, they can be considered a potential new treatment alternative.

Pterocarpanquinone LQB-118 induces apoptosis in Leishmania (Viannia) braziliensis and controls lesions in infected hamsters.

PubMed

Costa, Luciana; Pinheiro, Roberta O; Dutra, Patrícia M L; Santos, Rosiane F; Cunha-Júnior, Edézio F; Torres-Santos, Eduardo C; da Silva, Alcides J M; Costa, Paulo R R; Da-Silva, Silvia A G

2014-01-01

Previous results demonstrate that the hybrid synthetic pterocarpanquinone LQB-118 presents antileishmanial activity against Leishmania amazonensis in a mouse model. The aim of the present study was to use a hamster model to investigate whether LQB-118 presents antileishmanial activity against Leishmania (Viannia) braziliensis, which is the major Leishmania species related to American tegumentary leishmaniasis. The in vitro antileishmanial activity of LQB-118 on L. braziliensis was tested on the promastigote and intracellular amastigote forms. The cell death induced by LQB-118 in the L. braziliensis promastigotes was analyzed using an annexin V-FITC/PI kit, the oxidative stress was evaluated by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and the ATP content by luminescence. In situ labeling of DNA fragments by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to investigate apoptosis in the intracellular amastigotes. L. braziliensis-infected hamsters were treated from the seventh day of infection with LQB-118 administered intralesionally (26 µg/kg/day, three times a week) or orally (4,3 mg/kg/day, five times a week) for eight weeks. LQB-118 was active against the L. braziliensis promastigotes and intracellular amastigotes, producing IC50 (50% inhibitory concentration) values of 3,4±0,1 and 7,5±0,8 µM, respectively. LQB-118 induced promastigote phosphatidylserine externalization accompanied by increased reactive oxygen species production and ATP depletion. Intracellular amastigote DNA fragmentation was also observed, without affecting the viability of macrophages. The treatment of L. braziliensis-infected hamsters with LQB-118, either orally or intralesionally, was effective in the control of lesion size, parasite load and increase intradermal reaction to parasite antigen. Taken together, these results show that the antileishmanial effect of LQB-118 extends to L. braziliensis in the hamster model, involves the induction of parasite apoptosis and shows promising therapeutic option by oral or local routes in leishmaniasis.

Pterocarpanquinone LQB-118 Induces Apoptosis in Leishmania (Viannia) braziliensis and Controls Lesions in Infected Hamsters

PubMed Central

Costa, Luciana; Pinheiro, Roberta O.; Dutra, Patrícia M. L.; Santos, Rosiane F.; Cunha-Júnior, Edézio F.; Torres-Santos, Eduardo C.; da Silva, Alcides J. M.; Costa, Paulo R. R.; Da-Silva, Silvia A. G.

2014-01-01

Previous results demonstrate that the hybrid synthetic pterocarpanquinone LQB-118 presents antileishmanial activity against Leishmania amazonensis in a mouse model. The aim of the present study was to use a hamster model to investigate whether LQB-118 presents antileishmanial activity against Leishmania (Viannia) braziliensis, which is the major Leishmania species related to American tegumentary leishmaniasis. The in vitro antileishmanial activity of LQB-118 on L. braziliensis was tested on the promastigote and intracellular amastigote forms. The cell death induced by LQB-118 in the L. braziliensis promastigotes was analyzed using an annexin V-FITC/PI kit, the oxidative stress was evaluated by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and the ATP content by luminescence. In situ labeling of DNA fragments by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to investigate apoptosis in the intracellular amastigotes. L. braziliensis-infected hamsters were treated from the seventh day of infection with LQB-118 administered intralesionally (26 µg/kg/day, three times a week) or orally (4,3 mg/kg/day, five times a week) for eight weeks. LQB-118 was active against the L. braziliensis promastigotes and intracellular amastigotes, producing IC50 (50% inhibitory concentration) values of 3,4±0,1 and 7,5±0,8 µM, respectively. LQB-118 induced promastigote phosphatidylserine externalization accompanied by increased reactive oxygen species production and ATP depletion. Intracellular amastigote DNA fragmentation was also observed, without affecting the viability of macrophages. The treatment of L. braziliensis-infected hamsters with LQB-118, either orally or intralesionally, was effective in the control of lesion size, parasite load and increase intradermal reaction to parasite antigen. Taken together, these results show that the antileishmanial effect of LQB-118 extends to L. braziliensis in the hamster model, involves the induction of parasite apoptosis and shows promising therapeutic option by oral or local routes in leishmaniasis. PMID:25340550

Participation of heparin binding proteins from the surface of Leishmania (Viannia) braziliensis promastigotes in the adhesion of parasites to Lutzomyia longipalpis cells (Lulo) in vitro

PubMed Central

2012-01-01

Background Leishmania (V.) braziliensis is a causative agent of cutaneous leishmaniasis in Brazil. During the parasite life cycle, the promastigotes adhere to the gut of sandflies, to avoid being eliminated with the dejection. The Lulo cell line, derived from Lutzomyia longipalpis (Diptera: Psychodidae), is a suitable in vitro study model to understand the features of parasite adhesion. Here, we analyze the role of glycosaminoglycans (GAGs) from Lulo cells and proteins from the parasites in this event. Methods Flagellar (Ff) and membrane (Mf) fractions from promastigotes were obtained by differential centrifugation and the purity of fractions confirmed by western blot assays, using specific antibodies for cellular compartments. Heparin-binding proteins (HBP) were isolated from both fractions using a HiTrap-Heparin column. In addition, binding of promastigotes to Lulo cells or to a heparin-coated surface was assessed by inhibition assays or surface plasmon resonance (SPR) analysis. Results The success of promastigotes subcellular fractionation led to the obtainment of Ff and Mf proteins, both of which presented two main protein bands (65.0 and 55.0kDa) with affinity to heparin. The contribution of HBPs in the adherence of promastigotes to Lulo cells was assessed through competition assays, using HS or the purified HBPs fractions. All tested samples presented a measurable inhibition rate when compared to control adhesion rate (17 ± 2.0% of culture cells with adhered parasites): 30% (for HS 20μg/ml) and 16% (for HS 10μg/ml); HBP Mf (35.2% for 10μg/ml and 25.4% for 20μg/ml) and HBP Ff (10.0% for 10μg/ml and 31.4% for 20μg/ml). Additionally, to verify the presence of sulfated GAGs in Lulo cells surface and intracellular compartment, metabolic labeling with radioactive sulfate was performed, indicating the presence of an HS and chondroitin sulfate in both cell sections. The SPR analysis performed further confirmed the presence of GAGs ligands on L. (V.) braziliensis promastigote surfaces. Conclusions The data presented here point to evidences that HBPs present on the surface of L. (V.) braziliensis promastigotes participate in adhesion of these parasites to Lulo cells through HS participation. PMID:22805335

Identification of chalcone-based antileishmanial agents targeting trypanothione reductase.

PubMed

Ortalli, Margherita; Ilari, Andrea; Colotti, Gianni; De Ionna, Ilenia; Battista, Theo; Bisi, Alessandra; Gobbi, Silvia; Rampa, Angela; Di Martino, Rita M C; Gentilomi, Giovanna A; Varani, Stefania; Belluti, Federica

2018-05-02

All currently used first-line and second-line drugs for the treatment of leishmaniasis exhibit several drawbacks including toxicity, high costs and route of administration. Furthermore, some drugs are associated with the emergence of drug resistance. Thus, the development of new treatments for leishmaniasis is a priority in the field of neglected tropical diseases. The present work highlights the use of natural derived products, i.e. chalcones, as potential source of antileishmanial agents. Thirty-one novel chalcone compounds have been synthesized and their activity has been evaluated against promastigotes of Leishmania donovani; 16 compounds resulted active against L. donovani in a range from 3.0 to 21.5 μM, showing low toxicity against mammalian cells. Among these molecules, 6 and 16 showed good inhibitory activity on both promastigotes and intracellular amastigotes, coupled with an high selectivity index. Furthermore, compounds 6 and 16 inhibited the promastigote growth of other leishmanial species, including L. tropica, L. major and L. infantum. Finally, 6 and 16 interacted with high affinity with trypanothione reductase (TR), an essential enzyme for the leishmanial parasite and compound 6 inhibited TR with sub-micromolar potency. Thus, the effective inhibitory activity against Leishmania, the lack of toxicity on mammalian cells and the ability to block a crucial parasite's enzyme, highlight the potential for compound 6 to be optimized as novel drug candidate against leishmaniasis. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

In Vitro Assessment of Plants Growing in Cuba Belonging to Solanaceae Family Against Leishmania amazonensis.

PubMed

Monzote, Lianet; Jiménez, Jenny; Cuesta-Rubio, Osmany; Márquez, Ingrid; Gutiérrez, Yamile; da Rocha, Cláudia Quintino; Marchi, Mary; Setzer, William N; Vilegas, Wagner

2016-11-01

In this study, an in vitro antileishmanial assessment of plant extracts from 12 genera and 46 species growing in Cuba belonging to Solanaceae family was performed. A total of 226 extracts were screened against promastigotes of Leishmania amazonensis, and cytotoxicity of active extracts [median inhibitory concentration (IC 50 ) promastigotes <100 µg/mL] was determined on peritoneal macrophage from BALB/c mice. Extracts that showed selective index >5 were then assayed against intracellular amastigote. Metabolomics analysis of promissory extracts was performed using chemical profile obtained by ultra performance liquid chromatography. Only 11 extracts (4.9%) from nine plants were selected as potentially actives: Brunfelsia cestroides A. Rich, Capsicum annuum L., Capsicum chinense Jacq., Cestrum nocturnum L., Nicotiana plumbaginifolia Viv., Solanum havanense Jacq., Solanum myriacanthum Dunal, Solanum nudum Dunal and Solanum seaforthianum And., with IC 50  < 50 µg/mL and selectivity index >5. Metabolomics analysis demonstrated significant differences in the chemical profiles with an average of 42.8 (range 31-88) compounds from m/z 104 to 1477, which demonstrated the complex mixture of compounds. In addition, no common markers among active extracts were identified. The results demonstrate the importance of the Solanaceae family to search new antileishmanial agents, particularly in unexplored species of this family. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

In vitro and in vivo antileishmanial and trypanocidal studies of new N-benzene- and N-naphthalenesulfonamide derivatives.

PubMed

Galiana-Roselló, Cristina; Bilbao-Ramos, Pablo; Dea-Ayuela, M Auxiliadora; Rolón, Miriam; Vega, Celeste; Bolás-Fernández, Francisco; García-España, Enrique; Alfonso, Jorge; Coronel, Cathia; González-Rosende, M Eugenia

2013-11-27

We report in vivo and in vitro antileishmanial and trypanocidal activities of a new series of N-substituted benzene and naphthalenesulfonamides 1-15. Compounds 1-15 were screened in vitro against Leishmania infantum , Leishmania braziliensis , Leishmania guyanensis , Leishmania amazonensis , and Trypanosoma cruzi . Sulfonamides 6e, 10b, and 10d displayed remarkable activity and selectivity toward T. cruzi epimastigotes and amastigotes. 6e showed significant trypanocidal activity on parasitemia in a murine model of acute Chagas disease. Moreover, 6e, 8c, 9c, 12c, and 14d displayed interesting IC50 values against Leishmania spp promastigotes as well as L. amazonensis and L. infantum amastigotes. 9c showed excellent in vivo activity (up to 97% inhibition of the parasite growth) in a short-term treatment murine model for acute infection by L. infantum. In addition, the effect of compounds 9c and 14d on tubulin as potential target was assessed by confocal microscopy analysis applied to L. infantum promastigotes.

Imaging host cell-Leishmania interaction dynamics implicates parasite motility, lysosome recruitment, and host cell wounding in the infection process.

PubMed

Forestier, Claire-Lise; Machu, Christophe; Loussert, Celine; Pescher, Pascale; Späth, Gerald F

2011-04-21

Leishmania donovani causes human visceral leishmaniasis. The parasite infectious cycle comprises extracellular flagellated promastigotes that proliferate inside the insect vector, and intracellular nonmotile amastigotes that multiply within infected host cells. Using primary macrophages infected with virulent metacyclic promastigotes and high spatiotemporal resolution microscopy, we dissect the dynamics of the early infection process. We find that motile promastigotes enter macrophages in a polarized manner through their flagellar tip and are engulfed into host lysosomal compartments. Persistent intracellular flagellar activity leads to reorientation of the parasite flagellum toward the host cell periphery and results in oscillatory parasite movement. The latter is associated with local lysosomal exocytosis and host cell plasma membrane wounding. These findings implicate lysosome recruitment followed by lysosome exocytosis, consistent with parasite-driven host cell injury, as key cellular events in Leishmania host cell infection. This work highlights the role of promastigote polarity and motility during parasite entry. Copyright © 2011 Elsevier Inc. All rights reserved.

Molecular Mechanisms of In vitro Betulin-Induced Apoptosis of Leishmania donovani

PubMed Central

Saudagar, Prakash; Dubey, Vikash Kumar

2014-01-01

Although leishmanial infections of humans occur globally, the major health impact lies in developing nations, thus, leishmaniases remain “neglected” diseases for new drugs development. Multidrug resistance has been documented in most countries where leishmaniases is endemic. Betulin is a widely available and affordable natural product exerting leishmanicidal activity at micromolar concentration. In this study, the molecular mechanisms of death that contribute to the anti-leishmanial activity of betulin are investigated. In promastigotes, betulin stimulated reactive oxygen species generation at micromolar concentrations in Leishmania. Apoptosis was observed in betulin-treated promastigotes using flow cytometric analysis of treated cells stained with annexin V-FITC and propidium iodide. Furthermore, betulin treatment of promastigotes led to mitochondrial membrane damage, activation of caspase-like proteases, and DNA fragmentation in Leishmania donovani promastigotes. Betulin treatment of amastigotes cultured within macrophages, resulted in a reduced number of amastigotes, with no substantive cytotoxic damage to the host macrophage cells at leishmanicidal drug concentrations. PMID:24420777

Leishmanicidal activity of saponins isolated from the leaves of Eclipta prostrata and Gymnema sylvestre.

PubMed

Khanna, Venkatesan Gopiesh; Kannabiran, Krishnan; Getti, Giulia

2009-02-01

To evaluate the leishmanicidal activity of saponin, dasyscyphin C of Eclipta prostrata and sapogenin, gymnemagenol from Gymnema sylvestre leaves under in vitro conditions. Dasyscyphin C/Gymnemagenol were dissolved in phosphate buffered saline (PBS) and diluted with liquid medium to obtain concentrations ranging from 1000 to 15 mug /ml. The leishmanicidal activity against leishmanial parasites, Leishmania major, Leishmania aethiopica and Leishmania tropica promastigotes was studied by the MTS assay. The Dasyscyphin C isolated from E. prostrata showed good leishmanicidal activity at 1000mug/ml concentration, with the IC(50) value of 450mug/ml against L. major promastigote and the percentage of parasitic death was 73; whereas, gymnemagenol of G. sylvestre showed only 52% parasitic death at 1000 mug/ml concentration. The other Leishmania species, L. aethiopica and L. tropica promastigotes, were less sensitive to the saponins of E. prostrata and G. sylvestre. From this study, it can be concluded that the dasyscyphin C of E. prostrata has significant leishmanicidal activity against L. major promastigote.

Activity of a novel sulfonamide compound 2-nitro-N-(pyridin-2-ylmethyl)benzenesulfonamide against Leishmania donovani

PubMed Central

Dikhit, Manas R; Purkait, Bidyut; Singh, Ruby; Sahoo, Bikash Ranjan; Kumar, Ashish; Kar, Rajiv K; Ansari, Md Yousuf; Saini, Savita; Abhishek, Kumar; Sahoo, Ganesh C; Das, Sushmita; Das, Pradeep

2016-01-01

New treatments for visceral leishmaniasis, caused by Leishmania donovani, are needed to overcome sustained toxicity, cost, and drug resistance. The aim of this study was to evaluate the therapeutic effects of 2-nitro-N-(pyridin-2-ylmethyl)benzenesulfonamide (2NB) against promastigote and amastigote forms of L. donovani and examine its effect in combination with amphotericin B (AmB) against AmB-resistant clinical isolates. Effects were assessed against extracellular promastigotes in vitro and intracellular amastigotes in L. donovani-infected macrophages. Levels of inducible nitric oxide and Th1 and Th2 cytokines were measured in infected 2NB-treated macrophages, and levels of reactive oxygen species and NO were measured in 2NB-treated macrophages. 2NB was active against promastigotes and intracellular amastigotes with 50% inhibitory concentration values of 38.5±1.5 µg/mL and 86.4±2.4 µg/mL, respectively. 2NB was not toxic to macrophages. Parasite titer was reduced by >85% in infected versus uninfected macrophages at a 2NB concentration of 120 µg/mL. The parasiticidal activity was associated with increased levels of Th1 cytokines, NO, and reactive oxygen species. Finally, 2NB increased the efficacy of AmB against AmB-resistant L. donovani. These results demonstrate 2NB to be an antileishmanial agent, opening up a new avenue for the development of alternative chemotherapies against visceral leishmaniasis. PMID:27307706

Efficacy of the photodynamic antimicrobial therapy (PACT) with the use of methylene blue associated with the λ660nm laser in Leishmania (Leishmania) amazonesis: in vitro study

NASA Astrophysics Data System (ADS)

Pires-Santos, Gustavo M.; Marques, Aparecida M. C.; Alves, Eliomara S. S.; Oliveira, Susana C. P. S.; Monteiro, Juliana S. C.; Rosa, Cristiane B.; Colombo, Fabio; Pinheiro, Antônio L. B.; Vannier-Santos, Marcos A.

2012-03-01

The present studied evaluated the in vitro effects of PDT on Leishmania (Leishmania) amazonensis promastigotes. For this examination L. amazonensis promastigotes, stain Josefa, were used and maintained in Warren media supplement with fetal bovine serum at 26°C for 96 hours. A viability curve was accomplished using different concentrations of methylene blue photosensitizer associated to red laser light in order to obtain the most effective interaction to inhibit the parasite's growth. Two pre-irradiation periods, 5 and 30 minutes, were evaluated and the promastigotes were counted by colorimetry. On fluorescence microscopy the autophagic processes and reactive oxygen species were detected. Promastigotes treated with Photodynamic Therapy (PDT) by concentrations of 5 and 0,315ug/mL, presented cellular proliferation inhibition when compared to the control. In the first condition, the cells had structural alterations such as truncated cells, cells with two flagella, bleb formation and cells body deformation, while none of these modifications could be visualized in the control group. When analyzed through fluorescence microscopy, the promastigotes treated were positives for free radicals immediately after light application and also 1 hour after treatment presenting signs of autophagia. PDT on L. (L.) amazonensis is effective causing alterations that can help elucidate the mechanisms of the parasite's death when treated with methilene

Licochalcone A, a novel antiparasitic agent with potent activity against human pathogenic protozoan species of Leishmania.

PubMed Central

Chen, M; Christensen, S B; Blom, J; Lemmich, E; Nadelmann, L; Fich, K; Theander, T G; Kharazmi, A

1993-01-01

Licochalcone A, an oxygenated chalcone isolated from the roots of Chinese licorice plant, inhibited the growth of both Leishmania major and Leishmania donovani promastigotes and amastigotes. The structure of the licochalcone A was established by mass and nuclear magnetic resonance spectroscopies and by synthesis, and its purity was verified by high-pressure liquid chromatography. The 50% inhibition of growth of logarithmic- and stationary-phase promastigotes of L. major, as measured by [3H]thymidine uptake, were 4 and 2.5 micrograms/ml, respectively. The growth of L. major promastigotes was totally inhibited after a 20-h incubation period with licochalcone A at 5 micrograms/ml. At a concentration of 0.5 microgram/ml, licochalcone A markedly reduced the infection rate of human peripheral blood monocyte-derived macrophages and U937 cells with L. major promastigotes and exhibited a strong intracellular killing of the parasite. These data show that intracellular Leishmania amastigotes are more susceptible than promastigotes to licochalcone A. Results of studies on the site of action of licochalcone A indicate that the target organelle appears to be the parasite mitochondria. These findings demonstrate that licochalcone A in concentrations that are nontoxic to host cells exhibits a strong antileishmanial activity and that appropriate substituted chalcones might be a new class of antileishmanial drugs. Images PMID:8109916

Ecto-Nucleotidase Activities of Promastigotes from Leishmania (Viannia) braziliensis Relates to Parasite Infectivity and Disease Clinical Outcome

PubMed Central

Leite, Pauline M.; Gomes, Rodrigo S.; Figueiredo, Amanda B.; Serafim, Tiago D.; Tafuri, Wagner L.; de Souza, Carolina C.; Moura, Sandra A. L.; Fietto, Juliana L. R.; Melo, Maria N.; Ribeiro-Dias, Fátima; Oliveira, Milton A. P.; Rabello, Ana; Afonso, Luís C. C.

2012-01-01

Background Leishmania (Viannia) braziliensis has been associated with a broad range of clinical manifestations ranging from a simple cutaneous ulcer to destructive mucosal lesions. Factors leading to this diversity of clinical presentations are not clear, but parasite factors have lately been recognized as important in determining disease progression. Given the fact that the activity of ecto-nucleotidases correlates with parasitism and the development of infection, we evaluated the activity of these enzymes in promastigotes from 23 L. braziliensis isolates as a possible parasite-related factor that could influence the clinical outcome of the disease. Methodology/Principal Findings Our results show that the isolates differ in their ability to hydrolyze adenine nucleotides. Furthermore, we observed a positive correlation between the time for peak of lesion development in C57BL/6J mice and enzymatic activity and clinical manifestation of the isolate. In addition, we found that L. (V.) braziliensis isolates obtained from mucosal lesions hydrolyze higher amounts of adenine nucleotides than isolates obtained from skin lesions. One isolate with high (PPS6m) and another with low (SSF) ecto-nucleotidase activity were chosen for further studies. Mice inoculated with PPS6m show delayed lesion development and present larger parasite loads than animals inoculated with the SSF isolate. In addition, PPS6m modulates the host immune response by inhibiting dendritic cell activation and NO production by activated J774 macrophages. Finally, we observed that the amastigote forms from PPS6m and SSF isolates present low enzymatic activity that does not interfere with NO production and parasite survival in macrophages. Conclusions/Significance Our data suggest that ecto-nucleotidases present on the promastigote forms of the parasite may interfere with the establishment of the immune response with consequent impaired ability to control parasite dissemination and this may be an important factor in determining the clinical outcome of leishmaniasis. PMID:23071853

Selective Effect of 2′,6′-Dihydroxy-4′-Methoxychalcone Isolated from Piper aduncum on Leishmania amazonensis

PubMed Central

Torres-Santos, Eduardo Caio; Moreira, Davyson Lima; Kaplan, Maria Auxiliadora C.; Meirelles, Maria Nazareth; Rossi-Bergmann, Bartira

1999-01-01

2′,6′-Dihydroxy-4′-methoxychalcone (DMC) was purified from the dichloromethane extract of Piper aduncum inflorescences. DMC showed significant activity in vitro against promastigotes and intracellular amastigotes of Leishmania amazonensis, with 50% effective doses of 0.5 and 24 μg/ml, respectively. Its inhibitory effect on amastigotes is apparently a direct effect on the parasites and is not due to activation of the nitrogen oxidative metabolism of macrophages, since the production of nitric oxide by both unstimulated and recombinant gamma interferon-stimulated macrophages was decreased rather than increased with DMC. The phagocytic activity of macrophages was functioning normally even with DMC concentrations as high as 80 μg/ml, as seen by electron microscopy and by the uptake of fluorescein isothiocyanate-labeled beads. Ultrastructural studies also showed that in the presence of DMC the mitochondria of promastigotes were enlarged and disorganized. Despite destruction of intracellular amastigotes, no disarrangement of macrophage organelles were observed, even at 80 μg of DMC/ml. These observations suggest that DMC is selectively toxic to the parasites. Its simple structure may well enable it to serve as a new lead compound for the synthesis of novel antileishmanial drugs. PMID:10223942

Antileishmanial Activity of Date (Phoenix dactylifera L) Fruit and Pit Extracts In Vitro.

PubMed

Albakhit, Sedighe; Khademvatan, Shahram; Doudi, Monir; Foroutan-Rad, Masoud

2016-10-01

Leishmaniasis is considered as a major public health problem worldwide. Current drugs in treatment of leishmaniasis have some limitations; thus, the current study was aimed to assess the methanolic extracts of pit and fruit of Phoenix dactylifera against Leishmania major promastigotes. L major promastigotes were cultured in RPMI 1640 and incubated at 25°C ± 1°C for 24, 48, and 72 hours. For obtaining the IC50 (half maximal inhibitory concentration) value, MTT assay was employed. Furthermore, promastigotes were examined in terms of morphology under light microscope. About 48 hours after treatment, IC50s were estimated 23 μg/mL and 500 mg/mL for methanolic extracts of pit and fruit of P dactylifera, respectively. Both extracts exhibited a dose and time-dependent antileishmanial activity against L major parasites. Also, some visible morphological changes were seen. This finding revealed both date fruit and pit, are effective against L major promastigotes. Further studies should be designed in future based on apoptosis induction in vitro and in vivo. © The Author(s) 2016.

Leishmanicidal activity of saponins isolated from the leaves of Eclipta prostrata and Gymnema sylvestre

PubMed Central

Khanna, Venkatesan Gopiesh; Kannabiran, Krishnan; Getti, Giulia

2009-01-01

Objective: To evaluate the leishmanicidal activity of saponin, dasyscyphin C of Eclipta prostrata and sapogenin, gymnemagenol from Gymnema sylvestre leaves under in vitro conditions. Materials and Methods: Dasyscyphin C/Gymnemagenol were dissolved in phosphate buffered saline (PBS) and diluted with liquid medium to obtain concentrations ranging from 1000 to 15 μg /ml. The leishmanicidal activity against leishmanial parasites, Leishmania major, Leishmania aethiopica and Leishmania tropica promastigotes was studied by the MTS assay. Result: The Dasyscyphin C isolated from E. prostrata showed good leishmanicidal activity at 1000μg/ml concentration, with the IC50 value of 450μg/ml against L. major promastigote and the percentage of parasitic death was 73; whereas, gymnemagenol of G. sylvestre showed only 52% parasitic death at 1000 μg/ml concentration. The other Leishmania species, L. aethiopica and L. tropica promastigotes, were less sensitive to the saponins of E. prostrata and G. sylvestre. Conclusion: From this study, it can be concluded that the dasyscyphin C of E. prostrata has significant leishmanicidal activity against L. major promastigote. PMID:20177579

Chemotherapy and Biochemistry of Leishmania

DTIC Science & Technology

1985-12-01

Leishmania- sis is initiated when sandflies inject the extracellular prcmastigate form of the parasite into the skin . The promastigotes are...precipitate from the protamine sulfate step was removed by centri- fugation at 15,000 rpm for 15 min at 40. The supernatant was dialyzed in 2 L of...Fig. 13. Only two protein bands could be detected when stained with commassie blue . RNA polymerase activity could not be detected when gels were

CrATP interferes in the promastigote-macrophage interaction in Leishmania amazonensis infection.

PubMed

Ennes-Vidal, V; Castro, R O S; Britto, C; Barrabin, H; D'Avila-Levy, C M; Moreira, O C

2011-07-01

Recent have shown the relationship between Ecto-Nucleoside-Triphosphate-Diphosphohydrolases (Ecto-NTPDases or ecto-nucleotidases) and virulence and infectivity in trypanosomatids. In this work, the inhibition of the ecto-ATPase activities and promastigote growth of Leishmania amazonensis by CrATP was characterized. Furthermore, this compound was used to investigate the role of ecto-nucleotidase in the interaction of L. amazonensis with resident peritoneal macrophages obtained from BALB/c mice. CrATP partially inhibits the ecto-ATPase activity, presenting Ki values of 575·7±199·1 and 383·5±79·0 μm, in the presence or absence of 5 mm MgCl2, respectively. The apparent Kms for ATP (2·9±0·5 mm to Mg2+-dependent ecto-ATPase and 0·4±0·2 mm to Mg2+-independent ecto-ATPase activities) are not significantly altered by CrATP, suggesting a reversible non-competitive inhibition of both enzymes. When CrATP was added to the cultivation medium at 500 μm, it drastically inhibited the cellular growth. The interaction of promastigote forms of L. amazonensis with BALB/c peritoneal macrophages is strongly affected by CrATP. When the parasites were treated with 500 μm CrATP before interacting with macrophages, the adhesion and endocytic indices were strongly reduced to 53·0±14·8% and 39·8±1·1%, respectively. These results indicate that ecto-nucleotidase plays an important role in the infection process caused by Leishmania amazonensis.

[Amyloidosis in infected Didelphis marsupialis].

PubMed

Roa, Diana Milena; Sarmiento, Ladys; Rodríguez, Gerzaín

2002-09-01

A male opossum, Didelphis marsupialis, captured in Teruel (Huila), Colombia, was inoculated intraperitoneally with 1 x 10(6) promastigotes of Leishmania chagasi (MHOM/CO/84/CL044B). The animal died 5 weeks after inoculation. Autopsy revealed signs of visceral leishmaniasis along with amastigote parasite form in Kupffer cells and spleen macrophages. Amyloid deposits in liver and spleen were demonstrated by histological staining and electron microscopy. The rapid death was considered a consequence of a secondary, reactive amyloidosis.

The impact of distinct culture media in Leishmania infantum biology and infectivity.

PubMed

Santarém, Nuno; Cunha, Joana; Silvestre, Ricardo; Silva, Cátia; Moreira, Diana; Ouellette, Marc; Cordeiro-DA-Silva, Anabela

2014-02-01

An ideal culture medium for Leishmania promastigotes should retain the basic characteristics of promastigotes found in sandflies (morphology and infectivity). Furthermore, the media should not create a bias in experimental settings, thus enabling the proper extrapolation of results. To assess this we studied several established media for promastigote growth. We analysed morphology, viability, cell cycle progression, metacyclic profile, capacity to differentiate into axenic amastigotes and infectivity. Furthermore, using a rational approach from the evaluated media we developed a simple serum-free medium (cRPMI). We report that parasites growing in different media present different biological characteristics and distinct in vitro and in vivo infectivities. The developed medium, cRPMI, proved to be a less expensive substitute for traditional serum-supplemented media for the in vitro maintenance of promastigotes. In fact, cRPMI is ideal for the maintenance of parasites in the laboratory, diminishing the expected loss of virulence over time typical of the parasite cultivation. Ultimately this report is a clear warning that the normalization of culture media should be a real concern in the field as media-specific phenomena are sufficient to induce biological bias with consequences in infectivity and general parasite biology.

In vitro selection of Phytomonas serpens cells resistant to the calpain inhibitor MDL28170: alterations in fitness and expression of the major peptidases and efflux pumps.

PubMed

Oliveira, Simone S C; Gonçalves, Inês C; Ennes-Vidal, Vitor; Lopes, Angela H C S; Menna-Barreto, Rubem F S; D'Ávila-Levy, Claudia M; Santos, André L S; Branquinha, Marta H

2018-03-01

The species Phytomonas serpens is known to express some molecules displaying similarity to those described in trypanosomatids pathogenic to humans, such as peptidases from Trypanosoma cruzi (cruzipain) and Leishmania spp. (gp63). In this work, a population of P. serpens resistant to the calpain inhibitor MDL28170 at 70 µ m (MDLR population) was selected by culturing promastigotes in increasing concentrations of the drug. The only relevant ultrastructural difference between wild-type (WT) and MDLR promastigotes was the presence of microvesicles within the flagellar pocket of the latter. MDLR population also showed an increased reactivity to anti-cruzipain antibody as well as a higher papain-like proteolytic activity, while the expression of calpain-like molecules cross-reactive to anti-Dm-calpain (from Drosophila melanogaster) antibody and calcium-dependent cysteine peptidase activity were decreased. Gp63-like molecules also presented a diminished expression in MDLR population, which is probably correlated to the reduction in the parasite adhesion to the salivary glands of the insect vector Oncopeltus fasciatus. A lower accumulation of Rhodamine 123 was detected in MDLR cells when compared with the WT population, a phenotype that was reversed when MDLR cells were treated with cyclosporin A and verapamil. Collectively, our results may help in the understanding of the roles of calpain inhibitors in trypanosomatids.

L-arginine availability and arginase activity: Characterization of amino acid permease 3 in Leishmania amazonensis.

PubMed

Aoki, Juliana Ide; Muxel, Sandra Marcia; Zampieri, Ricardo Andrade; Acuña, Stephanie Maia; Fernandes, Juliane Cristina Ribeiro; Vanderlinde, Rubia Heloisa; Sales, Maria Carmen Oliveira de Pinho; Floeter-Winter, Lucile Maria

2017-10-01

Leishmania uses the amino acid L-arginine as a substrate for arginase, enzyme that produces urea and ornithine, last precursor of polyamine pathway. This pathway is used by the parasite to replicate and it is essential to establish the infection in the mammalian host. L-arginine is not synthesized by the parasite, so its uptake occurs through the amino acid permease 3 (AAP3). AAP3 is codified by two copies genes (5.1 and 4.7 copies), organized in tandem in the parasite genome. One copy presents the expression regulated by L-arginine availability. RNA-seq data revealed 14 amino acid transporters differentially expressed in the comparison of La-WT vs. La-arg- promastigotes and axenic amastigotes. The 5.1 and 4.7 aap3 transcripts were down-regulated in La-WT promastigotes vs. axenic amastigotes, and in La-WT vs. La-arg- promastigotes. In contrast, transcripts of other transporters were up-regulated in the same comparisons. The amount of 5.1 and 4.7 aap3 mRNA of intracellular amastigotes was also determined in sample preparations from macrophages, obtained from BALB/c and C57BL/6 mice and the human THP-1 lineage infected with La-WT or La-arg-, revealing that the genetic host background is also important. We also determined the aap3 mRNA and AAP3 protein amounts of promastigotes and axenic amastigotes in different environmental growth conditions, varying pH, temperature and L-arginine availability. Interestingly, the increase of temperature increased the AAP3 level in plasma membrane and consequently the L-arginine uptake, independently of pH and L-arginine availability. In addition, we demonstrated that besides the plasma membrane localization, AAP3 was also localized in the glycosome of L. amazonensis promastigotes and axenic amastigotes. In this report, we described the differential transcriptional profiling of amino acids transporters from La-WT and La-arg- promastigotes and axenic amastigotes. We also showed the increased AAP3 levels under amino acid starvation or its decrease in L-arginine supplementation. The differential AAP3 expression was determined in the differentiation of promastigotes to amastigotes conditions, as well as the detection of AAP3 in the plasma membrane reflecting in the L-arginine uptake. Our data suggest that depending on the amino acid pool and arginase activity, Leishmania senses and could use an alternative route for the amino acid transport in response to stress signaling.

L-arginine availability and arginase activity: Characterization of amino acid permease 3 in Leishmania amazonensis

PubMed Central

Aoki, Juliana Ide; Muxel, Sandra Marcia; Zampieri, Ricardo Andrade; Acuña, Stephanie Maia; Fernandes, Juliane Cristina Ribeiro; Vanderlinde, Rubia Heloisa; Sales, Maria Carmen Oliveira de Pinho

2017-01-01

Background Leishmania uses the amino acid L-arginine as a substrate for arginase, enzyme that produces urea and ornithine, last precursor of polyamine pathway. This pathway is used by the parasite to replicate and it is essential to establish the infection in the mammalian host. L-arginine is not synthesized by the parasite, so its uptake occurs through the amino acid permease 3 (AAP3). AAP3 is codified by two copies genes (5.1 and 4.7 copies), organized in tandem in the parasite genome. One copy presents the expression regulated by L-arginine availability. Methodology/Principal findings RNA-seq data revealed 14 amino acid transporters differentially expressed in the comparison of La-WT vs. La-arg- promastigotes and axenic amastigotes. The 5.1 and 4.7 aap3 transcripts were down-regulated in La-WT promastigotes vs. axenic amastigotes, and in La-WT vs. La-arg- promastigotes. In contrast, transcripts of other transporters were up-regulated in the same comparisons. The amount of 5.1 and 4.7 aap3 mRNA of intracellular amastigotes was also determined in sample preparations from macrophages, obtained from BALB/c and C57BL/6 mice and the human THP-1 lineage infected with La-WT or La-arg-, revealing that the genetic host background is also important. We also determined the aap3 mRNA and AAP3 protein amounts of promastigotes and axenic amastigotes in different environmental growth conditions, varying pH, temperature and L-arginine availability. Interestingly, the increase of temperature increased the AAP3 level in plasma membrane and consequently the L-arginine uptake, independently of pH and L-arginine availability. In addition, we demonstrated that besides the plasma membrane localization, AAP3 was also localized in the glycosome of L. amazonensis promastigotes and axenic amastigotes. Conclusions/Significance In this report, we described the differential transcriptional profiling of amino acids transporters from La-WT and La-arg- promastigotes and axenic amastigotes. We also showed the increased AAP3 levels under amino acid starvation or its decrease in L-arginine supplementation. The differential AAP3 expression was determined in the differentiation of promastigotes to amastigotes conditions, as well as the detection of AAP3 in the plasma membrane reflecting in the L-arginine uptake. Our data suggest that depending on the amino acid pool and arginase activity, Leishmania senses and could use an alternative route for the amino acid transport in response to stress signaling. PMID:29073150

Targeting Leishmania major parasite with peptides derived from a combinatorial phage display library.

PubMed

Rhaiem, Rafik Ben; Houimel, Mehdi

2016-07-01

Cutaneous leishmaniasis (CL) is a global problem caused by intracellular protozoan pathogens of the genus Leishmania for which there are no suitable vaccine or chemotherapy options. Thus, de novo identification of small molecules binding to the Leishmania parasites by direct screening is a promising and appropriate alternative strategy for the development of new drugs. In this study, we used a random linear hexapeptide library fused to the gene III protein of M13 filamentous bacteriophage to select binding peptides to metacyclic promastigotes from a highly virulent strain of Leishmania major (Zymodeme MON-25; MHOM/TN/94/GLC94). After four rounds of stringent selection and amplification, polyclonal and monoclonal phage-peptides directed against L. major metacyclic promastigotes were assessed by ELISA, and the optimal phage-peptides were grown individually and characterized for binding to L. major by monoclonal phage ELISA. The DNA of 42 phage-peptides clones was amplified by PCR, sequenced, and their amino acid sequences deduced. Six different peptide sequences were obtained with frequencies of occurrence ranging from 2.3% to 85.7%. The biological effect of the peptides was assessed in vitro on human monocytes infected with L. major metacyclic promastigotes, and in vivo on susceptible parasite-infected BALB/c mice. The development of cutaneous lesions in the right hind footpads of infected mice after 13 weeks post-infection showed a protection rate of 81.94% with the injected peptide P2. Moreover, Western blots revealed that the P2 peptide interacted with the major surface protease gp63, a protein of 63kDa molecular weight. Moreover, bioinformatics were used to predict the interaction between peptides and the major surface molecule of the L. major. The molecular docking showed that the P2 peptide has the minimum interaction energy and maximum shape complimentarity with the L. major gp63 active site. Our study demonstrated that the P2 peptide occurs at high frequency during the screening procedure, best inhibits L. major growth kinetics in vitro, and reduces cutaneous lesions in BALB/c mice, thus showing great promise in the development of new therapeutic molecules. Copyright © 2016 Elsevier B.V. All rights reserved.

Impact of Continuous Axenic Cultivation in Leishmania infantum Virulence

PubMed Central

Loureiro, Inês; Tavares, Joana; Silva, Ana Marta; Amorim, Ana Marina; Ouaissi, Ali; Cordeiro-da-Silva, Anabela; Silvestre, Ricardo

2012-01-01

Experimental infections with visceral Leishmania spp. are frequently performed referring to stationary parasite cultures that are comprised of a mixture of metacyclic and non-metacyclic parasites often with little regard to time of culture and metacyclic purification. This may lead to misleading or irreproducible experimental data. It is known that the maintenance of Leishmania spp. in vitro results in a progressive loss of virulence that can be reverted by passage in a mammalian host. In the present study, we aimed to characterize the loss of virulence in culture comparing the in vitro and in vivo infection and immunological profile of L. infantum stationary promastigotes submitted to successive periods of in vitro cultivation. To evaluate the effect of axenic in vitro culture in parasite virulence, we submitted L. infantum promastigotes to 4, 21 or 31 successive in vitro passages. Our results demonstrated a rapid and significant loss of parasite virulence when parasites are sustained in axenic culture. Strikingly, the parasite capacity to modulate macrophage activation decreased significantly with the augmentation of the number of in vitro passages. We validated these in vitro observations using an experimental murine model of infection. A significant correlation was found between higher parasite burdens and lower number of in vitro passages in infected Balb/c mice. Furthermore, we have demonstrated that the virulence deficit caused by successive in vitro passages results from an inadequate capacity to differentiate into amastigote forms. In conclusion, our data demonstrated that the use of parasites with distinct periods of axenic in vitro culture induce distinct infection rates and immunological responses and correlated this phenotype with a rapid loss of promastigote differentiation capacity. These results highlight the need for a standard operating protocol (SOP) when studying Leishmania species. PMID:22292094

Glycyrrhizic acid attenuates growth of Leishmania donovani by depleting ergosterol levels.

PubMed

Dinesh, Neeradi; Neelagiri, Soumya; Kumar, Vinay; Singh, Sushma

2017-05-01

In the present study, glycyrrhizic acid (GA) the main component of Glycyrrhiza glabra was evaluated for its efficacy as antileishmanial agent and its mode of action explored. GA inhibits promastigotes and intracellular amastigotes in a dose dependent manner at an IC 50 value of 34 ± 3.0 μM and 20 ± 4.2 μM respectively. GA was non-toxic against THP-1 macrophage host cell line. GA was found to inhibit recombinant Leishmania donovani HMG-CoA reductase (LdHMGR) enzyme at the half-maximum inhibitory concentration of 24 ± 4.3 μM indicating the sensitivity and specificity of GA towards the enzyme. However, GA could cause only 30% reduction in HMGR activity when measured in Leishmania promastigotes treated with 34 μM of GA. Interestingly western blot analysis revealed fivefold reduced HMGR expression in GLA treated promastigotes. To further study the mode of action of GA, we used transgenic parasites overexpressing LdHMGR. Results indicated that ∼2 fold resistance was exhibited by LdHMGR overexpressing promastigotes to GA with an IC 50 value of 74 μM compared to the wild type parasite. This explained the specific binding of GA to LdHMGR enzyme. There was ∼2 fold depletion in ergosterol levels in wild type promastigotes compared to the HMGR overexpressors. This data was further validated by exogenous supplementation of GA treated cells with ergosterol and 40% reversal of growth inhibition was observed. The results obtained suggested that GA kills the parasite by affecting sterol biosynthetic pathway, especially by inhibiting the L. donovani HMGR and altering ergosterol levels. The finding from the current study shows that GA is a potential antileishmanial chemotherapeutic agent. Copyright © 2017 Elsevier Inc. All rights reserved.

Screening of the in vitro antileishmanial activities of compounds and secondary metabolites isolated from Maytenus guianensis Klotzsch ex Reissek (Celastraceae) chichuá Amazon.

PubMed

Meneguetti, Dionatas Ulises de Oliveira; Lima, Renato Abreu; Hurtado, Fernanda Bay; Passarini, Guilherme Matos; Macedo, Sharon Rose Aragão; Barros, Neuza Biguinati de; Oliveira, Flávio Augusto de Souza; Medeiros, Patrícia Soares de Maria de; Militão, Júlio Sancho Linhares Teixeira; Nicolete, Roberto; Facundo, Valdir Alves

2016-01-01

Maytenus guianensis is a member of the Celastraceae family that is used in traditional medicine, particularly for its anti-parasitic and anti-cancer effects. To explore the ethnopharmacological potential of this plant, the present study was designed to screen the in vitro antileishmanial activities of extracts and compounds isolated from M. guianensis. Maytenus guianensis stems and leaves were extracted in acetone, followed by the preparation of eluates and isolation of secondary metabolites using chromatography on a glass column with silica gel as the fixed phase. The chemical components were identified using spectroscopic methods, including one- and two-dimensional nuclear magnetic resonance of hydrogen-1 and carbon-13, mass spectroscopy, and infrared spectroscopy. The anti-Leishmania amazonensis activities of these eluates and compounds were evaluated by direct promastigote counting and viability assays. It was found that the hexane bark eluate produced the strongest anti-L. amazonensis effect, with 90-100% inhibition of the promastigote form. The isolated metabolite that produced the best result was tingenone B, followed by a compound formed by the union of tingenone and tingenone B (80-90% inhibition). Maytenus guianensis shows anti-parasite activity that warrants further investigation to determine the mechanisms underlying this antileishmanial effect and to evaluate the pharmacological potential of these eluates and isolated secondary metabolites, while minimizing any adverse effects.

Antileishmanial Phenylpropanoids from the Leaves of Hyptis pectinata (L.) Poit

PubMed Central

Falcao, Rosangela A.; do Nascimento, Patricia L. A.; de Souza, Silvana A.; da Silva, Telma M. G.; de Queiroz, Aline C.; da Matta, Carolina B. B.; Moreira, Magna S. A.; Camara, Celso A.; Silva, Tania M. S.

2013-01-01

Hyptis pectinata, popularly known in Brazil as “sambacaitá” or “canudinho,” is an aromatic shrub largely grown in the northeast of Brazil. The leaves and bark are used in an infusion for the treatment of throat and skin inflammations, bacterial infections, pain, and cancer. Analogues of rosmarinic acid and flavonoids were obtained from the leaves of Hyptis pectinata and consisted of two new compounds, sambacaitaric acid (1) and 3-O-methyl-sambacaitaric acid (2), and nine known compounds, rosmarinic acid (3), 3-O-methyl-rosmarinic acid (4), ethyl caffeate (5), nepetoidin A (6), nepetoidin B (7), cirsiliol (8), circimaritin (9), 7-O-methylluteolin (10), and genkwanin (11). The structures of these compounds were determined by spectroscopic methods. Compounds 1–5, and 7 were evaluated in vitro against the promastigote form of L. braziliensis, and the ethanol extract. The hexane, ethyl acetate, and methanol-water fractions were also evaluated. The EtOH extract, the hexane extract, EtOAc, MeOH:H2O fractions; and compounds 1, 2 and 4 exhibited antileishmanial activity, and compound 1 was as potent as pentamidine. In contrast, compounds 3, 5, and 7 did not present activity against the promastigote form of L. braziliensis below 100 µM. To our knowledge, compounds 1 and 2 are being described for the first time. PMID:23983783

Arginase activity of Leishmania isolated from patients with cutaneous leishmaniasis.

PubMed

Badirzadeh, A; Taheri, T; Abedi-Astaneh, F; Taslimi, Y; Abdossamadi, Z; Montakhab-Yeganeh, H; Aghashahi, M; Niyyati, M; Rafati, S

2017-09-01

Cutaneous leishmaniasis (CL) is one of the most important vector-borne parasitic diseases, highly endemic in Iran, and its prevalence is increasing all over the country. Arginase (ARG) activity in isolated Leishmania parasites from CL patients is yet to be explored. This study aimed to compare the ARG activity of isolated Leishmania promastigotes from CL patients with a standard strain of Leishmania major and its influences on the disease pathogenesis. We recruited 16 confirmed CL patients from Qom Province, in central Iran; after detection of Leishmania species using PCR-RFLP, we assessed the levels of ARG in the isolated promastigotes and determined the parasites' growth rate. Only L. major was identified from CL patients. The level of ARG activity in the isolated Leishmania promastigotes from CL patients was significantly higher than that obtained from the standard strain of L. major. No significant correlations between ARG activity and lesion size, number or duration were observed; in contrast, a significant negative correlation was seen between ARG level and Leishmania' growth rate. The obtained results suggest that increased ARG expression and activity in the isolated Leishmania promastigotes might contribute to the higher parasite infectivity and play a major role in the pathogenicity of the CL. © 2017 John Wiley & Sons Ltd.

Antileishmanial activity and ultrastructural alterations of Leishmania (L.) chagasi treated with the calcium channel blocker nimodipine.

PubMed

Tempone, André Gustavo; Taniwaki, Noemi Nosomi; Reimão, Juliana Quero

2009-08-01

In a search for novel antileishmanial drugs, we investigated the activity of the calcium channel blocker nimodipine against Leishmania spp. and explored the ultrastructural damages of parasites induced by nimodipine after a short period of incubation. Nimodipine was highly effective against promastigotes and intracellular amastigotes of Leishmania (L.) chagasi, with 50% inhibitory concentration values of 81.2 and 21.5 muM, respectively. Nimodipine was about fourfold more effective than the standard pentavalent antimony against amastigotes and showed a Selectivity Index of 4.4 considering its mammalian cells toxicity. Leishmania (L.) amazonensis and Leishmania (L.) major promastigotes were also susceptible to nimodipine in a range concentration between 31 and 128 muM. Ultrastructural studies of L. (L.) chagasi revealed intense mitochondria damage and plasma membrane blebbing, resulting in a leishmanicidal effect as demonstrated by the lack of mitochondrial oxidative metabolism. The amastigote-killing effect suggests other mechanism than macrophage activation, as no upregulation of nitric oxide was seen. This calcium channel blocker is an effective in vitro antileishmanial compound and if adequately studied could be used as a novel drug candidate or as a novel drug lead compound for drug design studies against leishmaniasis.

Leishmania hijacking of the macrophage intracellular compartments.

PubMed

Liévin-Le Moal, Vanessa; Loiseau, Philippe M

2016-02-01

Leishmania spp., transmitted to humans by the bite of the sandfly vector, are responsible for the three major forms of leishmaniasis, cutaneous, diffuse mucocutaneous and visceral. Leishmania spp. interact with membrane receptors of neutrophils and macrophages. In macrophages, the parasite is internalized within a parasitophorous vacuole and engages in a particular intracellular lifestyle in which the flagellated, motile Leishmania promastigote metacyclic form differentiates into non-motile, metacyclic amastigote form. This phenomenon is induced by Leishmania-triggered events leading to the fusion of the parasitophorous vacuole with vesicular members of the host cell endocytic pathway including recycling endosomes, late endosomes and the endoplasmic reticulum. Maturation of the parasitophorous vacuole leads to the intracellular proliferation of the Leishmania amastigote forms by acquisition of host cell nutrients while escaping host defense responses. © 2015 FEBS.

Preparation of highly infective Leishmania promastigotes by cultivation on SNB-9 biphasic medium.

PubMed

Grekov, Igor; Svobodová, Milena; Nohýnková, Eva; Lipoldová, Marie

2011-12-01

Protozoan hemoflagellates Leishmania are causative agents of leishmaniases and an important biological model for study of host-pathogen interaction. A wide range of methods of Leishmania cultivation on both biphasic and liquid media is available. Biphasic media are considered to be superior for initial isolation of the parasites and obtaining high promastigote infectivity; however, liquid media are more suitable for large-scale experiments. The aim of the present study was the adaptation and optimization of the cultivation of Leishmania promastigotes on a biphasic SNB-9 (saline-neopeptone-blood 9) medium that was originally developed for Trypanosoma cultivation and combines the advantages of biphasic and liquid media. SNB-9 medium is characterized with a large volume of the liquid phase, which facilitates the manipulation with the culture and provides parasite yields comparable to parasite yields on such liquid medium as Schneider's Insect Medium. We demonstrate that SNB-9 very considerably surpasses Schneider's Insect Medium in in vitro infectivity of the parasites. Additionally, we show that the ratio of apoptotic parasites, which are important for the infectivity of the inoculum, in Leishmania culture in SNB-9 is higher than in Leishmania culture in Schneider's Insect Medium. Thus, we demonstrate that the cultivation of Leishmania on SNB-9 reliably yields highly infective promastigotes suitable for experimental infection. Copyright © 2011 Elsevier B.V. All rights reserved.

Ultrastructural Changes and Death of Leishmania infantum Promastigotes Induced by Morinda citrifolia Linn. Fruit (Noni) Juice Treatment

PubMed Central

Almeida-Souza, Fernando; Taniwaki, Noemi Nosomi; Amaral, Ana Cláudia Fernandes; de Souza, Celeste da Silva Freitas; Calabrese, Kátia da Silva; Abreu-Silva, Ana Lúcia

2016-01-01

The search for new treatments against leishmaniasis has increased due to high frequency of drug resistance registered in endemics areas, side effects, and complications caused by coinfection with HIV. Morinda citrifolia Linn., commonly known as Noni, has a rich chemical composition and various therapeutic effects have been described in the literature. Studies have shown the leishmanicidal activity of M. citrifolia; however, its action on the parasite has not yet been elucidated. In this work, we analyzed leishmanicidal activity and ultrastructural changes in Leishmania infantum promastigotes caused by M. citrifolia fruit juice treatment. M. citrifolia fruit extract showed a yield of 6.31% and high performance liquid chromatography identified phenolic and aromatic compounds as the major constituents. IC50 values were 260.5 µg/mL for promastigotes and 201.3 µg/mL for intracellular amastigotes of L. infantum treated with M. citrifolia. Cytotoxicity assay with J774.G8 macrophages showed that M. citrifolia fruit juice was not toxic up to 2 mg/mL. Transmission electron microscopy showed cytoplasmic vacuolization, lipid inclusion, increased exocytosis activity, and autophagosome-like vesicles in L. infantum promastigotes treated with M. citrifolia fruit juice. M. citrifolia fruit juice was active against L. infantum in the in vitro model used here causing ultrastructural changes and has a future potential for treatment against leishmaniasis. PMID:27313649

Ultrastructural Changes and Death of Leishmania infantum Promastigotes Induced by Morinda citrifolia Linn. Fruit (Noni) Juice Treatment.

PubMed

Almeida-Souza, Fernando; Taniwaki, Noemi Nosomi; Amaral, Ana Cláudia Fernandes; de Souza, Celeste da Silva Freitas; Calabrese, Kátia da Silva; Abreu-Silva, Ana Lúcia

2016-01-01

The search for new treatments against leishmaniasis has increased due to high frequency of drug resistance registered in endemics areas, side effects, and complications caused by coinfection with HIV. Morinda citrifolia Linn., commonly known as Noni, has a rich chemical composition and various therapeutic effects have been described in the literature. Studies have shown the leishmanicidal activity of M. citrifolia; however, its action on the parasite has not yet been elucidated. In this work, we analyzed leishmanicidal activity and ultrastructural changes in Leishmania infantum promastigotes caused by M. citrifolia fruit juice treatment. M. citrifolia fruit extract showed a yield of 6.31% and high performance liquid chromatography identified phenolic and aromatic compounds as the major constituents. IC50 values were 260.5 µg/mL for promastigotes and 201.3 µg/mL for intracellular amastigotes of L. infantum treated with M. citrifolia. Cytotoxicity assay with J774.G8 macrophages showed that M. citrifolia fruit juice was not toxic up to 2 mg/mL. Transmission electron microscopy showed cytoplasmic vacuolization, lipid inclusion, increased exocytosis activity, and autophagosome-like vesicles in L. infantum promastigotes treated with M. citrifolia fruit juice. M. citrifolia fruit juice was active against L. infantum in the in vitro model used here causing ultrastructural changes and has a future potential for treatment against leishmaniasis.

Leishmania mexicana: promastigotes and amastigotes secrete protein phosphatases and this correlates with the production of inflammatory cytokines in macrophages.

PubMed

Escalona-Montaño, A R; Ortiz-Lozano, D M; Rojas-Bernabé, A; Wilkins-Rodriguez, A A; Torres-Guerrero, H; Mondragón-Flores, R; Mondragón-Gonzalez, R; Becker, I; Gutiérrez-Kobeh, L; Aguirre-Garcia, M M

2016-09-01

Phosphatase activity of Leishmania spp. has been shown to deregulate the signalling pathways of the host cell. We here show that Leishmania mexicana promastigotes and amastigotes secrete proteins with phosphatase activity to the culture medium, which was higher in the Promastigote Secretion Medium (PSM) as compared with the Amastigote Secretion Medium (ASM) and was not due to cell lysis, since parasite viability was not affected by the secretion process. The biochemical characterization showed that the phosphatase activity present in PSM was higher in dephosphorylating the peptide END (pY) INASL as compared with the peptide RRA (pT)VA. In contrast, the phosphatase activity in ASM showed little dephosphorylating capacity for both peptides. Inhibition assays demonstrated that the phosphatase activity of both PSM and ASM was sensible only to protein tyrosine phosphatases inhibitors. An antibody against a protein phosphatase 2C (PP2C) of Leishmania major cross-reacted with a 44·9 kDa molecule in different cellular fractions of L. mexicana promastigotes and amastigotes, however, in PSM and ASM, the antibody recognized a protein about 70 kDa. By electron microscopy, the PP2C was localized in the flagellar pocket of amastigotes. PSM and ASM induced the production of tumor necrosis factor alpha, IL-1β, IL-12p70 and IL-10 in human macrophages.

Gluconeogenesis in Leishmania mexicana

PubMed Central

Rodriguez-Contreras, Dayana; Hamilton, Nicklas

2014-01-01

Gluconeogenesis is an active pathway in Leishmania amastigotes and is essential for their survival within the mammalian cells. However, our knowledge about this pathway in trypanosomatids is very limited. We investigated the role of glycerol kinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate phosphate dikinase (PPDK) in gluconeogenesis by generating the respective Leishmania mexicana Δgk, Δpepck, and Δppdk null mutants. Our results demonstrated that indeed GK, PEPCK, and PPDK are key players in the gluconeogenesis pathway in Leishmania, although stage-specific differences in their contribution to this pathway were found. GK participates in the entry of glycerol in promastigotes and amastigotes; PEPCK participates in the entry of aspartate in promastigotes, and PPDK is involved in the entry of alanine in amastigotes. Furthermore, the majority of alanine enters into the pathway via decarboxylation of pyruvate in promastigotes, whereas pathway redundancy is suggested for the entry of aspartate in amastigotes. Interestingly, we also found that l-lactate, an abundant glucogenic precursor in mammals, was used by Leishmania amastigotes to synthesize mannogen, entering the pathway through PPDK. On the basis of these new results, we propose a revision in the current model of gluconeogenesis in Leishmania, emphasizing the differences between amastigotes and promastigotes. This work underlines the importance of studying the trypanosomatid intracellular life cycle stages to gain a better understanding of the pathologies caused in humans. PMID:25288791

Arginase expression modulates nitric oxide production in Leishmania (Leishmania) amazonensis.

PubMed

Acuña, Stephanie Maia; Aoki, Juliana Ide; Laranjeira-Silva, Maria Fernanda; Zampieri, Ricardo Andrade; Fernandes, Juliane Cristina Ribeiro; Muxel, Sandra Marcia; Floeter-Winter, Lucile Maria

2017-01-01

Arginase is an enzyme that converts L-arginine to urea and L-ornithine, an essential substrate for the polyamine pathway supporting Leishmania (Leishmania) amazonensis replication and its survival in the mammalian host. L-arginine is also the substrate of macrophage nitric oxide synthase 2 (NOS2) to produce nitric oxide (NO) that kills the parasite. This competition can define the fate of Leishmania infection. The transcriptomic profiling identified a family of oxidoreductases in L. (L.) amazonensis wild-type (La-WT) and L. (L.) amazonensis arginase knockout (La-arg-) promastigotes and axenic amastigotes. We highlighted the identification of an oxidoreductase that could act as nitric oxide synthase-like (NOS-like), due to the following evidences: conserved domain composition, the participation of NO production during the time course of promastigotes growth and during the axenic amastigotes differentiation, regulation dependence on arginase activity, as well as reduction of NO amount through the NOS activity inhibition. NO quantification was measured by DAF-FM labeling analysis in a flow cytometry. We described an arginase-dependent NOS-like activity in L. (L.) amazonensis and its role in the parasite growth. The increased detection of NO production in the mid-stationary and late-stationary growth phases of La-WT promastigotes could suggest that this production is an important factor to metacyclogenesis triggering. On the other hand, La-arg- showed an earlier increase in NO production compared to La-WT, suggesting that NO production can be arginase-dependent. Interestingly, La-WT and La-arg- axenic amastigotes produced higher levels of NO than those observed in promastigotes. As a conclusion, our work suggested that NOS-like is expressed in Leishmania in the stationary growth phase promastigotes and amastigotes, and could be correlated to metacyclogenesis and amastigotes growth in a dependent way to the internal pool of L-arginine and arginase activity.

Large-scale investigation of Leishmania interaction networks with host extracellular matrix by surface plasmon resonance imaging.

PubMed

Fatoux-Ardore, Marie; Peysselon, Franck; Weiss, Anthony; Bastien, Patrick; Pratlong, Francine; Ricard-Blum, Sylvie

2014-02-01

We have set up an assay to study the interactions of live pathogens with their hosts by using protein and glycosaminoglycan arrays probed by surface plasmon resonance imaging. We have used this assay to characterize the interactions of Leishmania promastigotes with ~70 mammalian host biomolecules (extracellular proteins, glycosaminoglycans, growth factors, cell surface receptors). We have identified, in total, 27 new partners (23 proteins, 4 glycosaminoglycans) of procyclic promastigotes of six Leishmania species and 18 partners (15 proteins, 3 glycosaminoglycans) of three species of stationary-phase promastigotes for all the strains tested. The diversity of the interaction repertoires of Leishmania parasites reflects their dynamic and complex interplay with their mammalian hosts, which depends mostly on the species and strains of Leishmania. Stationary-phase Leishmania parasites target extracellular matrix proteins and glycosaminoglycans, which are highly connected in the extracellular interaction network. Heparin and heparan sulfate bind to most Leishmania strains tested, and 6-O-sulfate groups play a crucial role in these interactions. Numerous Leishmania strains bind to tropoelastin, and some strains are even able to degrade it. Several strains interact with collagen VI, which is expressed by macrophages. Most Leishmania promastigotes interact with several regulators of angiogenesis, including antiangiogenic factors (endostatin, anastellin) and proangiogenic factors (ECM-1, VEGF, and TEM8 [also known as anthrax toxin receptor 1]), which are regulated by hypoxia. Since hypoxia modulates the infection of macrophages by the parasites, these interactions might influence the infection of host cells by Leishmania.

Large-Scale Investigation of Leishmania Interaction Networks with Host Extracellular Matrix by Surface Plasmon Resonance Imaging

PubMed Central

Fatoux-Ardore, Marie; Peysselon, Franck; Weiss, Anthony; Bastien, Patrick; Pratlong, Francine

2014-01-01

We have set up an assay to study the interactions of live pathogens with their hosts by using protein and glycosaminoglycan arrays probed by surface plasmon resonance imaging. We have used this assay to characterize the interactions of Leishmania promastigotes with ∼70 mammalian host biomolecules (extracellular proteins, glycosaminoglycans, growth factors, cell surface receptors). We have identified, in total, 27 new partners (23 proteins, 4 glycosaminoglycans) of procyclic promastigotes of six Leishmania species and 18 partners (15 proteins, 3 glycosaminoglycans) of three species of stationary-phase promastigotes for all the strains tested. The diversity of the interaction repertoires of Leishmania parasites reflects their dynamic and complex interplay with their mammalian hosts, which depends mostly on the species and strains of Leishmania. Stationary-phase Leishmania parasites target extracellular matrix proteins and glycosaminoglycans, which are highly connected in the extracellular interaction network. Heparin and heparan sulfate bind to most Leishmania strains tested, and 6-O-sulfate groups play a crucial role in these interactions. Numerous Leishmania strains bind to tropoelastin, and some strains are even able to degrade it. Several strains interact with collagen VI, which is expressed by macrophages. Most Leishmania promastigotes interact with several regulators of angiogenesis, including antiangiogenic factors (endostatin, anastellin) and proangiogenic factors (ECM-1, VEGF, and TEM8 [also known as anthrax toxin receptor 1]), which are regulated by hypoxia. Since hypoxia modulates the infection of macrophages by the parasites, these interactions might influence the infection of host cells by Leishmania. PMID:24478075

Prophylactic immunization against experimental leishmaniasis. III. Protection against fatal Leishmania tropica infection induced by irradiated promastigotes involves Lyt-1/sup +/2/sup -/ T cells that do not mediate cutaneous DTH

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liew, F.Y.; Howard, J.G.; Hale, C.

1984-01-01

Protective immunity against fatal L. tropica infection in genetically vulnerable BALB/c mice can be induced by prophylactic immunization with irradiated promastigotes even when heat-killed. Such immunity is adoptively transferable transiently into intact or durably into sub-lethally irradiated (200 or 550 rad) syngeneic recipients by splenic T but not B cells. The effector T cells are of the Lyt-1/sup +/2/sup -/ phenotype, devoid of demonstrable cytotoxic activity. The immune splenic T cell population expresses specific helper activity for antibody synthesis. A causal role for helper T cells in this capacity, however, seems unlikely, because it was shown that antibody does notmore » determine the protective immunity against L. tropica. The immunized donors show no detectable cutaneous DTH or its early memory recall in response to live or killed promastigotes or a soluble L. tropica antigen preparation. Spleen, lymph node, and peritoneal exudate cells from protectively immunized donors similarly fail to transfer DTH locally or systemically. These cells also lack demonstrable suppressive activity against the expression or induction of DTH to L. tropica. Thus, protection against L. tropica induced by prophylactic i.v. immunization with irradiated promastigotes appears to be conferred by Lyt-1/sup +/2/sup -/ T cells that are distinguishable from T cells mediating either both DTH and T help, or cytotoxicity.« less

Gluconeogenesis in Leishmania mexicana: contribution of glycerol kinase, phosphoenolpyruvate carboxykinase, and pyruvate phosphate dikinase.

PubMed

Rodriguez-Contreras, Dayana; Hamilton, Nicklas

2014-11-21

Gluconeogenesis is an active pathway in Leishmania amastigotes and is essential for their survival within the mammalian cells. However, our knowledge about this pathway in trypanosomatids is very limited. We investigated the role of glycerol kinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate phosphate dikinase (PPDK) in gluconeogenesis by generating the respective Leishmania mexicana Δgk, Δpepck, and Δppdk null mutants. Our results demonstrated that indeed GK, PEPCK, and PPDK are key players in the gluconeogenesis pathway in Leishmania, although stage-specific differences in their contribution to this pathway were found. GK participates in the entry of glycerol in promastigotes and amastigotes; PEPCK participates in the entry of aspartate in promastigotes, and PPDK is involved in the entry of alanine in amastigotes. Furthermore, the majority of alanine enters into the pathway via decarboxylation of pyruvate in promastigotes, whereas pathway redundancy is suggested for the entry of aspartate in amastigotes. Interestingly, we also found that l-lactate, an abundant glucogenic precursor in mammals, was used by Leishmania amastigotes to synthesize mannogen, entering the pathway through PPDK. On the basis of these new results, we propose a revision in the current model of gluconeogenesis in Leishmania, emphasizing the differences between amastigotes and promastigotes. This work underlines the importance of studying the trypanosomatid intracellular life cycle stages to gain a better understanding of the pathologies caused in humans. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

In Silico Discovery of a Substituted 6-Methoxy-quinalidine with Leishmanicidal Activity in Leishmania infantum.

PubMed

Stevanović, Strahinja; Perdih, Andrej; Senćanski, Milan; Glišić, Sanja; Duarte, Margarida; Tomás, Ana M; Sena, Filipa V; Sousa, Filipe M; Pereira, Manuela M; Solmajer, Tom

2018-03-27

There is an urgent need for the discovery of new antileishmanial drugs with a new mechanism of action. Type 2 NADH dehydrogenase from Leishmania infantum ( Li NDH2) is an enzyme of the parasite's respiratory system, which catalyzes the electron transfer from NADH to ubiquinone without coupled proton pumping. In previous studies of the related NADH: ubiquinone oxidoreductase crystal structure from Saccharomyces cerevisiae , two ubiquinone-binding sites (UQ I and UQ II ) were identified and shown to play an important role in the NDH-2-catalyzed oxidoreduction reaction. Based on the available structural data, we developed a three-dimensional structural model of Li NDH2 using homology detection methods and performed an in silico virtual screening campaign to search for potential inhibitors targeting the Li NDH2 ubiquinone-binding site 1-UQ I . Selected compounds displaying favorable properties in the computational screening experiments were assayed for inhibitory activity in the structurally similar recombinant NDH-2 from S. aureus and leishmanicidal activity was determined in the wild-type axenic amastigotes and promastigotes of L. infantum . The identified compound, a substituted 6-methoxy-quinalidine, showed promising nanomolar leishmanicidal activity on wild-type axenic promastigotes and amastigotes of L. infantum and the potential for further development.

[Dog (Canis familiaris) infectivity to Lutzomyia youngi in Trujillo, Venezuela].

PubMed

Hernández, Dalila; Rojas, Elina; Scorza, José Vicente; Jorquera, Alicia

2006-10-01

In Trujillo, Venezuela the prevalence for American tegumentary leishmaniasis (ATL) is 38 per 100,000 inhabitants. In a periurban, rural settlement of the capital city Trujillo, we studied the potential capability of the domestic dog (Canis familiaris) as a source of infection for Lutzomyia youngi, a phlebotomine sand fly species abundant in the study area and whose domestic vectorial activity has been proven. Dogs with dermal lesions suggestive of ATL and parasitological confirmation of infection, were selected for xenodiagnosis by allowing sylvatic phlebotomines from a ATL free area, to feed ad libitum over each animal's entire body surface. The insects' intestinal tracts were dissected 5 days after the blood meal in order to look for flagellate forms. When these were found, parasitological identification was performed by the multiplex-PCR technique. Four hundred and fifty five sand flies engorged over two dogs in three different assays; promastigotes were found in 4 (0.88%) of the specimens on only one occasion. PCR identified DNA of the Leishmania Viannia subgenus. The household dog has the potential of being a domestic risk factor in the ATL transmission cycle.

In vitro study of the photodynamic antimicrobial therapy (PACT) against promastigotes form of the leishmania (viannia) braziliensis: in vitro study

NASA Astrophysics Data System (ADS)

Barbosa, Artur F. S.; Sangiorgi, Bruno B.; Galdino, Suely L.; Pitta, Ivan R.; Barral-Netto, Manoel; Pinheiro, Antônio L. B.

2013-03-01

Leishmaniasis, a protozoan parasitic disease that remains a major worldwide health problem with high endemicity in developing countries. Treatment of cutaneous Leishmaniasis (CL) should be decided by the clinical lesions, etiological species and its potential to develop into mucosal Leishmaniasis. High cost, systemic toxicity, and diminished efficacy due to development of parasite resistance are the serious drawbacks of current treatment options. Thus, identifying new, effective, and safer anti-leishmanial drug(s) is of paramount importance. The aim of this study was to verify the effectiveness of PACT in vitro, as a new technique for the treatment of Leishmaniasis. For this, semiconductor laser (λ = 660nm, 40mW, 8.4J/cm2, CW) associated to phenothiazine's derivatives (5 and 10 μg/ml, TBO, Methylene Blue or Phenothiazine) on the promastigotes form of Leishmania braziliensis in a single session was used. Viability of the parasites was assessed in quadruplicates of each group. The samples were removed and analyzed in a hemocytometer 72h after PACT. We found an important decrease in the number of viable parasites on all treated groups in comparison to their controls. The results of present study showed significant percentage of lethality (above 92%) of the protocol. The 98.33% of lethality was achieved with 10 μg/ml of FTZ. No lethality was seen on groups treated neither with laser nor with each compounds separately. The results are promising and indicative that the use of PACT may be a powerful treatment of Leishmaniasis when compared to already available ones.

In vitro evaluation of cytotoxicity and leishmanicidal activity of phthalimido-thiazole derivatives.

PubMed

Aliança, Amanda Silva Dos Santos; Oliveira, Arsênio Rodrigues; Feitosa, Ana Paula Sampaio; Ribeiro, Karla Raíza Cardoso; de Castro, Maria Carolina Accioly Brelaz; Leite, Ana Cristina Lima; Alves, Luiz Carlos; Brayner, Fábio André

2017-07-15

It is estimated that the worldwide prevalence of leishmaniasis is around 12 million individuals in 80 countries, with 400,000new cases per year. In the search for new leishmanicidal agents, the hybrid phthalimido-thiazoles have been identified as an important scaffold for drug design and discovery. The present study thus reports the in vitro activity of a series of phthalimido-thiazole derivatives. Cytotoxicity against a strain of L. infantum, Vero cells, J774 macrophages and peritoneal macrophages was evaluated, as well as nitric oxide (NO) production. Activity against amastigote and promastigote forms of L. infantum and microscopic changes in the parasite and intracellular targets of the parasite were achieved. The results show that the compounds arising from hybridization of phthalimide and 1,3-thiazole exhibit promising leishmanicidal activity. Compounds 2j and 2m were the most potent of the series tested and the parasites treated with these compounds exhibited ultrastructural changes, such as cell body shrinkage, loss of cellular membrane integrity, vacuolization of cytoplasm, membrane profiles surrounding organelles and swelling of mitochondria. The data showed that these compounds reduced the survival of intracellular amastigotes and presented low toxicity for mammalian cells. The compounds produced increased NO production compared to untreated cells in non-infected macrophages. Treated promastigote forms showed an increase in the number of cells stained with propidium iodide. The compounds brought about significant changes in mitochondrial membrane potential. According to the present study, phthalimido-thiazole compounds exhibit leishmanicidal activity and could be used to develop novel antileishmaniasis drugs and explore potential molecular targets. Copyright © 2017 Elsevier B.V. All rights reserved.

The Leishmania donovani histidine acid ecto-phosphatase LdMAcP: insight into its structure and function.

PubMed

Papadaki, Amalia; Politou, Anastasia S; Smirlis, Despina; Kotini, Maria P; Kourou, Konstadina; Papamarcaki, Thomais; Boleti, Haralabia

2015-05-01

Acid ecto-phosphatase activity has been implicated in Leishmania donovani promastigote virulence. In the present study, we report data contributing to the molecular/structural and functional characterization of the L. donovani LdMAcP (L. donovani membrane acid phosphatase), member of the histidine acid phosphatase (HAcP) family. LdMAcP is membrane-anchored and shares high sequence identity with the major secreted L. donovani acid phosphatases (LdSAcPs). Sequence comparison of the LdMAcP orthologues in Leishmania sp. revealed strain polymorphism and species specificity for the L. donovani complex, responsible for visceral leishmaniasis (Khala azar), proposing thus a potential value of LdMAcP as an epidemiological or diagnostic tool. The extracellular orientation of the LdMAcP catalytic domain was confirmed in L. donovani promastigotes, wild-type (wt) and transgenic overexpressing a recombinant LdMAcP-mRFP1 (monomeric RFP1) chimera, as well as in transiently transfected mammalian cells expressing rLdMAcP-His. For the first time it is demonstrated in the present study that LdMAcP confers tartrate resistant acid ecto-phosphatase activity in live L. donovani promastigotes. The latter confirmed the long sought molecular identity of at least one enzyme contributing to this activity. Interestingly, the L. donovani rLdMAcP-mRFP1 promastigotes generated in this study, showed significantly higher infectivity and virulence indexes than control parasites in the infection of J774 mouse macrophages highlighting thereby a role for LdMAcP in the parasite's virulence.

Major Surface Protease of Trypanosomatids: One Size Fits All? ▿

PubMed Central

Yao, Chaoqun

2010-01-01

Major surface protease (MSP or GP63) is the most abundant glycoprotein localized to the plasma membrane of Leishmania promastigotes. MSP plays several important roles in the pathogenesis of leishmaniasis, including but not limited to (i) evasion of complement-mediated lysis, (ii) facilitation of macrophage (Mø) phagocytosis of promastigotes, (iii) interaction with the extracellular matrix, (iv) inhibition of natural killer cellular functions, (v) resistance to antimicrobial peptide killing, (vi) degradation of Mø and fibroblast cytosolic proteins, and (vii) promotion of survival of intracellular amastigotes. MSP homologues have been found in all other trypanosomatids studied to date including heteroxenous members of Trypanosoma cruzi, the extracellular Trypanosoma brucei, unusual intraerythrocytic Endotrypanum spp., phytoparasitic Phytomonas spp., and numerous monoxenous species. These proteins are likely to perform roles different from those described for Leishmania spp. Multiple MSPs in individual cells may play distinct roles at some time points in trypanosomatid life cycles and collaborative or redundant roles at others. The cellular locations and the extracellular release of MSPs are also discussed in connection with MSP functions in leishmanial promastigotes. PMID:19858295

Leishmania major: effect of protein kinase A and phosphodiesterase activity on infectivity and proliferation of promastigotes.

PubMed

Malki-Feldman, Laura; Jaffe, Charles L

2009-09-01

Effect of modulators on protein kinase A (PKA) activity, promastigote growth and their ability to infect peritoneal macrophages was monitored. PKA inhibitors reduced [Protein Kinase Inhibitor (PKI) - 56%; H89 - 54.5%] kemptide phosphorylation by Leishmania major promastigote lysates, while activators increased phosphorylation (8-CPT-cAMP - 88%; Sp-cAMPS-AM - 152%). Activation was specifically inhibited by PKI. Phosphodiesterase inhibitors also increased kemptide phosphorylation (dipyridamole - 171%; rolipram - 106%; and 3-isobutyl-1-methyl-xanthine - 154%). Parasite proliferation was significantly retarded (200 nM H89; 100 microM myristoylated-PKI) or completely inhibited (500 nM H89) by culturing with PKA inhibitors. Incubation with dipyridamole or Sp-cAMPS-AM also inhibited proliferation. Brief treatment (2h) with either H89, myristoylated-PKI, dipyridamole or Sp-cAMPS-AM reduced initial macrophage infection at days 1 and 2 (>40%) and on day 3 (>78% only for 100 microM myr-PKI). Characterization of leishmanial cAMP mediated signal transduction pathways will serve as the basis for the new drug design.

In vitro evaluation of newly synthesised [1,2,4]triazolo[1,5a]pyrimidine derivatives against Trypanosoma cruzi, Leishmania donovani and Phytomonas staheli.

PubMed

Luque, F; Fernández-Ramos, C; Entrala, E; Rosales, M J; Navarro, J A; Romero, M A; Salas, J M; Sánchez-Moreno, M

2000-05-01

The antiprotozoal activity of newly synthesised compounds, all [1,2,4]triazolo [1,5a]pyrimidine derivatives, was tested against the protozoan parasites Trypanosoma cruzi, Leishmania donovani and Phytotmonas staheli. Six of these compounds significantly inhibited in vitro cell growth of the epimastigote forms of T. cruzi, and the promastigote forms of L. donovani and P. staheli. Some of the compounds reached complete growth inhibition at 1 microg/ml for 48 h of parasite/drug interaction. None of the compounds tested showed significant toxicity against cells of Aedes albopictus, mouse macrophages J-774A.1 and Lycopersicum esculentum at dosages five times greater than used against parasites.

Evaluation of antimycobacterial, leishmanicidal and antibacterial activity of three medicinal orchids of Arunachal Pradesh, India.

PubMed

Bhatnagar, Manisha; Sarkar, Nandan; Gandharv, Nigam; Apang, Ona; Singh, Sarman; Ghosal, Sabari

2017-08-01

The ethnic population of Arunachal Pradesh uses a number of orchids as such, or in decoction for various ailments. Three untapped orchids namely, Rhynchostylis retusa, Tropidia curculioides and Satyrium nepalense, traditionally used in tuberculosis, asthma and cold stage of malaria in folk medicine, were selected for the present study. Dried material of each plant was divided into three parts. Solvent extraction and fractionation afforded altogether 30 extracts and fractions, which were evaluated against Mycobacterium tuberculosis (H37Rv and MDR strain) for antimycobacterial activity; promastigotes and amastigotes of Leishmania donovani for leishmanicidal activity and two gram positive and three gram negative clinical isolates for antibacterial activity. The most significant antimycobacterial activity was observed with n-hexane fraction of the flower of Satyrium nepalense with MIC of 15.7 μg/mL. The most promising leishmanicidal activity was observed with diethyl ether fraction of the roots of Rhynchostylis retusa with IC 50 values of 56.04 and 18.4 μg/mL against promastigotes and intracellular amastigotes respectively. Evaluation of antibacterial activity identified S. nepalense flower n-hexane and R. retusa roots diethyl ether as potential fractions with MIC values of ≤100 μg/mL against selected clinical isolates. This is the first report of the plants possessing antimycobacterial and leishmanicidal activity. The investigation resulted in identification of S. nepalense as the most promising plant, which possessed all three activities in significant proportion. This laboratory outcome could be translated to marketable pharmaceutical products and also to produce maximum benefits to the local of nearby area. Antimycobacterial and leishmanicidal activity of medicinal orchids.

Trypanocidal and Leishmanicidal Activities of Sesquiterpene Lactones from Ambrosia tenuifolia Sprengel (Asteraceae) ▿

PubMed Central

Sülsen, Valeria P.; Frank, Fernanda M.; Cazorla, Silvia I.; Anesini, Claudia A.; Malchiodi, Emilio L.; Freixa, Blanca; Vila, Roser; Muschietti, Liliana V.; Martino, Virginia S.

2008-01-01

Bioassay-guided fractionation of the organic extract of Ambrosia tenuifolia Sprengel (Asteraceae) led to the isolation of two bioactive sesquiterpene lactones with significant trypanocidal and leishmanicidal activities. By spectroscopic methods (1H- and 13C-nuclear magnetic resonance, distortionless enhancement by polarization transfer, correlated spectroscopy, heteronuclear multiple-quantum coherence, electron impact-mass spectrometry, and infrared spectroscopy), these compounds were identified as psilostachyin and peruvin. Both compounds showed a marked in vitro trypanocidal activity against Trypanosoma cruzi epimastigotes with 50% inhibitory concentration (IC50) values of less than 2 μg/ml. Psilostachyin exerted a significant in vitro activity against the trypomastigote forms of T. cruzi (IC50, 0.76 μg/ml) and was selected for in vivo testing. Psilostachyin-treated mice had a survival of 100% and lower parasitemia values than control mice. Both compounds were also tested on Leishmania sp. promastigotes: psilostachyin (IC50, 0.12 μg/ml) and peruvin (IC50, 0.39 μg/ml) exerted significant leishmanicidal activities. This is the first time that the trypanocidal and leishmanicidal activities of these compounds have been reported. The selectivity index (SI) was employed to evaluate the cytotoxic effect of lactones on T lymphocytes. Although the SIs of both compounds were high for T. cruzi epimastigotes, psilostachyin was more selective against trypomastigotes (SI, 33.8) while peruvin showed no specificity for this parasite. Both compounds presented high selectivity for Leishmania spp. The results shown herein suggest that psilostachyin and peruvin could be considered potential candidates for the development of new antiprotozoal agents against Chagas' disease and leishmaniasis. PMID:18443111

Leishmania HASP and SHERP Genes Are Required for In Vivo Differentiation, Parasite Transmission and Virulence Attenuation in the Host

PubMed Central

Doehl, Johannes S. P.; Sádlová, Jovana; Aslan, Hamide; Pružinová, Kateřina; Votýpka, Jan; Kamhawi, Shaden; Volf, Petr

2017-01-01

Differentiation of extracellular Leishmania promastigotes within their sand fly vector, termed metacyclogenesis, is considered to be essential for parasites to regain mammalian host infectivity. Metacyclogenesis is accompanied by changes in the local parasite environment, including secretion of complex glycoconjugates within the promastigote secretory gel and colonization and degradation of the sand fly stomodeal valve. Deletion of the stage-regulated HASP and SHERP genes on chromosome 23 of Leishmania major is known to stall metacyclogenesis in the sand fly but not in in vitro culture. Here, parasite mutants deficient in specific genes within the HASP/SHERP chromosomal region have been used to investigate their role in metacyclogenesis, parasite transmission and establishment of infection. Metacyclogenesis was stalled in HASP/SHERP mutants in vivo and, although still capable of osmotaxis, these mutants failed to secrete promastigote secretory gel, correlating with a lack of parasite accumulation in the thoracic midgut and failure to colonise the stomodeal valve. These defects prevented parasite transmission to a new mammalian host. Sand fly midgut homogenates modulated parasite behaviour in vitro, suggesting a role for molecular interactions between parasite and vector in Leishmania development within the sand fly. For the first time, stage-regulated expression of the small HASPA proteins in Leishmania (Leishmania) has been demonstrated: HASPA2 is expressed only in extracellular promastigotes and HASPA1 only in intracellular amastigotes. Despite its lack of expression in amastigotes, replacement of HASPA2 into the null locus background delays onset of pathology in BALB/c mice. This HASPA2-dependent effect is reversed by HASPA1 gene addition, suggesting that the HASPAs may have a role in host immunomodulation. PMID:28095465

Evaluation of photodynamic antimicrobial therapy (PACT) against promastigotes form of the Leishmania (Viannia) braziliensis: in vitro study

NASA Astrophysics Data System (ADS)

Barbosa, Artur F. S.; Sangiorgi, Bruno B.; Galdino, Suely L.; Pitta, Ivan R.; Barral Netto, Manoel; Correia, Neandder A.; Pinheiro, Antônio L. B.

2012-03-01

Leishmaniasis is a complex disease that affects more than 12 million people in 88 countries worldwide. Leishmania (Viannia) braziliensis is the most common species in the Americas and the most important causative agent of cutaneous and mucocutaneous leishmaniasis in Brazil. The therapeutic arsenal routinely employed to treat patients with leishmaniasis is limited and unsatisfactory. For cutaneous leishmaniasis, pentavalent antimonials are the first line therapeutic scheme recommended by the WHO. These compounds are highly toxic, poorly tolerated and their effectiveness highly variable. In this work, a technique with, so far, an unknown disadvantage is discussed. The aim of this study was to verify the effectiveness of PACT in vitro, as a new technique for the treatment of Leishmaniasis. For this, semiconductor laser (λ = 660nm, 40mW, 4.2J/cm2, CW) associated to phenothiazine's derivatives (5 and 10 μg/ml, TBO, Methylene Blue or Phenothiazine) on the promastigotes form of Leishmania braziliensis in a single session was used. Viability of the parasites was assessed in quadruplicates of each group. The samples were removed and analyzed in a hemocytometer 72h after PACT. We found an important decrease in the number of viable parasites on all treated groups in comparison to their controls. The results of present study showed significant percentage of lethality (above 95%) of the protocol. The 99.23% of lethality was achieved with 10 μg/ml of TBO. No lethality was seen on groups treated neither with laser nor with each compounds separately. The results are promising and indicative that the use of PACT may be a powerful treatment of leishmaniasis when compared to already available ones.

Antileishmanial and immunomodulatory activities of lupeol, a triterpene compound isolated from Sterculia villosa.

PubMed

Das, Antu; Jawed, Junaid Jibran; Das, Manash C; Sandhu, Padmani; De, Utpal C; Dinda, Biswanath; Akhter, Yusuf; Bhattacharjee, Surajit

2017-10-01

Visceral leishmaniasis (VL) is one of the most severe forms of leishmaniasis, caused by the protozoan parasite Leishmania donovani. Nowadays there is a growing interest in the therapeutic use of natural products to treat parasitic diseases. Sterculia villosa is an ethnomedicinally important plant. A triterpenoid was isolated from this plant and was screened for its antileishmanial and immunomodulatory activities in vitro and in vivo. Biochemical colour test and spectroscopic data confirmed that the isolated pure compound was lupeol. Lupeol exhibited significant antileishmanial activity, with IC 50 values of 65 ± 0.41 µg/mL and 15 ± 0.45 µg/mL against promastigote and amastigote forms, respectively. Lupeol caused maximum cytoplasmic membrane damage of L. donovani promastigote at its IC 50 dose. It is well known that during infection the Leishmania parasite exerts its pathogenicity in the host by suppressing nitric oxide (NO) production and inhibiting pro-inflammatory responses. It was observed that lupeol induces NO generation in L. donovani-infected macrophages, followed by upregulation of pro-inflammatory cytokines and downregulation of anti-inflammatory cytokines. Lupeol was also found to reduce the hepatic and splenic parasite burden through upregulation of the pro-inflammatory response in L. donovani-infected BALB/c mice. Strong binding affinity of lupeol was observed for four major potential drug targets, namely pteridine reductase 1, adenine phosphoribosyltransferase, lipophosphoglycan biosynthetic protein and glycoprotein 63 of L. donovani, which also supported its antileishmanial and immunomodulatory activities. Therefore, the present study highlights the antileishmanial and immunomodulatory activities of lupeol in an in vitro and in vivo model of VL. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Verbascoside Inhibits Promastigote Growth and Arginase Activity of Leishmania amazonensis.

PubMed

Maquiaveli, Claudia C; Lucon-Júnior, João F; Brogi, Simone; Campiani, Giuseppe; Gemma, Sandra; Vieira, Paulo C; Silva, Edson R

2016-05-27

Verbascoside (1) is a phenylethanoid glycoside that has antileishmanial activity against Leishmania infantum and Leishmania donovani. In this study, we verified the activity of 1 on Leishmania amazonensis and arginase inhibition. Compound 1 showed an EC50 of 19 μM against L. amazonensis promastigotes and is a competitive arginase inhibitor (Ki = 0.7 μM). Docking studies were performed to assess the interaction of 1 with arginase at the molecular level. Arginase is an enzyme of the polyamine biosynthesis pathway that is important to parasite infectivity, and the results of our study suggest that 1 could be useful to develop new approaches for treating leishmaniasis.

Specific immunization of mice against Leishmania mexicana amazonensis using solubilized promastigotes

NASA Technical Reports Server (NTRS)

Barral-Netto, M.; Sadigursky, M.; Reed, S. G.; Sonnenfeld, G.

1987-01-01

In this work, it was demonstrated that mice (BALB/c strain) highly susceptible to Leishmania mexicana amazonensis can be protected against infection by this parasite by being preimmunized with whole solubilized (in a buffer that contained EDTA, NP-40, and SDS) promastigotes; the use of adjuvant or intact inactivated parasite cells is shown to be not necessary. The best immunization schedule consisted of three intravenous injections of 5 x 10 to the 7th parasite equivalents, administered one to eight weeks before infection. Immunized mice exhibited a marked inhibition of primary lesion development, reduced numbers of parasites in the spleen, and reduced death rate.

Cryopreservation of Leishmania Species in Manisa Province.

PubMed

Çavuş, İbrahim; Ocak, Fulya; Kaya, Tuğba; Özbilgin, Ahmet

2017-09-01

It was aimed to assess the success of the cryopreservation process which is carried out in order to preserve the genetic material and the virulence of the Leishmania species that are an important health problem in our region. Leishmania tropica, L. infantum, L. major, and L. donovani strains in Novy-MacNeal-Nicolle (NNN) medium in MCBU were used. Promastigotes cultured in the NNN medium were transferred to RPMI 1640 medium; promastigotes in the logarithmic phase were washed three times with PBS, and 15% dimethylsulfoxide (DMSO) was added. Leishmania species were transferred to 12 separate tubes. The tubes were stored at -86°C for one night by placing them in Coolcell boxes. The tubes were transferred into a liquid nitrogen tank. One cryotube per Leishmania strain is thawed monthly and cultured in NNN medium. For the duration of study it was observed that each Leishmania isolate preserved 60-65% of their viability and entered the logarithmic phase on the 7th day following the inoculation in the NNN medium. Abnormalities in the structures and movements of the promastigotes were not observed in microscopic examinations. The following conclusions were made: cryopreservation is important for studies planned related to leishmaniasis and cryopreservation with DMSO is successful.

In Vitro and In Vivo Antileishmanial Effects of Pistacia khinjuk against Leishmania tropica and Leishmania major

PubMed Central

Saedi Dezaki, Ebrahim; Mahmoudvand, Hossein; Azadpour, Mojgan; Ezzatkhah, Fatemeh

2015-01-01

The present study aims to evaluate the in vitro and in vivo antileishmanial activities of Pistacia khinjuk Stocks (Anacardiaceae) alcoholic extract and to compare its efficacy with a reference drug, meglumine antimoniate (MA, Glucantime), against Leishmania tropica and Leishmania major. This extract (0–100 µg/mL) was evaluated in vitro against promastigote and intracellular amastigote forms of L. tropica (MRHO/IR/75/ER) and then tested on cutaneous leishmaniasis (CL) in male BALB/c mice with L. major to reproduce the antileishmanial activity topically. In vitro, P. khinjuk extract significantly (P < 0.05) inhibited the growth rate of promastigote (IC50 58.6 ± 3.2 µg/mL) and intramacrophage amastigotes (37.3 ± 2.5 µg/mL) of L. tropica as a dose-dependent response. In the in vivo assay, after 30 days of treatment, 75% recovery was observed in the infected mice treated with 30% extract. After treatment of the subgroups with the concentration of 20 and 30% of P. khinjuk extract, mean diameter of lesions was significantly (P < 0.05) reduced. To conclude, the present investigation demonstrated that P. vera extract had in vitro and in vivo effectiveness against L. major. Obtained findings also provide the scientific evidences that natural plants could be used in the traditional medicine for the prevention and treatment of CL. PMID:25815025

Germacranolide-type sesquiterpene lactones from Smallanthus sonchifolius with promising activity against Leishmania mexicana and Trypanosoma cruzi.

PubMed

Ulloa, Jerónimo L; Spina, Renata; Casasco, Agustina; Petray, Patricia B; Martino, Virginia; Sosa, Miguel A; Frank, Fernanda M; Muschietti, Liliana V

2017-11-13

Leishmaniasis and Chagas disease are life-threatening illnesses caused by the protozoan parasites Leishmania spp. and Trypanosoma cruzi, respectively. They are known as "neglected diseases" due to the lack of effective drug treatments and the scarcity of research work devoted to them. Therefore, the development of novel and effective drugs is an important and urgent need. Natural products are an important source of bioactive molecules for the development of new drugs. In this study, we evaluated the activity of enhydrin, uvedalin and polymatin B, three sesquiterpene lactones (STLs) isolated from Smallanthus sonchifolius, on Leishmania mexicana (MNYC/BZ/62/M) and Trypanosoma cruzi (Dm28c). In addition, the in vivo trypanocidal activity of enhydrin and uvedalin and the effects of these STLs on parasites' ultrastructure were evaluated. The inhibitory effect of the three STLs on the growth of L. mexicana amastigotes and promastigotes as well as T. cruzi epimastigotes was evaluated in vitro. The changes produced by the STLs on the ultrastructure of parasites were examined by transmission electron microscopy (TEM). Enhydrin and uvedalin were also studied in a murine model of acute T. cruzi infection (RA strain). Serum activities of the hepatic enzymes alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase were used as biochemical markers of hepatotoxicity. The three compounds exhibited leishmanicidal activity on both parasite forms with IC 50 values of 0.42-0.54 μg/ml for promastigotes and 0.85-1.64 μg/ml for intracellular amastigotes. Similar results were observed on T. cruzi epimastigotes (IC 50 0.35-0.60 μg/ml). The TEM evaluation showed marked ultrastructural alterations, such as an intense vacuolization and mitochondrial swelling in both L. mexicana promastigotes and T. cruzi epimastigotes exposed to the STLs. In the in vivo study, enhydrin and uvedalin displayed a significant decrease in circulating parasites (50-71%) and no signs of hepatotoxicity were detected. Enhydrin, uvedalin and polymatin B possess significant leishmanicidal and trypanocidal activity on different parasite stages. These results show that these compounds may provide valuable leads for the development of new drugs against these neglected parasitic diseases.

Activity of Pt(II) and Ru(III) Triazolopyrimidine Complexes Against Parasites of the Genus Leishmania, Trypanosomas and Phytomonas.

PubMed

Salas, J M; Quirós, M; Abul Haj, M; Magán, R; Marín, C; Sáchez-Moreno, M; Faure, R

2001-01-01

The synthesis and characterization of two Pt(II) Complexes with the isomeric ligands 4,5-dihydro-5-oxo- [1,2,4]triazolo-[ 1,5-a]pyrimidine (5HtpO) and 4,7-dihydro-7-oxo-[ 1,2,4]-triazolo-[ 1,5-a]pyrimidine (7HtpO) are described, as well as a Ru(III) complex with 7HtpO. The crystal structure of cis-[PtCl(2)(7HtpO)(2)].2H(2)O has been solved by X-ray diffraction analysis. In vitro activity of the new isolated complexes against the epimastigote form of T. cruzi, procyclic form of T. b. brucei and promastigote form of L. donnovani and P. characias has also been studied. The three complexes markedly affect the growth of the parasites and none of them shows cytotoxicity against macrophage of the J774.2 line at the heaviest dosages used.

Activity of Pt(II) and Ru(III) Triazolopyrimidine Complexes Against Parasites of the Genus Leishmania, Trypanosomas and Phytomonas

PubMed Central

Quirós, Miguel; Abul Haj, Mohammad; Magán, Rosa; Marín, Clotilde; Sáchez-Moreno, Manuel; Faure, René

2001-01-01

The synthesis and characterization of two Pt(II) Complexes with the isomeric ligands 4,5-dihydro-5-oxo- [1,2,4]triazolo-[ 1,5-a]pyrimidine (5HtpO) and 4,7-dihydro-7-oxo-[ 1,2,4]-triazolo-[ 1,5-a]pyrimidine (7HtpO) are described, as well as a Ru(III) complex with 7HtpO. The crystal structure of cis-[PtCl2(7HtpO)2].2H2O has been solved by X-ray diffraction analysis. In vitro activity of the new isolated complexes against the epimastigote form of T. cruzi, procyclic form of T. b. brucei and promastigote form of L. donnovani and P. characias has also been studied. The three complexes markedly affect the growth of the parasites and none of them shows cytotoxicity against macrophage of the J774.2 line at the heaviest dosages used. PMID:18475985

Antileishmanial activity of the hydroalcoholic extract of Miconia langsdorffii, isolated compounds, and semi-synthetic derivatives.

PubMed

Peixoto, Juliana A; Andrade E Silva, Márcio Luis; Crotti, Antônio E M; Cassio Sola Veneziani, Rodrigo; Gimenez, Valéria M M; Januário, Ana H; Groppo, Milton; Magalhães, Lizandra G; Dos Santos, Fransérgio F; Albuquerque, Sérgio; da Silva Filho, Ademar A; Cunha, Wilson R

2011-02-22

The in vitro activity of the crude hydroalcoholic extract of the aerial parts of Miconia langsdorffii Cogn. was evaluated against the promastigote forms of L. amazonensis, the causative agent of cutaneous leishmaniasis in humans. The bioassay-guided fractionation of this extract led to identification of the triterpenes ursolic acid and oleanolic acid as the major compounds in the fraction that displayed the highest activity. Several ursolic acid semi-synthetic derivatives were prepared, to find out whether more active compounds could be obtained. Among these ursolic acid-derived substances, the C-28 methyl ester derivative exhibited the best antileishmanial activity.

Antileishmanial compounds from Cordia fragrantissima collected in Burma (Myanmar).

PubMed

Mori, Kanami; Kawano, Marii; Fuchino, Hiroyuki; Ooi, Takashi; Satake, Motoyoshi; Agatsuma, Yutaka; Kusumi, Takenori; Sekita, Setsuko

2008-01-01

A methanol extract of the wood of Cordia fragrantissima, collected in Burma (Myanmar), was found to exhibit significant activity against Leishmania major. Bioassay-guided fractionation of this extract using several chromatographic techniques afforded three new compounds (1-3) and five known compounds (4-8). The structures of the new compounds were revealed on the basis of spectroscopic data interpretation and by X-ray crystallographic analysis. Interestingly, the new compounds, despite the presence of asymmetric carbons, were found to be racemates. The activities of the isolates from C. fragrantissima and several derivatives were evaluated against the promastigote forms of Leishmania major, L. panamensis, and L. guyanensis.

Consequences of acute oxidative stress in Leishmania amazonensis: From telomere shortening to the selection of the fittest parasites.

PubMed

da Silva, Marcelo Santos; Segatto, Marcela; Pavani, Raphael Souza; Gutierrez-Rodrigues, Fernanda; Bispo, Vanderson da Silva; de Medeiros, Marisa Helena Gennari; Calado, Rodrigo Tocantins; Elias, Maria Carolina; Cano, Maria Isabel Nogueira

2017-01-01

Leishmaniasis is a spectrum of diseases caused by parasites of the genus Leishmania that affects millions of people around the world. During infection, the parasites use different strategies to survive the host's defenses, including overcoming exposure to reactive oxidant species (ROS), responsible for causing damage to lipids, proteins and DNA. This damage especially affects telomeres, which frequently results in genome instability, senescence and cell death. Telomeres are the physical ends of the chromosomes composed of repetitive DNA coupled with proteins, whose function is to protect the chromosomes termini and avoid end-fusion and nucleolytic degradation. In this work, we induced acute oxidative stress in promastigote forms of Leishmania amazonensis by treating parasites with 2mM hydrogen peroxide (H 2 O 2 ) for 1h, which was able to increase intracellular ROS levels. In addition, oxidative stress induced DNA damage, as confirmed by 8-oxodGuo quantification and TUNEL assays and the dissociation of LaRPA-1 from the 3' G-overhang, leading to telomere shortening. Moreover, LaRPA-1 was observed to interact with newly formed C-rich single-stranded telomeric DNA, probably as a consequence of the DNA damage response. Nonetheless, acute oxidative stress caused the death of some of the L. amazonensis population and induced cell cycle arrest at the G2/M phase in survivor parasites, which were able to continue proliferating and replicating DNA and became more resistant to oxidative stress. Taken together, these results suggest that adaptation occurs through the selection of the fittest parasites in terms of repairing oxidative DNA damage at telomeres and maintaining genome stability in a stressful environment. Copyright © 2016 Elsevier B.V. All rights reserved.

Parasite Killing of Leishmania (V) braziliensis by Standardized Propolis Extracts

PubMed Central

Rebouças-Silva, Jéssica; Celes, Fabiana S.; Lima, Jonilson Berlink

2017-01-01

Treatments based on antimonials to cutaneous leishmaniasis (CL) entail a range of toxic side effects. Propolis, a natural compound widely used in traditional medical applications, exhibits a range of biological effects, including activity against infectious agents. The aim of this study was to test the potential leishmanicidal effects of different propolis extracts against Leishmania (Viannia) braziliensis promastigotes and intracellular amastigotes in vitro. Stationary-phase L. (V) braziliensis promastigotes were incubated with medium alone or treated with dry, alcoholic, or glycolic propolis extract (10, 50, or 100 μg/mL) for 96 h. Our data showed that all extracts exhibited a dose-dependent effect on the viability of L. (V) braziliensis promastigotes, while controlling the parasite burden inside infected macrophages. Dry propolis extract significantly modified the inflammatory profile of murine macrophages by downmodulating TGF-β and IL-10 production, while upmodulating TNF-α. All three types of propolis extract were found to reduce nitric oxide and superoxide levels in activated L. braziliensis-infected macrophages. Altogether, our results showed that propolis extracts exhibited a leishmanicidal effect against both stages of L. (V) braziliensis. The low cell toxicity and efficient microbicidal effect of alcoholic or glycolic propolis extracts make them candidates to an additive treatment for cutaneous leishmaniasis. PMID:28690662

Identification of Proteins in Promastigote and Amastigote-like Leishmania Using an Immunoproteomic Approach

PubMed Central

Coelho, Vinicio T. S.; Oliveira, Jamil S.; Valadares, Diogo G.; Chávez-Fumagalli, Miguel A.; Duarte, Mariana C.; Lage, Paula S.; Soto, Manuel; Santoro, Marcelo M.; Tavares, Carlos A. P.; Fernandes, Ana Paula; Coelho, Eduardo A. F.

2012-01-01

Background The present study aims to identify antigens in protein extracts of promastigote and amastigote-like Leishmania (Leishmania) chagasi syn. L. (L.) infantum recognized by antibodies present in the sera of dogs with asymptomatic and symptomatic visceral leishmaniasis (VL). Methodology/Principal Findings Proteins recognized by sera samples were separated by two-dimensional electrophoresis (2DE) and identified by mass spectrometry. A total of 550 spots were observed in the 2DE gels, and approximately 104 proteins were identified. Several stage-specific proteins could be identified by either or both classes of sera, including, as expected, previously known proteins identified as diagnosis, virulence factors, drug targets, or vaccine candidates. Three, seven, and five hypothetical proteins could be identified in promastigote antigenic extracts; while two, eleven, and three hypothetical proteins could be identified in amastigote-like antigenic extracts by asymptomatic and symptomatic sera, as well as a combination of both, respectively. Conclusions/Significance The present study represents a significant contribution not only in identifying stage-specific L. infantum molecules, but also in revealing the expression of a large number of hypothetical proteins. Moreover, when combined, the identified proteins constitute a significant source of information for the improvement of diagnostic tools and/or vaccine development to VL. PMID:22272364

Serum Removal from Culture Induces Growth Arrest, Ploidy Alteration, Decrease in Infectivity and Differential Expression of Crucial Genes in Leishmania infantum Promastigotes.

PubMed

Alcolea, Pedro J; Alonso, Ana; Moreno-Izquierdo, Miguel A; Degayón, María A; Moreno, Inmaculada; Larraga, Vicente

2016-01-01

Leishmania infantum is one of the species responsible for visceral leishmaniasis. This species is distributed basically in the Mediterranean basin. A recent outbreak in humans has been reported in Spain. Axenic cultures are performed for most procedures with Leishmania spp. promastigotes. This model is stable and reproducible and mimics the conditions of the gut of the sand fly host, which is the natural environment of promastigote development. Culture media are undefined because they contain mammalian serum, which is a rich source of complex lipids and proteins. Serum deprivation slows down the growth kinetics and therefore, yield in biomass. In fact, we have confirmed that the growth rate decreases, as well as infectivity. Ploidy is also affected. Regarding the transcriptome, a high-throughput approach has revealed a low differential expression rate but important differentially regulated genes. The most remarkable profiles are: up-regulation of the GINS Psf3, the fatty acyl-CoA synthase (FAS1), the glyoxylase I (GLO1), the hydrophilic surface protein B (HASPB), the methylmalonyl-CoA epimerase (MMCE) and an amastin gene; and down-regulation of the gPEPCK and the arginase. Implications for metabolic adaptations, differentiation and infectivity are discussed herein.

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity, a process that is counteracted by sandfly saliva

PubMed Central

Rochael, Natalia Cadaxo; Lima, Luize Gonçalves; de Oliveira, Sandra Maria Pereira; Barcinski, Marcello André; Saraiva, Elvira Maria; Monteiro, Robson Queiroz; Pinto-da-Silva, Lucia Helena

2013-01-01

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host's immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva. PMID:24037188

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhaskar,; Kumari, Neeti; Goyal, Neena, E-mail: neenacdri@yahoo.com

Highlights: Black-Right-Pointing-Pointer The study presents cloning and characterization of TCP1{gamma} gene from L. donovani. Black-Right-Pointing-Pointer TCP1{gamma} is a subunit of T-complex protein-1 (TCP1), a chaperonin class of protein. Black-Right-Pointing-Pointer LdTCP{gamma} exhibited differential expression in different stages of promastigotes. Black-Right-Pointing-Pointer LdTCP{gamma} co-localized with actin, a cytoskeleton protein. Black-Right-Pointing-Pointer The data suggests that this gene may have a role in differentiation/biogenesis. Black-Right-Pointing-Pointer First report on this chapronin in Leishmania. -- Abstract: T-complex protein-1 (TCP1) complex, a chaperonin class of protein, ubiquitous in all genera of life, is involved in intracellular assembly and folding of various proteins. The gamma subunit of TCP1 complexmore » (TCP1{gamma}), plays a pivotal role in the folding and assembly of cytoskeleton protein(s) as an individual or complexed with other subunits. Here, we report for the first time cloning, characterization and expression of the TCP1{gamma} of Leishmania donovani (LdTCP1{gamma}), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1{gamma} revealed the presence of all the characteristic features of TCP1{gamma}. However, leishmanial TCP1{gamma} represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1{gamma} exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1{gamma} as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1{gamma} was studied in log phase promastigotes by employing indirect immunofluorescence microscopy. The protein was present not only in cytoplasm but it was also localized in nucleus, peri-nuclear region, flagella, flagellar pocket and apical region. Co-localization of LdTCP1{gamma} with actin suggests that, this gene may have a role in maintaining the structural dynamics of cytoskeleton of parasite.« less

Experimental infection of Leishmania (L.) chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae).

PubMed

Bello, Felio J; Mejía, Astrid J; Corena, María del Pilar; Ayala, Martha; Sarmiento, Ladys; Zuñiga, Claudio; Palau, María T

2005-10-01

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37 masculineC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6%) and on day 4 in the J774 cells (51%). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.

Assessment of the antiprotozoal activity of Pulicaria inuloides extracts, an Algerian medicinal plant: leishmanicidal bioguided fractionation.

PubMed

Fadel, Hamza; Sifaoui, Ines; López-Arencibia, Atteneri; Reyes-Batlle, María; Hajaji, Soumaya; Chiboub, Olfa; Jiménez, Ignacio A; Bazzocchi, Isabel L; Lorenzo-Morales, Jacob; Benayache, Samir; Piñero, José E

2018-02-01

The lack of an effective chemotherapy for treatment of protozoan disease urges a wide investigation for active compounds, and plant-derived compounds continue to provide key leads for therapeutic agents. The current study reports the in vitro antiprotozoal evaluation of the Algerian medicinal plant Pulicaria inuloides against Leishmania amazonensis, Trypanosoma cruzi, and Acanthamoeba castellanii str. Neff. All the extracts from the aerial part showed to be present a higher leishmanicidal activity than anti-Acanthamoeba or Trypanosoma. Therefore, bioguided fractionation of the active CHCl 3 extract led to the isolation and characterization of the flavonol, quercetagetin-3,5,7,3'-tetramethyl ether (1) as the main component. The structure of compound 1 was established by extensive 1D and 2D NMR spectroscopic analysis (COSY, HSQC, HMBC, and ROESY experiments), chemical transformation (derivatives 2 and 3), and comparison with data in the literature. Compound 1 and derivatives 2 and 3 were further evaluated against the promastigote and amastigote stage of L. amazonensis. Compounds 1-3 exhibited moderate leishmanicidal activity with IC 50 values ranging from 0.234 to 0.484 mM and from 0.006 to 0.017 mM for the promastigote and amastigote forms, respectively, as well as low toxicity levels on macrophages (CC 50 ranging from 0.365 to 0.664 mM). This study represents the first report of the antiprotozoal evaluation of Pulicaria inuloides, and the results highlight this species as a promising source of leishmanicidal agents.

First report of natural infection of a bush dog (Speothos venaticus) with Leishmania (Leishmania) chagasi in Brazil.

PubMed

Figueiredo, F B; Gremião, I D F; Pereira, S A; Fedulo, L P; Menezes, R C; Balthazar, D A; Schubach, T M P; Madeira, M F

2008-02-01

We report here the first known case of natural infection of a bush dog with Leishmania (Leishmania) chagasi in Brazil. The specimen was captured in the wild in the State of Mato Grosso and is currently being held in captivity at Fundação Jardim Zoológico, Rio de Janeiro, Brazil. The leishmaniasis was diagnosed by culture of promastigote forms in intact skin fragments and their characterization by isoenzyme electrophoresis. This report calls attention to the parasitological and etiological control of certain zoonoses, such as leishmaniasis, in wild animals kept in captivity, especially when animals are exchanged between zoos in Brazil.

Isopropyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives induce regulated necrosis-like cell death on Leishmania (Leishmania) mexicana.

PubMed

Chacón-Vargas, Karla Fabiola; Andrade-Ochoa, Sergio; Nogueda-Torres, Benjamín; Juárez-Ramírez, Dulce Carolina; Lara-Ramírez, Edgar E; Mondragón-Flores, Ricardo; Monge, Antonio; Rivera, Gildardo; Sánchez-Torres, Luvia Enid

2018-01-01

Leishmaniasis is a neglected tropical disease caused by the parasite of the genus Leishmania. About 13 million people are infected worldwide, and it is estimated that 350 million are at risk of infection. Clinical manifestations depend on the parasite species and factors related to the host such as the immune system, nutrition, housing, and financial resources. Available treatments have severe side effects; therefore, research currently focuses on finding more active and less toxic compounds. Quinoxalines have been described as promising alternatives. In this context, 17 isopropyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives were evaluated as potential leishmanicidal agents. Their effect on the cell metabolism of Leishmania mexicana promastigotes and their cytotoxic effects on the J774.A1 cell line and on erythrocytes were evaluated, and their selectivity index was calculated. Compounds T-069 (IC 50  = 1.49 μg/mL), T-070 (IC 50  = 1.71 μg/mL), T-072 (IC 50  = 6.62 μg/mL), T-073 (IC 50  = 1.25 μg/mL), T-085 (IC 50  = 0.74 μg/mL), and T-116 (IC 50  = 0.88 μg/mL) were the most active against L. mexicana promastigotes and their mechanism of action was characterized by flow cytometry and microscopy. Compound T-073, the most selective quinoxaline derivative, induced cell membrane damage, phosphatidylserine exposition, reactive oxygen species production, disruption of the mitochondrion membrane potential, and DNA fragmentation, all in a dose-dependent manner, indicating the induction of regulated necrosis. Light and transmission electron microscopy showed the drastic morphological changes induced and the mitochondrion as the most sensitive organelle in response to T-073. This study describes the mechanism by which active isopropyl quinoxaline-7-carboxylate 1,4-di-N-oxide quinoxalines affect the parasite.

Deprivation of L-Arginine Induces Oxidative Stress Mediated Apoptosis in Leishmania donovani Promastigotes: Contribution of the Polyamine Pathway

PubMed Central

Mandal, Abhishek; Das, Sushmita; Roy, Saptarshi; Ghosh, Ayan Kumar; Sardar, Abul Hasan; Verma, Sudha; Saini, Savita; Singh, Ruby; Abhishek, Kumar; Kumar, Ajay; Mandal, Chitra; Das, Pradeep

2016-01-01

The growth and survival of intracellular parasites depends on the availability of extracellular nutrients. Deprivation of nutrients viz glucose or amino acid alters redox balance in mammalian cells as well as some lower organisms. To further understand the relationship, the mechanistic role of L-arginine in regulation of redox mediated survival of Leishmania donovani promastigotes was investigated. L-arginine deprivation from the culture medium was found to inhibit cell growth, reduce proliferation and increase L-arginine uptake. Relative expression of enzymes, involved in L-arginine metabolism, which leads to polyamine and trypanothione biosynthesis, were downregulated causing decreased production of polyamines in L-arginine deprived parasites and cell death. The resultant increase in reactive oxygen species (ROS), due to L-arginine deprivation, correlated with increased NADP+/NADPH ratio, decreased superoxide dismutase (SOD) level, increased lipid peroxidation and reduced thiol content. A deficiency of L-arginine triggered phosphatidyl serine externalization, a change in mitochondrial membrane potential, release of intracellular calcium and cytochrome-c. This finally led to DNA damage in Leishmania promastigotes. In summary, the growth and survival of Leishmania depends on the availability of extracellular L-arginine. In its absence the parasite undergoes ROS mediated, caspase-independent apoptosis-like cell death. Therefore, L-arginine metabolism pathway could be a probable target for controlling the growth of Leishmania parasites and disease pathogenesis. PMID:26808657

Identification of Leishmania spp. promastigotes in the intestines, ovaries, and salivary glands of Rhipicephalus sanguineus actively infesting dogs.

PubMed

Viol, Milena Araúz; Guerrero, Felix D; de Oliveira, Bruno César Miranda; de Aquino, Monally Conceição Costa; Loiola, Saulo Hudson; de Melo, Guilherme Dias; de Souza Gomes, Aparecida Helena; Kanamura, Cristina Takami; Garcia, Marcos Valério; Andreotti, Renato; de Lima, Valéria Marçal Félix; Bresciani, Katia Denise Saraiva

2016-09-01

Sand flies are recognized as the major vector of canine visceral leishmaniasis. However, in some areas of Brazil where sand flies do not occur, this disease is found in humans and dogs. There has been speculation that ticks might play a role in transmission of canine visceral leishmaniasis and the DNA of Leishmania spp. has been reported in whole ticks. We investigated the presence of Leishmania spp. promastigotes in the intestines, ovaries, and salivary glands of Rhipicephalus sanguineus ticks collected from tick-infested dogs in two cities of Brazil. We used 66 dogs that tested positive and 33 that tested negative for Leishmania spp. according to direct cytological examination assays. Ten ticks were collected from each dog and dissected to collect the intestines, ovaries, and salivary glands for immunohistochemistry (IHC) and diagnostic real-time polymerase chain reaction (RT-PCR). IHC results showed Leishmania spp. in 98, 14, and 8 % of the intestines, ovaries, and salivary glands, respectively. Real-time PCR showed that 89, 41, and 33 % of the tick intestine, ovary, and salivary glands, respectively, were positive for Leishmania spp. The verification of promastigotes of Leishmania spp. by two independent techniques in ticks collected from these urban region dogs showed that there is need for clarification of the role of ticks in the transmission of canine visceral leishmaniasis in Brazil.

Evaluation of methylene blue as photosensitizer in promastigotes of Leishmania major and Leishmania braziliensis.

PubMed

Pinto, Juliana Guerra; Martins, Jaciara Fagundes de Souza; Pereira, André Henrique Correia; Mittmann, Josane; Raniero, Leandro José; Ferreira-Strixino, Juliana

2017-06-01

The cutaneous leishmaniasis is caused by the protozoan of the genus Leishmania. It is considered by WHO as a public health issue and a neglected disease, which affects rural workers and it is also a risk to travelers in endemic areas. The conventional treatment is toxic and leads to severe side effects. The photodynamic therapy has been studied as an alternative treatment to cutaneous leishmaniasis. This study aimed to evaluate the methylene blue internalization and the impact of the PDT in the viability and morphology of Leishmania major and Leishmania braziliensis promastigote in culture medium. The fluorescence microscopy was used to determine the MB localization. To evaluate the mitochondrial activity (MTT), viability (Trypan blue test) and the morphological alterations both species were incubated with the MB in concentrations starting in 500μg/ml, in serial dilution, until 7,8μg/ml. The fluorescence microscopy demonstrated that the MB is internalized by both species after one hour of incubation. The MB presented low toxicity at the dark and the PDT was capable of decreasing the viability in more than 70% in the higher concentrations tested. The PDT also triggered significant morphological alterations in the Leishmania promastigotes. The results presented in this study are an indicative that the MB is a photosensitizer with promising potential to clinical application, besides its low cost. Copyright © 2017 Elsevier B.V. All rights reserved.

In vitro and in vivo leishmanicidal activity of Astronium fraxinifolium (Schott) and Plectranthus amboinicus (Lour.) Spreng against Leishmania (Viannia) braziliensis.

PubMed

de Lima, Silvio César Gomes; Teixeira, Maria Jania; Lopes, José Evaldo Gonçalves; de Morais, Selene Maia; Torres, Alba Fabiola; Braga, Milena Aguiar; Rodrigues, Raphael Oliveira; Santiago, Gilvandete Maria Pinheiro; Martins, Alice Costa; Nagao-Dias, Aparecida Tiemi

2014-01-01

The aim of the present work was to evaluate antileishmanial activity of Astronium fraxinifolium and Plectranthus amboinicus. For the in vitro tests, essential oil of P. amboinicus (OEPA) and ethanolic extracts from A. fraxinifolium (EEAF) were incubated with 10(6) promastigotes of L. (Viannia) braziliensis. The OEPA was able to reduce the parasite growth after 48 h; nonetheless, all the EEAFs could totally abolish the parasite growth. For the in vivo studies, BALB/c mice were infected subcutaneously (s.c.) with 10(7) L. braziliensis promastigotes. Treatment was done by administering OEPA intralesionally (i.l.) for 14 days. No difference was found in lesion thickness when those animals were compared with the untreated animals. Further, golden hamsters were infected s.c. with 10(6) L. braziliensis promastigotes. The first protocol of treatment consisted of ethanolic leaf extract from A. fraxinifolium (ELEAF) administered i.l. for 4 days and a booster dose at the 7th day. The animals showed a significant reduction of lesion thickness in the 6th week, but it was not comparable to the animals treated with Glucantime. The second protocol consisted of 15 daily intralesional injections. The profiles of lesion thickness were similar to the standard treatment. In conclusion, in vivo studies showed a high efficacy when the infected animals were intralesionally treated with leaf ethanolic extract from A. fraxinifolium.

Altered expression of an RBP-associated arginine methyltransferase 7 in Leishmania major affects parasite infection.

PubMed

Ferreira, Tiago R; Alves-Ferreira, Eliza V C; Defina, Tania P A; Walrad, Pegine; Papadopoulou, Barbara; Cruz, Angela K

2014-10-08

Protein arginine methylation is a widely conserved post-translational modification performed by arginine methyltransferases (PRMTs). However, its functional role in parasitic protozoa is still under-explored. The Leishmania major genome encodes five PRMT homologs, including PRMT7. Here we show that LmjPRMT7 expression and arginine monomethylation are tightly regulated in a lifecycle stage-dependent manner. LmjPRMT7 levels are higher during the early promastigote logarithmic phase, negligible at stationary and late-stationary phases and rise once more post-differentiation to intracellular amastigotes. Immunofluorescence and co-immunoprecipitation studies demonstrate that LmjPRMT7 is a cytosolic protein associated with several RNA-binding proteins (RBPs) from which Alba20 is monomethylated only in LmjPRMT7-expressing promastigote stages. In addition, Alba20 protein levels are significantly altered in stationary promastigotes of the LmjPRMT7 knockout mutant. Considering RBPs are well-known mammalian PRMT substrates, our data suggest that arginine methylation via LmjPRMT7 may modulate RBP function during Leishmania spp. lifecycle progression. Importantly, genomic deletion of the LmjPRMT7 gene leads to an increase in parasite infectivity both in vitro and in vivo, while lesion progression is significantly reduced in LmjPRMT7-overexpressing parasites. This study is the first to describe a role of Leishmania protein arginine methylation in host-parasite interactions. © 2014 John Wiley & Sons Ltd.

Preparation of live attenuated leishmania parasites by using laser technology

NASA Astrophysics Data System (ADS)

Hussain, Nabiha; Alkhouri, Hassan; Haddad, Shaden

2018-05-01

Leishmaniasis is a parasitic disease of humans, affecting the skin, mucosal and/or internal organs, caused by flagellate protozoa Leishmania of the Trypanosomatidae family. Leishmania would be one for which a vaccine could be developed with relative ease. Many studies mount an effective response that resolves the infection and confers solid immunity to reinfection and suggesting that infection may be a prerequisite for immunological memory. Genetically altered live attenuated parasites with controlled infectivity could achieve such immunological memory. Recent concepts include use of genetically modified live-attenuated Leishmania parasites, and proteomics approach for the search of a cross-protective leishmanial vaccine that would ideally protect against both cutaneous and visceral forms of the disease. No licensed vaccine is available till date against any form of leishmaniasis. The present study evaluated role of laser technology in development of a safe live Leishmania vaccine, a vaccine is a biological preparation that improves immunity to a particular disease, and is often made from weakened or killed forms of LPs. The parasite culture was expanded in RPMI 1640 medium with 10% fetal calf serum (FCS) and grown until stationary phase for experiments. 80 samples of leishmania promastigotes (Culture media of LPs) were exposed to Nd:YAG laser (wavelength 1064 nm, single spot or double) with different outputs powers (7w, 100 Hz, 99.03w/cm2, 0.99 J/cm2 and 8 w, 100 Hz, 113.18w/cm2 1.13J/cm2)) for suitable exposer times. The effect of semiconductor laser (wavelength 810 nm, 7w, 2000 Hz, 99.03w/cm2, 0.05 J/cm2) or (7 w, 500 Hz, 99.03 w/cm2, 0.2J/cm2) single spot or double with long exposure times. The viability of Leishmania parasites was measured using XTT method; viable parasites were decreased with long exposure times. XTT test referred both these wavelengths were effective in killing percentage of Leishmania promastigotes, the remaining were devoid flagellum that may lost their ability to cause infection and could be used as vaccine.

Antileishmanial activity of berenil and methylglyoxal bis (guanylhydrazone) and its correlation with S-adenosylmethionine decarboxylase and polyamines.

PubMed

Mukhopadhyay, R; Madhubala, R

1995-01-01

Leishmania donovani S-adenosyl-L-methionine (AdoMet) decarboxylase was found to show a growth related pattern. Methylglyoxal bis (guanylhydrazone) (MGBG) and Berenil inhibited the growth of Leishmania donovani promastigotes (strain UR6) in a dose dependent manner. The concentrations of MGBG and Berenil required for 50% inhibition of rate of growth were 67 and 47 microM, respectively. The growth inhibition of MGBG was partially reversed by spermidine (100 microM) and spermine (100 microM). Berenil inhibition of promastigote growth was partially reversed by 100 microM spermidine whereas 100 microM spermine did not result in any reversal of growth. The reduction in parasitemia in vitro by these inhibitors was accompanied by inhibition of AdoMet decarboxylase activity and spermidine levels.

Assessment of amphotericin B susceptibility in Leishmania infantum promastigotes by flow cytometric membrane potential assay.

PubMed

Azas, N; Di Giorgio, C; Delmas, F; Gasquet, M; Timon-David, P

1997-06-01

Flow cytometry was used for measuring the effects of amphotericin B on the membrane of Leishmania infantum strains. The technique was adapted from the rapid flow cytometric membrane potential assay developed by Ordonez and Wehman (Cytometry 22:154-157, 1995) for evaluating antibiotic-susceptibility of Candida species. The study consisted of measuring membrane potential changes induced by amphotericin B in 3 initial strains and 12 laboratory-generated variants adapted to grow with amphotericin B. Results showed that, after 3 h of incubation, amphotericin B induced a dose-related decrease of membrane potential that reached its maximal level at the same concentrations that inhibited parasite growth. These results suggest that the flow cytometric membrane potential assay could be used to assess the susceptibility of Leishmania promastigotes to amphotericin B.

Essential oils: in vitro activity against Leishmania amazonensis, cytotoxicity and chemical composition.

PubMed

Andrade, Milene Aparecida; Azevedo, Clênia Dos Santos; Motta, Flávia Nader; Santos, Maria Lucília Dos; Silva, Camila Lasse; Santana, Jaime Martins de; Bastos, Izabela M D

2016-11-08

The current chemotherapy for cutaneous leishmaniosis (CL) has a series of drug limitations such as toxic side effects, long duration, high costs and drug resistance, which requires the development of new drugs or effective alternatives to the CL treatment. Essential oils (EOs) are complex mixtures of secondary metabolites from various plants. It has been shown that several EOs, or their constituents, have inhibitory activity against protozoa. Thus, this study aims to evaluate the biological activity of different essential oils (EOs) on Leishmania (L.) amazonensis promastigotes forms, as well as their cytotoxicity on mammalian cells and chemical composition. Sixteen EOs were evaluated by mean of IC 50 /24 h and cytotoxicity against L6 cells (CC 50 /24 h) using Resazurin assay. Only those EOs that presented better results for IC 50 /24 h were submitted to GC-MS analysis to determine their chemical constitution. The EO from Cinnamodendron dinisii, Matricaria chamomilla, Myroxylon peruiferum, Salvia sclarea, Bulnesia sarmientoi, Ferula galbaniflua, Siparuna guianensis and Melissa officinalis were the most active against L. amazonensis with IC50/24 h ranging from 54.05 to 162.25 μg/mL. Analysis of EOs by GC-MS showed mainly the presence of β-farnesene (52.73 %) and bisabolol oxide (12.09 %) for M. chamomilla; α-copaene (13.41 %), safrole (8.35 %) and δ-cadinene (7.08 %) for M. peruiferum; linalool (28.80 %) and linalyl acetate (60.08 %) for S. sclarea; guaiol (48.29 %) and 2-undecanone (19.49 %) for B. sarmientoi; ethyl phthalate (13.09 %) and methyl-8-pimaren-18-oate (41.82 %) for F. galbaniflua; and neral (37.18 %) and citral (5.02 %) for M. officinalis. The EO from F. galbaniflua showed to be effective against L. amazonensis promastigotes forms and presented low cytotoxic activity against L6 cells. Thus, it represents a strong candidate for future studies aiming its molecular activity on these pathogenic parasites.

In vitro activity of new N-benzyl-1H-benzimidazol-2-amine derivatives against cutaneous, mucocutaneous and visceral Leishmania species.

PubMed

Nieto-Meneses, Rocío; Castillo, Rafael; Hernández-Campos, Alicia; Maldonado-Rangel, Armando; Matius-Ruiz, Jeferson B; Trejo-Soto, Pedro Josué; Nogueda-Torres, Benjamín; Dea-Ayuela, Ma Auxiliadora; Bolás-Fernández, Francisco; Méndez-Cuesta, Carlos; Yépez-Mulia, Lilián

2018-01-01

The identification of specific therapeutic targets and the development of new drugs against leishmaniasis are urgently needed, since chemotherapy currently available for its treatment has several problems including many adverse side effects. In an effort to develop new antileishmanial drugs, in the present study a series of 28 N-benzyl-1H-benzimidazol-2-amine derivatives was synthesized and evaluated in vitro against Leishmania mexicana promastigotes. Compounds 7 and 8 with the highest antileishmanial activity (micromolar) and lower cytotoxicity than miltefosine and amphotericin B were selected to evaluate their activity against L. braziliensis 9and L. donovani, species causative of mucocutaneous and visceral leishmaniasis, respectively. Compound 7 showed significantly higher activity against L. braziliensis promastigotes than compound 8 and slightly lower than miltefosine. Compounds 7 and 8 had IC 50 values in the micromolar range against the amastigote of L. mexicana and L. braziliensis. However, both compounds did not show better activity against L. donovani than miltefosine. Compound 8 showed the highest SI against both parasite stages of L. mexicana. In addition, compound 8 inhibited 68.27% the activity of recombinant L. mexicana arginase (LmARG), a therapeutic target for the treatment of leishmaniasis. Docking studies were also performed in order to establish the possible mechanism of action by which this compound exerts its inhibitory effect. Compound 8 shows promising potential for the development of more potent antileishmanial benzimidazole derivatives. Copyright © 2017 Elsevier Inc. All rights reserved.

Leishmania cell surface prohibitin: role in host-parasite interaction.

PubMed

Jain, Rohit; Ghoshal, Angana; Mandal, Chitra; Shaha, Chandrima

2010-04-01

Proteins selectively upregulated in infective parasitic forms could be critical for disease pathogenesis. A mammalian prohibitin orthologue is upregulated in infective metacyclic promastigotes of Leishmania donovani, a parasite that causes visceral leishmaniasis. Leishmania donovani prohibitin shares 41% similarity with mammalian prohibitin and 95-100% within the genus. Prohibitin is concentrated at the surface of the flagellar and the aflagellar pole, the aflagellar pole being a region through which host-parasite interactions occur. Prohibitin is attached to the membrane through a GPI anchor. Overexpression of wild-type prohibitin increases protein surface density resulting in parasites with higher infectivity. However, parasites overexpressing a mutant prohibitin with an amino acid substitution at the GPI anchor site to prevent surface expression through GPI-link show lesser surface expression and lower infective abilities. Furthermore, the presence of anti-prohibitin antibodies during macrophage-Leishmania interaction in vitro reduces infection. The cognate binding partner for Leishmania prohibitin on the host cell appears to be macrophage surface HSP70, siRNA mediated downregulation of which abrogates the capability of the macrophage to bind to parasites. Leishmania prohibitin is able to generate a strong humoral response in visceral leishmaniasis patients. The above observations suggest that prohibitin plays an important role in events leading to Leishmania-host interaction.

Gene disruptions indicate an essential function for the LmmCRK1 cdc2-related kinase of Leishmania mexicana.

PubMed

Mottram, J C; McCready, B P; Brown, K G; Grant, K M

1996-11-01

The generation of homozygous null mutants for the crk1 Cdc2-Related Kinase of Leishmania mexicana was attempted using targeted gene disruption. Promastigote mutants heterozygous for crk1 were readily isolated with a hyg-targeting fragment, but attempts to create null mutants by second-round transfections with a bie-targeting fragment yielded two classes of mutant, neither of which was null. First, the transfected fragment formed an episome; second, the cloned transfectants were found to contain wild-type crk1 alleles as well as hyg and ble integrations. DNA-content analysis revealed that these mutants were triploid or tetraploid. Plasticity in chromosome number following targeting has been proposed as a means by which Leishmania avoids deletion of essential genes. These data support this theory and implicate crk1 as an essential gene, validating CRK1 as a potential drug target. L mexicana transfected with a Trypanosoma brucel homologue, tbcrk1, was shown to be viable in an immcrk1 null background, thus showing complementation of function between these trypanosomatid genes. The expression of crk1 was further manipulated by engineering a six-histidine tag at the C-terminus of the kinase, allowing purification of the active complex by affinity selection on Nl(2+)-nitriloacetic acid (NTA) agarose.

Th1-stimulatory polyproteins of soluble Leishmania donovani promastigotes ranging from 89.9 to 97.1 kDa offers long-lasting protection against experimental visceral leishmaniasis.

PubMed

Kumari, Shraddha; Samant, Mukesh; Misra, Pragya; Khare, Prashant; Sisodia, Brijesh; Shasany, Ajit K; Dube, Anuradha

2008-10-23

Our earlier studies identified a fraction (F2) of Leishmania donovani soluble promastigote antigen belonging to 97.4-68 kDa for its ability to stimulate Th1-type cellular responses in cured visceral leishmaniasis (VL) patients as well as in cured hamsters. A further fractionation of F2-fraction into seven subfractions (F2.1-F2.7) and re-assessment for their immunostimulatory responses revealed that out of these, only four (F2.4-F2.7) belonging to 89.9-97.1 kDa, stimulated remarkable Th1-type cellular responses either individually or in a pooled form (P4-7). In this study these potential subfractions were further assessed for their prophylactic potential in combination with BCG against L. donovani challenge in hamsters. Optimum parasite inhibition ( approximately 99%) was obtained in hamsters vaccinated with pooled subfractions and they survived for 1 year. The protection was further supported by remarkable lymphoproliferative, IFN-gamma and IL-12 responses along with profound delayed type hypersensitivity and increased levels of Leishmania-specific IgG2 antibody as observed on days 45, 90 and 120 post-challenge suggesting that a successful subunit vaccine against VL may require multiple Th1-immunostimulatory proteins. MALDI-TOF-MS/MS analysis of these subfractions further revealed that of the 19 identified immunostimulatory proteins, Elongation factor-2, p45, Heat shock protein-70/83, Aldolase, Enolase, Triosephosphate isomerase, Disulfideisomerase and Calreticulin were the major ones in these subfractions.

Physalis angulata induces death of promastigotes and amastigotes of Leishmania (Leishmania) amazonensis via the generation of reactive oxygen species.

PubMed

Da Silva, B J M; Da Silva, R R P; Rodrigues, A P D; Farias, L H S; Do Nascimento, J L M; Silva, E O

2016-03-01

Leishmaniasis are a neglected group of emerging diseases that have been found in 98 countries and are caused by protozoa of the genus Leishmania. The therapy for leishmaniasis causes several side effects and leads to drug-resistant strains. Natural products from plants have exhibited activities against Leishmania in various experimental models. Physalis angulata is a widely used plant in popular medicine, and in the literature it has well-documented leishmanicidal activity. However, its mechanism of action is still unknown. Thus, this study aims to evaluate the mechanism driving the leishmanicidal activity of an aqueous extract of P. angulata root (AEPa). AEPa was effective against both promastigotes and intracellular amastigote forms of Leishmania amazonensis. This effect was mediated by an increase of reactive oxygen species (ROS), but not of nitric oxide (NO). The increased production of ROS induces cell death by phenotypes seems by apoptosis cell death in Leishmania, but not autophagy or necrosis. In addition, morphological analysis of macrophages showed that AEPa induced a high number of cytoplasmic projections, increased the volume of cytoplasm and number of vacuoles, caused cytoskeleton alterations and resulted in high spreading ability. AEPa also promoted superoxide anion (O2(-)) production in both uninfected macrophages and those infected with Leishmania. Therefore, these results revealed that AEPa causes cell death by phenotypes seems by apoptosis cell death in L. amazonensis and modulates macrophage activation through morphofunctional alterations and O2(-) generation to induce Leishmania death. Copyright © 2015 Elsevier Ltd. All rights reserved.

Morinda citrifolia Linn. fruit (Noni) juice induces an increase in NO production and death of Leishmania amazonensis amastigotes in peritoneal macrophages from BALB/c.

PubMed

Almeida-Souza, Fernando; de Souza, Celeste da Silva Freitas; Taniwaki, Noemi Nosomi; Silva, João José Mendes; de Oliveira, Renata Mondêgo; Abreu-Silva, Ana Lúcia; Calabrese, Kátia da Silva

2016-08-31

Leishmaniasis is a complex disease that is considered a serious public health problem. Due to the absence of an effective vaccine and debilitating chemotherapy better therapies are urgently needed. This situation has stimulated the search for alternative treatments such as the use of herbal medicines. Several studies conducted with Morinda citrifolia Linn. have shown various biological activities such as antitumor, immunomodulation and antileishmanial activity, however its mechanisms of action are still unknown. This study aimed to analyze the activity of M. citrifolia fruit juice against Leishmania amazonensis and its action on peritoneal macrophages from BALB/c infected with L. amazonensis. Activity against the promastigote forms showed IC50 at 275.3 μg/mL. Transmission electron microscopy was used to evaluate the ultrastructural alterations in the promastigotes treated with the juice and the results showed cytoplasmic vacuolization, lipid inclusion and increased activity of exocytosis. The juice treatment presented an IC50 at 208.4 μg/mL against intracellular amastigotes and led to an increased nitrite production in infected and non-infected macrophages. When macrophages were pre-treated with iNOS inhibitors, aminoguanidine or 1400W, the intracellular amastigotes increased, demonstrating the important role of NO production in M. citrifolia fruit activity. In conclusion, our results reveal that treatment with M. citrifolia fruit juice can increase NO production in peritoneal macrophages and this ability has an important role in the killing of L. amazonensis intracellular amastigotes. Copyright © 2016 Elsevier Inc. All rights reserved.

Syzygium cumini (L.) Skeels essential oil and its major constituent α-pinene exhibit anti-Leishmania activity through immunomodulation in vitro.

PubMed

Rodrigues, Klinger Antonio da Franca; Amorim, Layane Valéria; Dias, Clarice Noleto; Moraes, Denise Fernandes Coutinho; Carneiro, Sabrina Maria Portela; Carvalho, Fernando Aécio de Amorim

2015-02-03

Syzygium cumini (L.) Skeels (Myrtaceae), commonly known as "jambolão" in Brazil is widely used in folk medicine against leishmaniasis, inflammation, chronic diarrhea, and ulcers. It is one of the most commonly used plants for the treatment of diabetes worldwide. In previous studies, Syzygium cumini was shown to possess antihyperlipidemic and anti-allergic properties, and to exhibit good performance as an antimicrobial agent against bacteria, fungi, and protozoa parasites of the genus Leishmania and Trypanosoma. This study was aimed at evaluating the effects of S. cumini essential oil (ScEO) and its major component α-pinene on Leishmania (Leishmania) amazonensis, as well as their cytotoxicity and possible mechanisms of action. To evaluate the anti-proliferative effect on Leishmania, effects on promastigote and axenic amastigote forms were assessed using tetrazolium salt (MTT) assay. The intramacrophagic amastigotes were exposed to ScEO and α-pinene to determine the survival index. To gain insight into the mechanism of action involved in the effect on the samples, we evaluated the modulation of macrophage activation state by observing structural (phagocytic and lysosomal activities) and cellular (nitric oxide increase) changes. To assess the safety profile of ScEO and α-pinene, murine macrophages and human red blood cells were treated with ScEO and α-pinene and the selectivity index was calculated for each treatment. α-Pinene was effective against Leishmania amazonensis promastigote forms, with a half-maximal inhibitory concentration (IC50) value of 19.7µg/mL. α-Pinene was more active (IC50 values of 16.1 and 15.6µg/mL against axenic and intracellular amastigotes, respectively) than ScEO (IC50 values of 43.9 and 38.1µg/mL against axenic and intracellular amastigotes, respectively). Our results showed that the anti-Leishmania effects were mediated by immunomodulatory activity, as evidenced by the observed increases in both phagocytic and lysosomal activity, and the elevated NO levels. ScEO and α-pinene exhibited low cytotoxicity against murine macrophages and human erythrocytes. The 50% cytotoxicity concentration (CC50) values for the macrophages in the MTT assay were 614.1 and 425.2µg/mL for ScEO and α-pinene, respectively, while the corresponding half-maximal hemolytic concentration (HC50) values were 874.3 and 233.3µg/mL. Taken together, the results demonstrate that ScEO and its major constituent α-pinene have significant anti-Leishmania activity, modulated by macrophage activation, with acceptable levels of cytotoxicity in murine macrophages and human erythrocytes. Further work is warranted, involving more in-depth mechanistic studies and in vivo investigations. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

In Vitro Sensitivity of Cutaneous Leishmania Promastigote Isolates Circulating in French Guiana to a Set of Drugs

PubMed Central

Ginouvès, Marine; Simon, Stéphane; Nacher, Mathieu; Demar, Magalie; Carme, Bernard; Couppié, Pierre; Prévot, Ghislaine

2017-01-01

Anti-leishmaniasis drug resistance is a common problem worldwide. The aim of this study was to inventory the general in vitro level of sensitivity of Leishmania isolates circulating in French Guiana and to highlight potential in vitro pentamidine-resistant isolates. This sensitivity study was conducted on 36 patient-promastigote isolates for seven drugs (amphotericin B, azithromycin, fluconazole, meglumine antimoniate, miltefosine, paromomycin, and pentamidine) using the Cell Counting Kit-8 viability test. The IC50 values obtained were heterogeneous. One isolate exhibited high IC50 values for almost all drugs tested. Pentamidine, which is the first-line treatment in French Guiana, showed efficacy at very low doses (mean of 0.0038 μg/mL). The concordance of the in vitro pentamidine results with the patients' clinical outcomes was 94% (K = 0.82). PMID:28167598

LABCG2, a New ABC Transporter Implicated in Phosphatidylserine Exposure, Is Involved in the Infectivity and Pathogenicity of Leishmania

PubMed Central

González-Rey, Elena; Delgado, Mario; Castanys, Santiago; Pérez-Victoria, José M.; Gamarro, Francisco

2013-01-01

Leishmaniasis is a neglected disease produced by the intracellular protozoan parasite Leishmania. In the present study, we show that LABCG2, a new ATP-binding cassette half-transporter (ABCG subfamily) from Leishmania, is involved in parasite virulence. Down-regulation of LABCG2 function upon expression of an inactive mutant version of this half-transporter (LABCG2K/M) is shown to reduce the translocation of short-chain analogues of phosphatidylserine (PS). This dominant-negative phenotype is specific for the headgroup of the phospholipid, as the movement of phospholipid analogues of phosphatidylcholine, phosphatidylethanolamine or sphingomyelin is not affected. In addition, promastigotes expressing LABCG2K/M expose less endogenous PS in the stationary phase than control parasites. Transient exposure of PS at the outer leaflet of the plasma membrane is known to be one of the mechanisms used by Leishmania to infect macrophages and to silence their immune response. Stationary phase/metacyclic promastigotes expressing LABCG2K/M are less infective for macrophages and show decreased pathogenesis in a mouse model of cutaneous leishmaniasis. Thus, mice infected with parasites expressing LABCG2K/M did not develop any lesion and showed significantly lower inflammation and parasite burden than mice infected with control parasites. Our results indicate that LABCG2 function is required for the externalization of PS in Leishmania promastigotes, a process that is involved in the virulence of the parasite. PMID:23638200

Internal and Surface-Localized Major Surface Proteases of Leishmania spp. and Their Differential Release from Promastigotes▿

PubMed Central

Yao, Chaoqun; Donelson, John E.; Wilson, Mary E.

2007-01-01

Major surface protease (MSP), also called GP63, is a virulence factor of Leishmania spp. protozoa. There are three pools of MSP, located either internally within the parasite, anchored to the surface membrane, or released into the extracellular environment. The regulation and biological functions of these MSP pools are unknown. We investigated here the trafficking and extrusion of surface versus internal MSPs. Virulent Leishmania chagasi undergo a growth-associated lengthening in the t1/2 of surface-localized MSP, but this did not occur in the attenuated L5 strain. The release of surface-localized MSP was enhanced in a dose-dependent manner by MβCD, which chelates membrane cholesterol-ergosterol. Furthermore, incubation of promastigotes at 37°C with Matrigel matrix, a soluble basement membrane extract of Engelbreth-Holm-Swarm tumor cells, stimulated the release of internal MSP but not of surface-located MSP. Taken together, these data indicate that MSP subpopulations in distinct cellular locations are released from the parasite under different environmental conditions. We hypothesize that the internal MSP with its lengthy t1/2 does not serve as a pool for promastigote surface MSP in the sand fly vector but that it instead functions as an MSP pool ready for quick release upon inoculation of metacyclic promastigotes into mammals. We present a model in which these different MSP pools are released under distinct life cycle-specific conditions. PMID:17693594

Antiprotozoal activity of quinonemethide triterpenes from Maytenus ilicifolia (Celastraceae).

PubMed

Dos Santos, Vania A F F M; Leite, Karoline M; da Costa Siqueira, Mariana; Regasini, Luis O; Martinez, Isabel; Nogueira, Camila T; Galuppo, Mariana Kolos; Stolf, Beatriz S; Pereira, Ana Maria Soares; Cicarelli, Regina M B; Furlan, Maysa; Graminha, Marcia A S

2013-01-15

The present study describes the leishmanicidal and trypanocidal activities of two quinonemethide triterpenes, maytenin (1) and pristimerin (2), isolated from Maytenus ilicifolia root barks (Celastraceae). The compounds were effective against the Trypanosomatidae Leishmania amazonensis and Leishmania chagasi and Trypanosoma cruzi, etiologic agents of leishmaniasis and Chagas' disease, respectively. The quinonemethide triterpenes 1 and 2 exhibited a marked in vitro leishmanicidal activity against promastigotes and amastigotes with 50% inhibitory concentration (IC(50)) values of less than 0.88 nM. Both compounds showed IC(50) lower than 0.3 nM against Trypanosoma cruzi epimastigotes. The selectivity indexes (SI) based on BALB/c macrophages for L. amazonensis and L. chagasi were 243.65 and 46.61 for (1) and 193.63 and 23.85 for (2) indicating that both compounds presented high selectivity for Leishmania sp. The data here presented suggests that these compounds should be considered in the development of new and more potent drugs for the treatment of leishmaniasis and Chagas' disease.

The 2',4'-dihydroxychalcone could be explored to develop new inhibitors against the glycerol-3-phosphate dehydrogenase from Leishmania species.

PubMed

Passalacqua, Thais G; Torres, Fábio A E; Nogueira, Camila T; de Almeida, Leticia; Del Cistia, Mayara L; dos Santos, Mariana B; Dutra, Luis A; Bolzani, Vanderlan da Silva; Regasini, Luis O; Graminha, Márcia A S; Marchetto, Reinaldo; Zottis, Aderson

2015-09-01

The enzyme glycerol-3-phosphate dehydrogenase (G3PDH) from Leishmania species is considered as an attractive target to design new antileishmanial drugs and a previous in silico study reported on the importance of chalcones to achieve its inhibition. Here, we report the identification of a synthetic chalcone in our in vitro assays with promastigote cells from Leishmania amazonensis, its biological activity in animal models, and docking followed by molecular dynamics simulation to investigate the molecular interactions and structural patterns that are crucial to achieve the inhibition complex between this compound and G3PDH. A molecular fragment of this natural product derivative can provide new inhibitors with increased potency and selectivity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Selective effects of Euterpe oleracea (açai) on Leishmania (Leishmania) amazonensis and Leishmania infantum.

PubMed

Da Silva, Bruno José Martins; Souza-Monteiro, José Rogério; Rogez, Herve; Crespo-López, Maria Elena; Do Nascimento, Jose Luiz M; Silva, Edilene O

2018-01-01

Leishmania (Leishmania) amazonensis and Leishmania infantum (=Leishmania chagasi) are protozoa that cause American cutaneous and visceral leishmaniasis, respectively. These diseases show a high incidence in developing countries such as Brazil. The treatments used for leishmaniasis are still limited due to their high cost and toxicity. Currently, some natural products are considered an important alternative source of new leishmanicidal agents. Euterpe oleracea Martius, a palm producing black fruits, is frequently consumed in the Amazon region, as a juice, known as açai, with potent antioxidant, anti-inflammatory and anticonvulsant properties. Interestingly, the biological activity of clarified açai juice (EO) on L. (L.) amazonensis and L. infantum (=L. chagasi) is unknown. Therefore, the mechanism of anti-leishmanial action of EO has been evaluated on L. (L.) amazonensis and L. infantum (=L. chagasi). EO reduced the number of promastigotes and caused morphological alterations, increased the production of reactive oxygen species (ROS) and induced cell death phenotypes probably seems by apoptosis in the promastigotes of L. (L.) amazonensis (IC 50  = 1:40) and L. infantum (=L. chagasi) (IC 50  = 1:38). EO also presented activity against Leishmania amastigotes. Treatment with EO for 72 h strongly reduced IL-17 cytokine levels at all tested concentrations and decreased the number of intracellular amastigotes in macrophages infected with L. (L.) amazonensis (IC 50  = 1:30) and L. infantum (=L. chagasi) (IC 50  = 1:38). Additionally, no cytotoxic effect was observed in murine macrophages treated with EO (72 h - CC 50  > 1:1). Our results demonstrated that EO has leishmanicidal activity against two different species that cause American visceral and cutaneous leishmaniasis without cytotoxic effects for the host cell. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Potential of 2-hydroxy-3-phenylsulfanylmethyl-[1,4]-naphthoquinones against Leishmania (L.) infantum: biological activity and structure-activity relationships.

PubMed

Pinto, Erika G; Santos, Isabela O; Schmidt, Thomas J; Borborema, Samanta E T; Ferreira, Vitor F; Rocha, David R; Tempone, Andre G

2014-01-01

Naphtoquinones have been used as promising scaffolds for drug design studies against protozoan parasites. Considering the highly toxic and limited therapeutic arsenal, the global negligence with tropical diseases and the elevated prevalence of co-morbidities especially in developing countries, the parasitic diseases caused by various Leishmania species (leishmaniasis) became a significant public health threat in 98 countries. The aim of this work was the evaluation of antileishmanial in vitro potential of thirty-six 2-hydroxy-3-phenylsulfanylmethyl-[1,4]-naphthoquinones obtained by a three component reaction of lawsone, the appropriate aldehyde and thiols adequately substituted, exploiting the in situ generation of o-quinonemethides (o-QM) via the Knoevenagel condensation. The antileishmanial activity of the naphthoquinone derivatives was evaluated against promastigotes and intracellular amastigotes of Leishmania (Leishmania) infantum and their cytotoxicity was verified in mammalian cells. Among the thirty-six compounds, twenty-seven were effective against promastigotes, with IC50 values ranging from 8 to 189 µM; fourteen compounds eliminated the intracellular amastigotes, with IC50 values ranging from 12 to 65 µM. The compounds containing the phenyl groups at R1 and R2 and with the fluorine substituent at the phenyl ring at R2, rendered the most promising activity, demonstrating a selectivity index higher than 15 against amastigotes. A QSAR (quantitative structure activity relationship) analysis yielded insights into general structural requirements for activity of most compounds in the series. Considering the in vitro antileishmanial potential of 2-hydroxy-3-phenylsulfanylmethyl-[1,4]-naphthoquinones and their structure-activity relationships, novel lead candidates could be exploited in future drug design studies for leishmaniasis.

Antileishmanial activities of dihydrochalcones from piper elongatum and synthetic related compounds. Structural requirements for activity.

PubMed

Hermoso, Alicia; Jiménez, Ignacio A; Mamani, Zulma A; Bazzocchi, Isabel L; Piñero, José E; Ravelo, Angel G; Valladares, Basilio

2003-09-01

Two dihydrochalcones (1 and 2) were isolated from Piper elongatum Vahl by activity-guided fractionation against extracellular promastigotes of Leishmania braziliensis in vitro. Their structures were elucidated by spectral analysis, including homonuclear and heteronuclear correlation NMR experiments. Derivatives 3-7 and 20 synthetic related compounds (8-27) were also assayed to establish the structural requirements for antileishmanial activity. Compounds 1-11 that proved to be more active that ketoconazol, used as positive control, were further assayed against promastigotes of Leishmania tropica and Leishmania infantum. Compounds 7 and 11, with a C(6)-C(3)-C(6) system, proved to be the most promising compounds, with IC(50) values of 2.98 and 3.65 microg/mL, respectively, and exhibited no toxic effect on macrophages (around 90% viability). Correlation between the molecular structures and antileishmanial activity is discussed in detail.

In Vitro Antileishmanial Activity of Sterols from Trametes versicolor (Bres. Rivarden).

PubMed

Leliebre-Lara, Vivian; Monzote Fidalgo, Lianet; Pferschy-Wenzig, Eva-Maria; Kunert, Olaf; Nogueiras Lima, Clara; Bauer, Rudolf

2016-08-10

Two ergostanes, 5α,8α-epidioxy-22E-ergosta-6,22-dien-3β-ol (1) and 5α-ergost-7,22-dien-3β-ol (2), and a lanostane, 3β-hydroxylanostan-8,24-diene-21-oic acid (trametenolic acid) (3), were isolated from an n-hexane extract prepared from the fruiting body of Trametes versicolor (Bres. Rivarden). The activity of the isolated sterols was evaluated against promastigotes and amastigotes of Leishmania amazonensis Lainson and Shaw, 1972. The lanostane, compound (3), showed the best inhibitory response (IC50 promastigotes 2.9 ± 0.1 μM and IC50 amastigotes 1.6 ± 0.1 μM). This effect was 25-fold higher compared with its cytotoxic effect on peritoneal macrophages from BALB/c mice. Therefore, trametenolic acid could be regarded as a promising lead for the synthesis of compounds with antileishmanial activity.

Studies on the protective efficacy of freeze thawed promastigote antigen of Leishmania donovani along with various adjuvants against visceral leishmaniasis infection in mice.

PubMed

Thakur, Ankita; Kaur, Harpreet; Kaur, Sukhbir

2015-09-01

Visceral leishmaniasis (VL) caused by Leishmania donovani persists as a major public health issue in tropical and subtropical areas of the world. Current treatment of this disease relies on use of drugs. It is doubtful that chemotherapy can alone eradicate the disease, so there is a need for an effective vaccine. Killed antigen candidates remain a good prospect considering their ease of formulation, stability, low cost and safety. To enhance the efficacy of killed vaccines suitable adjuvant and delivery system are needed. Therefore, the current study was conducted to determine the protective efficacy of freeze-thawed L. donovani antigen in combination with different adjuvants against experimental infection of VL. For this, BALB/c mice were immunized thrice at an interval of two weeks. Challenge infection was given two weeks after last immunization. Mice were sacrificed after last immunization and on different post challenge/infection days. Immunized mice showed significant reduction in parasite burden, enhanced DTH responses with increased levels of Th1 cytokines and lower levels of Th2 cytokines, thus indicating the development of a protective Th1 response. Maximum protection was achieved with liposome encapsulated freeze thawed promastigote (FTP) antigen of L. donovani and it was followed by group immunized with FTP+MPL-A, FTP+saponin, FTP+alum and FTP antigen (alone). The present study highlights greater efficacy of freeze thawed promastigote antigen as a potential vaccine candidate along with effective adjuvant formulations against experimental VL infection. Copyright © 2015 Elsevier GmbH. All rights reserved.

Antileishmanial Activity of Ezetimibe: Inhibition of Sterol Biosynthesis, In Vitro Synergy with Azoles, and Efficacy in Experimental Cutaneous Leishmaniasis

PubMed Central

Andrade-Neto, Valter Viana; Cunha-Júnior, Edézio Ferreira; do Canto-Cavalheiro, Marilene Marcuzzo; Atella, Geórgia Correa; Fernandes, Talita de Almeida; Costa, Paulo Roberto Ribeiro

2016-01-01

Leishmaniasis affects mainly low-income populations in tropical regions. Radical innovation in drug discovery is time-consuming and expensive, imposing severe restrictions on the ability to launch new chemical entities for the treatment of neglected diseases. Drug repositioning is an attractive strategy for addressing a specific demand more easily. In this project, we have evaluated the antileishmanial activities of 30 drugs currently in clinical use for various morbidities. Ezetimibe, clinically used to reduce intestinal cholesterol absorption in dyslipidemic patients, killed Leishmania amazonensis promastigotes with a 50% inhibitory concentration (IC50) of 30 μM. Morphological analysis revealed that ezetimibe caused the parasites to become rounded, with multiple nuclei and flagella. Analysis by gas chromatography (GC)-mass spectrometry (MS) showed that promastigotes treated with ezetimibe had smaller amounts of C-14-demethylated sterols, and accumulated more cholesterol and lanosterol, than untreated promastigotes. We then evaluated the combination of ezetimibe with well-known antileishmanial azoles. The fractional inhibitory concentration index (FICI) indicated synergy when ezetimibe was combined with ketoconazole or miconazole. The activity of ezetimibe against intracellular amastigotes was confirmed, with an IC50 of 20 μM, and ezetimibe reduced the IC90s of ketoconazole and miconazole from 11.3 and 11.5 μM to 4.14 and 8.25 μM, respectively. Subsequently, we confirmed the activity of ezetimibe in vivo, showing that it decreased lesion development and parasite loads in murine cutaneous leishmaniasis. We concluded that ezetimibe has promising antileishmanial activity and should be considered in combination with azoles in further preclinical and clinical studies. PMID:27600041

Epoxy-α-Lapachone Has In Vitro and In Vivo Anti-Leishmania (Leishmania) amazonensis Effects and Inhibits Serine Proteinase Activity in This Parasite

PubMed Central

Souza-Silva, Franklin; Bourguignon, Saulo Cabral; Pereira, Bernardo Acácio Santini; Côrtes, Luzia Monteiro de Castro; de Oliveira, Luiz Filipe Gonçalves; Henriques-Pons, Andrea; Finkelstein, Lea Cysne; Ferreira, Vitor Francisco; Carneiro, Paula Fernandes; de Pinho, Rosa Teixeira; Caffarena, Ernesto Raul

2015-01-01

Leishmania (Leishmania) amazonensis is a protozoan that causes infections with a broad spectrum of clinical manifestations. The currently available chemotherapeutic treatments present many problems, such as several adverse side effects and the development of resistant strains. Natural compounds have been investigated as potential antileishmanial agents, and the effects of epoxy-α-lapachone on L. (L.) amazonensis were analyzed in the present study. This compound was able to cause measurable effects on promastigote and amastigote forms of the parasite, affecting plasma membrane organization and leading to death after 3 h of exposure. This compound also had an effect in experimentally infected BALB/c mice, causing reductions in paw lesions 6 weeks after treatment with 0.44 mM epoxy-α-lapachone (mean lesion area, 24.9 ± 2.0 mm2), compared to untreated animals (mean lesion area, 30.8 ± 2.6 mm2) or animals treated with Glucantime (mean lesion area, 28.3 ± 1.5 mm2). In addition, the effects of this compound on the serine proteinase activities of the parasite were evaluated. Serine proteinase-enriched fractions were extracted from both promastigotes and amastigotes and were shown to act on specific serine proteinase substrates and to be sensitive to classic serine proteinase inhibitors (phenylmethylsulfonyl fluoride, aprotinin, and antipain). These fractions were also affected by epoxy-α-lapachone. Furthermore, in silico simulations indicated that epoxy-α-lapachone can bind to oligopeptidase B (OPB) of L. (L.) amazonensis, a serine proteinase, in a manner similar to that of antipain, interacting with an S1 binding site. This evidence suggests that OPB may be a potential target for epoxy-α-lapachone and, as such, may be related to the compound's effects on the parasite. PMID:25583728

In vitro biological action of aqueous extract from roots of Physalis angulata against Leishmania (Leishmania) amazonensis.

PubMed

da Silva, Raquel Raick P; da Silva, Bruno J M; Rodrigues, Ana Paula D; Farias, Luis Henrique S; da Silva, Milton N; Alves, Danila Teresa V; Bastos, Gilmara N T; do Nascimento, José Luiz M; Silva, Edilene O

2015-07-24

Leishmaniasis is an infectious disease caused by various species of the protozoan parasites of the Leishmania genus and transmitted by phlebotomine sandflies. The protozoa multiply in phagocytic cells, mainly macrophages, which play an important role defending the organism from pathogens. The most effective treatment for leishmaniasis is the chemotherapy and besides the high cost, these drugs are toxic and require a long period of treatment. Currently, some herbal products are considered an important alternative source of a new leishmanicidal agent, which includes the plant Physalis angulata, . We evaluated effects of an aqueous extract from roots of Physalis angulata (AEPa) on Leishmania proliferation, morphology and also determined whether physalins were present in the extract contributing to the knowledge of its pharmacological efficacy. Morphological alterations were determined by light microscopy, transmission and scanning electron microscopy. Host cell viability was evaluated by MTT, and propidium iodide. AEPa were submitted in full HRESITOF analysis. AEPa promoted a dose-dependent reduction on promastigotes (IC50 = 39.5 μg/mL ± 5.1) and amastigotes (IC50 = 43.4 μg/mL ± 10.1) growth. This growth inhibition was associated with several morphological alterations observed in promastigote forms. No cytotoxic effect in mammalian cells was detected (IC50 > 4000 μg/mL). Furthemore, the presence of physalins A, B, D, E, F, G and H were described, for the first time, in the P. angulata root. Results demonstrate that AEPa effectively promotes antileishmanial activity with several important morphological alterations and has no cytotoxic effects on host cells.

Crystal structure and theoretical calculations of Julocrotine, a natural product with antileishmanial activity

NASA Astrophysics Data System (ADS)

Moreira, Rafael Y. O.; Brasil, Davi S. B.; Alves, Cláudio N.; Guilhon, Giselle M. S. P.; Santos, Lourivaldo S.; Arruda, Mara S. P.; Müller, Adolfo H.; Barbosa, Patrícia S.; Abreu, Alcicley S.; Silva, Edilene O.; Rumjanek, Victor M.; Souza, Jaime, Jr.; da Silva, Albérico B. F.; Santos, Regina H. De A.

Julocrotine, N-(2,6-dioxo-1-phenethyl-piperidin-3-yl)-2-methyl-butyramide, is a potent antiproliferative agent against the promastigote and amastigote forms of Leishmania amazonensis (L.). In this work, the crystal structure of Julocrotine was solved by X-ray diffraction, and its geometrical parameters were compared with theoretical calculations at the B3LYP and HF level of theory. IR and NMR spectra also have been obtained and compared with theoretical calculations. IR absorptions calculated with the B3LYP level of theory employed together with the 6-311G+(d,p) basis set, are close to those observed experimentally. Theoretical NMR calculations show little deviation from experimental results. The results show that the theory is in accordance with the experimental data.0

3D-Quantitative structure-activity relationships of synthetic antileishmanial ring-substituted ether phospholipids.

PubMed

Kapou, Agnes; Benetis, Nikolas P; Avlonitis, Nikos; Calogeropoulou, Theodora; Koufaki, Maria; Scoulica, Efi; Nikolaropoulos, Sotiris S; Mavromoustakos, Thomas

2007-02-01

The application of 2D-NMR spectroscopy and Molecular Modeling in determining the active conformation of flexible molecules in 3D-QSAR was demonstrated in the present study. In particular, a series of 33 flexible synthetic phospholipids, either 2-(4-alkylidene-cyclohexyloxy)ethyl- or omega-cycloalkylidene-substituted ether phospholipids were systematically evaluated for their in vitro antileishmanial activity against the promastigote forms of Leishmania infantum and Leishmania donovani by CoMFA and CoMSIA 3D-QSAR studies. Steric and hydrophobic properties of the phospholipids under study appear to govern their antileishmanial activity against both strains, while the electrostatic properties have no significant contribution. The acknowledgment of these important properties of the pharmacophore will aid in the rational design of new analogues with higher activity.

Leishmanicidal and cytotoxic activity from plants used in Tacana traditional medicine (Bolivia).

PubMed

Arévalo-Lopéz, Diandra; Nina, Nélida; Ticona, Juan C; Limachi, Ivan; Salamanca, Efrain; Udaeta, Enrique; Paredes, Crispin; Espinoza, Boris; Serato, Alcides; Garnica, David; Limachi, Abigail; Coaquira, Dayana; Salazar, Sarah; Flores, Ninoska; Sterner, Olov; Giménez, Alberto

2018-04-24

Thirty-eight Tacana medicinal plant species used to treat skin problems, including leishmania ulcers, skin infections, inflammation and wound healing, were collected in the community of Buena Vista, Bolivia, with the Tacana people. Twenty two species are documented for the first time as medicinal plants for this ethnic group living in the northern area of the Department of La Paz. To evaluate the leishmanicidal effect (IC 50 ) and cytotoxicity (LD 50 ) of the selected plants. To carry out bioguided studies on the active extracts. To assess the potential of Bolivian plant biodiversity associated with traditional knowledge in the discovery of alternative sources to fight leishmaniasis. Seventy three ethanol extracts were prepared from 38 species by maceration and were evaluated in vitro against promastigotes of Leishmania amazonensis and L. braziliensis. Active extracts (IC 50 ≤ 50 μg/mL) were fractionated by chromatography on Silica gel column and the fractions were assessed against the two Leishmania strains. The most active fractions and the crude extracts were evaluated against reference strains of L. amazonensis, L. braziliensis, L. aethiopica, two native strains (L. Lainsoni and L. braziliensis) and for cytotoxicity against HeLa cells. The chromatographic profile of the active fractions was obtained by reverse phase chromatography using HPLC. From the 73 extracts, 39 extracts (53.4%) were inactive and 34 showed activity. Thirteen species were sselected for bioguided studies. The crude extracts and their 36 fractions were evaluated against two Leishmania strains. The most active fraction were tested in a panel of five leishmania strains and for cytotoxicity. The Selective Index (SI = LD 50 /IC 50 ) was calculated, and were generally low. Retention time and UV spectra were recorded for the active fractions by HPLC-DAD using a reverse phase column. Profiles were very different from each other, showing the presence of different compounds. Bolivian traditional knowledge from the Tacanba was useful to identify plants with effect on Leishmania promastigotes. Chromatographic bioguided studies showed stronger leishmanicidal and cytotoxic activity for the medium polar fraction. HPLC analysis showed different chromatographic profiles of the active fractions. Copyright © 2018 Elsevier B.V. All rights reserved.

Target Oriented Drugs against Leishmania.

DTIC Science & Technology

1980-01-31

present in very significant amounts and there is a possibility of arabinose - normally found only as a component of plant glycoproteins. 6...as does EF. This is also demonstrated by our experiments on promastigote agglutination by PNA (§ 2a). These oligosaccharide substituents, containing

Chemotherapy of leishmaniasis part-VIII: synthesis and bioevaluation of novel chalcones.

PubMed

Suryawanshi, S N; Chandra, Naveen; Kumar, Pawan; Porwal, Jyoti; Gupta, Suman

2008-11-01

Some novel dihydro-alpha-ionone based chalcones have been synthesized and evaluated for their in vitro antileishmanial activity in promastigote and amastigote model. Some of the compounds showed 100% inhibition at 5 and 2 microm/ml concentration.

Evaluation of the leishmanicidal and cytotoxic potential of essential oils derived from ten colombian plants.

PubMed

Sanchez-Suarez, Jf; Riveros, I; Delgado, G

2013-01-01

The leishmanicidal and cytotoxic activity of ten essential oils obtained from ten plant specimens were evaluated. Essential oils were obtained by the steam distillation of plant leaves without any prior processing. Cytotoxicity was tested on J774 macrophages and leishmanicidal activity was assessed against four species of Leishmania associated with cutaneous leishmaniasis. Seven essential oils exhibited activity against Leishmania parasites, five of which were toxic against J774 macrophages. Selectivity indices of >6 and 13 were calculated for the essential oils of Ocimum basilicum and Origanum vulgare, respectively. The essential oil of Ocimum basilicum was active against promastigotes of Leishmania and innocuous to J774 macrophages at concentrations up to 1600 µg/mL and should be further investigated for leishmanicidal activity in others in vitro and in vivo experimental models.

Cutaneous Leishmaniasis

DTIC Science & Technology

2011-06-01

macrophage. Note also a few extracellular organisms (arrows). Giemsa x750 Figure 4.3 Female sandfly ( Lutzomyia longipalpis) taking blood meal from...Old World; Lutzomyia sp (Fig 4.3) and Psychodopygus sp in the New World) and transmitted to a host during a blood meal (Fig 4.4). Promastigotes

Anti-Leishmania activity of new ruthenium(II) complexes: Effect on parasite-host interaction.

PubMed

Costa, Mônica S; Gonçalves, Yasmim G; Nunes, Débora C O; Napolitano, Danielle R; Maia, Pedro I S; Rodrigues, Renata S; Rodrigues, Veridiana M; Von Poelhsitz, Gustavo; Yoneyama, Kelly A G

2017-10-01

Leishmaniasis is a parasitic disease caused by protozoa of the genus Leishmania. The many complications presented by the current treatment - including high toxicity, high cost and parasite resistance - make the development of new therapeutic agents indispensable. The present study aims to evaluate the anti-Leishmania potential of new ruthenium(II) complexes, cis‑[Ru II (η 2 -O 2 CR)(dppm) 2 ]PF 6 , with dppm=bis(diphenylphosphino)methane and R=4-butylbenzoate (bbato) 1, 4-(methylthio)benzoate (mtbato) 2 and 3-hydroxy-4-methoxybenzoate (hmxbato) 3, in promastigote cytotoxicity and their effect on parasite-host interaction. The cytotoxicity of complexes was analyzed by MTT assay against Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, Leishmania (Leishmania) infantum promastigotes and the murine macrophage (RAW 264.7). The effect of complexes on parasite-host interaction was evaluated by in vitro infectivity assay performed in the presence of two different concentrations of each complex: the promastigote IC 50 value and the concentration nontoxic to 90% of RAW 264.7 macrophages. Complexes 1-3 exhibited potent cytotoxic activity against all Leishmania species assayed. The IC 50 values ranged from 7.52-12.59μM (complex 1); 0.70-3.28μM (complex 2) and 0.52-1.75μM (complex 3). All complexes significantly inhibited the infectivity index at both tested concentrations. The infectivity inhibitions ranged from 37 to 85%. Interestingly, the infectivity inhibitions due to complex action did not differ significantly at either of the tested concentrations, except for the complex 1 against Leishmania (Leishmania) infantum. The infectivity inhibitions resulted from reductions in both percentage of infected macrophages and number of parasites per macrophage. Taken together the results suggest remarkable leishmanicidal activity in vitro by these new ruthenium(II) complexes. Copyright © 2017 Elsevier Inc. All rights reserved.

Antileishmanial Activity of Ezetimibe: Inhibition of Sterol Biosynthesis, In Vitro Synergy with Azoles, and Efficacy in Experimental Cutaneous Leishmaniasis.

PubMed

Andrade-Neto, Valter Viana; Cunha-Júnior, Edézio Ferreira; Canto-Cavalheiro, Marilene Marcuzzo do; Atella, Geórgia Correa; Fernandes, Talita de Almeida; Costa, Paulo Roberto Ribeiro; Torres-Santos, Eduardo Caio

2016-11-01

Leishmaniasis affects mainly low-income populations in tropical regions. Radical innovation in drug discovery is time-consuming and expensive, imposing severe restrictions on the ability to launch new chemical entities for the treatment of neglected diseases. Drug repositioning is an attractive strategy for addressing a specific demand more easily. In this project, we have evaluated the antileishmanial activities of 30 drugs currently in clinical use for various morbidities. Ezetimibe, clinically used to reduce intestinal cholesterol absorption in dyslipidemic patients, killed Leishmania amazonensis promastigotes with a 50% inhibitory concentration (IC 50 ) of 30 μM. Morphological analysis revealed that ezetimibe caused the parasites to become rounded, with multiple nuclei and flagella. Analysis by gas chromatography (GC)-mass spectrometry (MS) showed that promastigotes treated with ezetimibe had smaller amounts of C-14-demethylated sterols, and accumulated more cholesterol and lanosterol, than untreated promastigotes. We then evaluated the combination of ezetimibe with well-known antileishmanial azoles. The fractional inhibitory concentration index (FICI) indicated synergy when ezetimibe was combined with ketoconazole or miconazole. The activity of ezetimibe against intracellular amastigotes was confirmed, with an IC 50 of 20 μM, and ezetimibe reduced the IC 90 s of ketoconazole and miconazole from 11.3 and 11.5 μM to 4.14 and 8.25 μM, respectively. Subsequently, we confirmed the activity of ezetimibe in vivo, showing that it decreased lesion development and parasite loads in murine cutaneous leishmaniasis. We concluded that ezetimibe has promising antileishmanial activity and should be considered in combination with azoles in further preclinical and clinical studies. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Castalagin from Anogeissus leiocarpus mediates the killing of Leishmania in vitro.

PubMed

Shuaibu, M N; Pandey, K; Wuyep, P A; Yanagi, T; Hirayama, K; Ichinose, A; Tanaka, T; Kouno, I

2008-11-01

Stem barks of Anogeissus leiocarpus and Terminalia avicennoides widely used in Africa for treatment of some parasitic diseases were collected and made into methanolic extracts. The extracts were tested on four strains of promastigote forms of Leishmania in vitro. Solvent fractionation in aqueous, butanolic, and ethyl acetate layer indicated butanol and aqueous fractions to have a superior leishmanicidal activity. Chromatographic separation of the butanolic fraction on Sephadex LH-20 followed by nuclear magnetic resonance and correlation high-performance liquid chromatography revealed the presence of known hydrolyzable tannins and some related compounds-with castalagin as the major compound. The observed activity ranged from 62.5 to > or =150, 112.5 to > or =500, and 55 to >150 microg/ml for the crude methanolic extract, different solvent fractions, and the isolated compounds, respectively, on the four different Leishmania strains.

Transmission of cutaneous leishmaniasis by sand flies is enhanced by regurgitation of fPPG.

PubMed

Rogers, Matthew E; Ilg, Thomas; Nikolaev, Andrei V; Ferguson, Michael A J; Bates, Paul A

2004-07-22

Sand flies are the exclusive vectors of the protozoan parasite Leishmania, but the mechanism of transmission by fly bite has not been determined nor incorporated into experimental models of infection. In sand flies with mature Leishmania infections the anterior midgut is blocked by a gel of parasite origin, the promastigote secretory gel. Here we analyse the inocula from Leishmania mexicana-infected Lutzomyia longipalpis sand flies. Analysis revealed the size of the infectious dose, the underlying mechanism of parasite delivery by regurgitation, and the novel contribution made to infection by filamentous proteophosphoglycan (fPPG), a component of promastigote secretory gel found to accompany the parasites during transmission. Collectively these results have important implications for understanding the relationship between the parasite and its vector, the pathology of cutaneous leishmaniasis in humans and also the development of effective vaccines and drugs. These findings emphasize that to fully understand transmission of vector-borne diseases the interaction between the parasite, its vector and the mammalian host must be considered together.

1,2,4-triazole derivative with Schiff base; thiol-thione tautomerism, DFT study and antileishmanial activity

NASA Astrophysics Data System (ADS)

Süleymanoğlu, Nevin; Ustabaş, Reşat; Direkel, Şahin; Alpaslan, Yelda Bingöl; Ünver, Yasemin

2017-12-01

Thiol-thione tautomerism of 1,2,4-triazole derivative with Schiff base was investigated by spectroscopic methods and quantum mechanical calculations. Theoretical study of thiol-thione tautomeric forms of 1,2,4-triazole derivative with Schiff base; 1,2,4-triazole-thiol form, 1-((5-mercapto-4-(thiophene-2-ylmethyleneamino)-4H-1,2,4-triazole-3-yl)methyl)-3-(thiophene-2-ylmethyl)-4-(thiophene-2-ylmethyleneamino)-1H-1,2,4-triazole-5(4H)-one (I) and 1,2,4-triazole-thione form, 3-(thiophene-2-ylmethyl)-4-(thiophene-2-ylmethyleneamino)-1-((4-(thiophene-2-ylmethyleneamino)-5-thioxo-4,5-dihydro-1H-1,2,4-triazole-3-yl)methyl)-1H-1,2,4-triazole-5(4H)-one (II) was performed by the density functional theory (DFT) method with 6-311++G(d,p) basis set. Structural parameters were obtained and spectral parameters of NMR, FTIR and UV-vis were compared with experimental ones to determine structural details. In vitro antileishmanial activity was studied against Leishmania infantum promastigots by microdilution broth assay with Alamar Blue Dye. The results indicate that 1,2,4-triazole derivative exists in both thiol and thione form and, can be evaluated as antiparasitic in term of antileishmanial activity.

Evaluation of the Leishmanicidal and Cytotoxic Potential of Essential Oils Derived From Ten Colombian Plants

PubMed Central

Sanchez-Suarez, JF; Riveros, I; Delgado, G

2013-01-01

Background The leishmanicidal and cytotoxic activity of ten essential oils obtained from ten plant specimens were evaluated. Methods Essential oils were obtained by the steam distillation of plant leaves without any prior processing. Cytotoxicity was tested on J774 macrophages and leishmanicidal activity was assessed against four species of Leishmania associated with cutaneous leishmaniasis. Results Seven essential oils exhibited activity against Leishmania parasites, five of which were toxic against J774 macrophages. Selectivity indices of >6 and 13 were calculated for the essential oils of Ocimum basilicum and Origanum vulgare, respectively. Conclusion The essential oil of Ocimum basilicum was active against promastigotes of Leishmania and innocuous to J774 macrophages at concentrations up to 1600 µg/mL and should be further investigated for leishmanicidal activity in others in vitro and in vivo experimental models. PMID:23682270

Trypanocidal and leishmanicidal activities of different antimicrobial peptides (AMPs) isolated from aquatic animals.

PubMed

Löfgren, S E; Miletti, L C; Steindel, M; Bachère, E; Barracco, M A

2008-02-01

Most of the available animal antimicrobial peptides (AMPs) have been tested against bacteria and fungi, but very few against protozoan parasites. In the present study, we investigated the antiparasitic activity of different AMPs isolated from aquatic animals: tachyplesin (Tach, from Tachypleus tridentatus), magainin (Mag, from Xenopus laevis), clavanin (Clav, from Styela clava), penaeidin (Pen, from Litopenaeus vannamei), mytilin (Myt, from Mytilus edulis) and anti-lipopolysaccharide factor (ALF, from Penaeus monodon). The antiparasitic activity was evaluated against the promastigote form of Leishmania braziliensis and epi and trypomastigote forms of Trypanosoma cruzi, through the MTT method. Tach was the most potent peptide, killing completely L. braziliensis and trypomastigote T. cruzi from 12.5microM, whereas Pen and Clav were weakly active against trypomastigotes and Myt against L. braziliensis, only at a high concentration (100microM). Tach and Mag were markedly hemolytic at high concentrations, whereas the other peptides caused only a slight hemolysis (<10% up to 50microM). Our results point to Tach as the only potential candidate for further investigation and potential application as a therapeutic agent.

Chemical composition, leishmanicidal and cytotoxic activities of the essential oils from Mangifera indica L. var. Rosa and Espada.

PubMed

Ramos, Eduardo H S; Moraes, Marcílio M; Nerys, Laís L de A; Nascimento, Silene C; Militão, Gardênia C G; de Figueiredo, Regina C B Q; da Câmara, Cláudio A G; Silva, Teresinha Gonçalves

2014-01-01

The essential oils from Mangifera indica var. Rosa and Espada latex were obtained by hydrodistillation and analyzed using GC-FID and GC-MS. Twenty-seven components were identified. The main compound in the essential oil from M. indica var. Espada (EOMiE) was terpinolene (73.6%). The essential oil of M. indica var. Rosa (EOMiR) was characterized by high amounts of β-pinene (40.7%) and terpinolene (28.3%). In the test for leishmanicidal activity against promastigotes forms of L. amazonensis, EOMiR and EOMiE showed IC50 (72 h) of 39.1 and 23.0 μg/mL, respectively. In macrophages, EOMiR and EOMiE showed CC50 of 142.84 and 158.65 μg/mL, respectively. However, both were more specific to the parasite than macrophages, with values of selectivity index of 6.91 for EOMiE and 3.66 for EOMiR. The essential oils were evaluated for their cytotoxicity against the human tumor cells HEp-2, HT-29, NCI-H292, and HL-60. The EOMiR and EOMiE were most effective against the HL-60, with IC50 values of 12.3 and 3.6 μg/mL, respectively. The results demonstrated that the essential oils of M. indica can destroy L. amazonensis and inhibit tumor cell growth. These findings contribute to the knowledge of the Brazilian biodiversity as a source of potential therapeutic agents.

New alkenyl derivative from Piper malacophyllum and analogues: Antiparasitic activity against Trypanosoma cruzi and Leishmania infantum.

PubMed

Varela, Marina T; Lima, Marta L; Galuppo, Mariana K; Tempone, Andre G; de Oliveira, Alberto; Lago, João Henrique G; Fernandes, João Paulo S

2017-11-01

Alkylphenols isolated from Piper malacophyllum (Piperaceae), gibbilimbols A and B, showed interesting activity against the parasites Trypanosoma cruzi and Leishmania infantum. In continuation to our previous work, a new natural product from the essential oil of the leaves of P. malacophyllum was isolated, the 5-[(3E)-oct-3-en-1-il]-1,3-benzodioxole, and also a new set of five compounds was prepared. The antiparasitic activity of the natural product was evaluated in vitro against these parasites, indicating potential against the promastigote/trypomastigote/amastigote forms (IC 50 32-83 μm) of the parasites and low toxicity (CC 50  > 200 μm) to mammalian cells. The results obtained to the synthetic compounds indicated that the new derivatives maintained the promising antiparasitic activity, but the cytotoxicity was considerably lowered. The amine derivative LINS03011 displayed the most potent IC 50 values (13.3 and 16.7 μm) against amastigotes of T. cruzi and L. infantum, respectively, indicating comparable activity to the phenolic prototype LINS03003, with threefold decreased (CC 50 73.5 μm) cytotoxicity, leading the selectivity index (SI) towards the parasites up to 24.5. In counterpart, LINS03011 has not shown membrane disruptor activity in SYTOX Green model. In summary, this new set showed the hydroxyl is not essential for the antiparasitic activity, and its substitution could decrease the toxicity to mammalian cells. © 2017 John Wiley & Sons A/S.

Valosin-containing protein VCP/p97 is essential for the intracellular development of Leishmania and its survival under heat stress.

PubMed

Guedes Aguiar, Bruno; Padmanabhan, Prasad K; Dumas, Carole; Papadopoulou, Barbara

2018-06-12

Valosin-containing protein (VCP)/p97/Cdc48 is one of the best-characterised type II cytosolic AAA+ ATPases most known for their role in ubiquitin-dependent protein quality control. Here, we provide functional insights into the role of the Leishmania VCP/p97 homologue (LiVCP) in the parasite intracellular development. We demonstrate that although LiVCP is an essential gene, Leishmania infantum promastigotes can grow with less VCP. In contrast, growth of axenic and intracellular amastigotes is dramatically affected upon decreased LiVCP levels in heterozygous and temperature sensitive (ts) LiVCP mutants or the expression of dominant negative mutants known to specifically target the second conserved VCP ATPase domain, a major contributor of the VCP overall ATPase activity. Interestingly, these VCP mutants are also unable to survive heat stress, and a ts VCP mutant is defective in amastigote growth. Consistent with LiVCP's essential function in amastigotes, LiVCP messenger ribonucleic acid undergoes 3'Untranslated Region (UTR)-mediated developmental regulation, resulting in higher VCP expression in amastigotes. Furthermore, we show that parasite mutant lines expressing lower VCP levels or dominant negative VCP forms exhibit high accumulation of polyubiquitinated proteins and increased sensitivity to proteotoxic stress, supporting the ubiquitin-selective chaperone function of LiVCP. Together, these results emphasise the crucial role LiVCP plays under heat stress and during the parasite intracellular development. © 2018 John Wiley & Sons Ltd.

Chemical Composition, Leishmanicidal and Cytotoxic Activities of the Essential Oils from Mangifera indica L. var. Rosa and Espada

PubMed Central

Ramos, Eduardo H. S.; Moraes, Marcílio M.; Nerys, Laís L. de A.; Nascimento, Silene C.; Militão, Gardênia C. G.; de Figueiredo, Regina C. B. Q.; da Câmara, Cláudio A. G.; Silva, Teresinha Gonçalves

2014-01-01

The essential oils from Mangifera indica var. Rosa and Espada latex were obtained by hydrodistillation and analyzed using GC-FID and GC-MS. Twenty-seven components were identified. The main compound in the essential oil from M. indica var. Espada (EOMiE) was terpinolene (73.6%). The essential oil of M. indica var. Rosa (EOMiR) was characterized by high amounts of β-pinene (40.7%) and terpinolene (28.3%). In the test for leishmanicidal activity against promastigotes forms of L. amazonensis, EOMiR and EOMiE showed IC50 (72 h) of 39.1 and 23.0 μg/mL, respectively. In macrophages, EOMiR and EOMiE showed CC50 of 142.84 and 158.65 μg/mL, respectively. However, both were more specific to the parasite than macrophages, with values of selectivity index of 6.91 for EOMiE and 3.66 for EOMiR. The essential oils were evaluated for their cytotoxicity against the human tumor cells HEp-2, HT-29, NCI-H292, and HL-60. The EOMiR and EOMiE were most effective against the HL-60, with IC50 values of 12.3 and 3.6 μg/mL, respectively. The results demonstrated that the essential oils of M. indica can destroy L. amazonensis and inhibit tumor cell growth. These findings contribute to the knowledge of the Brazilian biodiversity as a source of potential therapeutic agents. PMID:25136617

Comparative Study of rK39 Leishmania Antigen for Serodiagnosis of Visceral Leishmaniasis: Systematic Review with Meta-Analysis

PubMed Central

Maia, Zuinara; Lírio, Monique; Mistro, Sóstenes; Mendes, Carlos Maurício Cardeal; Mehta, Sanjay R.; Badaro, Roberto

2012-01-01

Background The rK39 recombinant protein is derived from a specific antigen produced by the Leishmania donovani complex, and has been used in the last two decades for the serodiagnosis of visceral leishmaniasis. We present here a systematic review and meta-analysis of studies evaluating serologic assays to diagnose visceral leishmaniasis to determine the accuracy of rK39 antigen in comparison to the use of other antigen preparations. Methodology/Principal Findings A systematic review with meta-analysis of the literature was performed to compare the rK39 strip-test and ELISA formats against serological tests using promastigote antigens derived from whole or soluble parasites for Direct Aglutination Test (DAT), Indirect Immunofluorescence test (IFAT) and ELISA with a promastigote antigen preparation (p-ELISA). Gold standard diagnosis was defined by the demonstration of amastigotes on hematological specimens. A database search was performed on Medline, Lilacs, Scopus, Isi Web of Science, and Cochrane Library. Quality of data was assessed using the QUADAS questionnaire. A search of the electronic databases found 352 papers of which only 14 fulfilled the selection criteria. Three evaluated the rK39 ELISA, while 13 evaluated the rK39 immunochromatographic strip test. The summarized sensitivity for the rK39-ELISA was 92% followed by IFAT 88% and p-ELISA 87%. The summarized specificity for the three diagnostic tests was 81%, 90%, and 77%. Studies comparing the rK39 strip test with DAT found a similar sensitivity of 94%, although the DAT had a slightly higher specificity. The rK39 strip test was more sensitive and specific than the IFAT and p-ELISA. We did not detect any difference in the sensitivity and specificity between strips produced by different manufacturers. Conclusions The rK39 protein used either in a strip test or in an ELISA, and the DAT are the best choices for implementation of rapid, easy and efficient test for serodiagnosis of VL. PMID:22303488

Comparative study of rK39 Leishmania antigen for serodiagnosis of visceral leishmaniasis: systematic review with meta-analysis.

PubMed

Maia, Zuinara; Lírio, Monique; Mistro, Sóstenes; Mendes, Carlos Maurício Cardeal; Mehta, Sanjay R; Badaro, Roberto

2012-01-01

The rK39 recombinant protein is derived from a specific antigen produced by the Leishmania donovani complex, and has been used in the last two decades for the serodiagnosis of visceral leishmaniasis. We present here a systematic review and meta-analysis of studies evaluating serologic assays to diagnose visceral leishmaniasis to determine the accuracy of rK39 antigen in comparison to the use of other antigen preparations. A systematic review with meta-analysis of the literature was performed to compare the rK39 strip-test and ELISA formats against serological tests using promastigote antigens derived from whole or soluble parasites for Direct Aglutination Test (DAT), Indirect Immunofluorescence test (IFAT) and ELISA with a promastigote antigen preparation (p-ELISA). Gold standard diagnosis was defined by the demonstration of amastigotes on hematological specimens. A database search was performed on Medline, Lilacs, Scopus, Isi Web of Science, and Cochrane Library. Quality of data was assessed using the QUADAS questionnaire. A search of the electronic databases found 352 papers of which only 14 fulfilled the selection criteria. Three evaluated the rK39 ELISA, while 13 evaluated the rK39 immunochromatographic strip test. The summarized sensitivity for the rK39-ELISA was 92% followed by IFAT 88% and p-ELISA 87%. The summarized specificity for the three diagnostic tests was 81%, 90%, and 77%. Studies comparing the rK39 strip test with DAT found a similar sensitivity of 94%, although the DAT had a slightly higher specificity. The rK39 strip test was more sensitive and specific than the IFAT and p-ELISA. We did not detect any difference in the sensitivity and specificity between strips produced by different manufacturers. The rK39 protein used either in a strip test or in an ELISA, and the DAT are the best choices for implementation of rapid, easy and efficient test for serodiagnosis of VL.

Trypanocidal and leishmanicidal activities of flavonoids isolated from Stevia satureiifolia var. satureiifolia.

PubMed

Beer, María Florencia; Frank, Fernanda Maria; Germán Elso, Orlando; Ernesto Bivona, Augusto; Cerny, Natacha; Giberti, Gustavo; Luis Malchiodi, Emilio; Susana Martino, Virginia; Alonso, María Rosario; Patricia Sülsen, Valeria; Cazorla, Silvia Ines

2016-10-01

Context Chagas' disease and leishmaniasis produce significant disability and mortality with great social and economic impact. The genus Stevia (Asteraceae) is a potential source of antiprotozoal compounds. Objective Aerial parts of four Stevia species were screened on Trypanosoma cruzi. Stevia satureiifolia (Lam.) Sch. Bip. var. satureiifolia (Asteraceae) dichloromethane extract was selected for a bioassay-guided fractionation in order to isolate its active compounds. Additionally, the antileishmanial activity and the cytotoxicity of these compounds on mammalian cells were assessed. Materials and methods The dichloromethane extract was fractionated by column chromatography. The isolated compounds were evaluated using concentrations of 0-100 μg/mL on T. cruzi epimastigotes and on Leishmania braziliensis promastigotes for 72 h, on trypomastigotes and amastigotes of T. cruzi for 24 h and 120 h, respectively. The compounds' cytotoxicity (12.5-500 μg/mL) was assessed on Vero cells by the MTT assay. The structure elucidation of each compound was performed by spectroscopic methods and HPLC analysis. Results The dichloromethane extracts of Stevia species showed significant activity on T. cruzi epimastigotes. The flavonoids eupatorin (1.3%), cirsimaritin (1.9%) and 5-desmethylsinensetin (1.5%) were isolated from S. satureiifolia var. satureiifolia extract. Eupatorin and 5-desmethylsinensetin showed IC50 values of 0.2 and 0.4 μg/mL on T. cruzi epimastigotes and 61.8 and 75.1 μg/mL on trypomastigotes, respectively. The flavonoid 5-desmethylsinensetin showed moderate activity against T. cruzi amastigotes (IC50  value = 78.7 μg/mL) and was the most active compound on L. braziliensis promastigotes (IC50  value = 37.0 μg/mL). Neither of the flavonoids showed cytotoxicity on Vero cells, up to a concentration of 500 μg/mL.

An effective in vitro and in vivo antileishmanial activity and mechanism of action of 8-hydroxyquinoline against Leishmania species causing visceral and tegumentary leishmaniasis.

PubMed

Costa Duarte, Mariana; dos Reis Lage, Letícia Martins; Lage, Daniela Pagliara; Mesquita, Juliana Tonini; Salles, Beatriz Cristina Silveira; Lavorato, Stefânia Neiva; Menezes-Souza, Daniel; Roatt, Bruno Mendes; Alves, Ricardo José; Tavares, Carlos Alberto Pereira; Tempone, André Gustavo; Coelho, Eduardo Antonio Ferraz

2016-02-15

The development of new therapeutic strategies to treat leishmaniasis has become a priority. In the present study, the antileishmanial activity of 8-hydroxyquinoline (8-HQN) was investigated against in vitro promastigotes and in vivo intra-macrophage amastigotes of three Leishmania species: Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis. Studies were performed to establish the 50% Leishmania inhibitory concentration (IC50) of 8-HQN, as well as its 50% cytotoxic concentration (CC50) on murine macrophages and in human red blood cells. The inhibition of macrophages infection was also evaluated using parasites that were pre-treated with 8-HQN. The effects of this compound on nitric oxide (NO) production and in the mitochondrial membrane potential were also evaluated. Finally, the therapeutic efficacy of 8-HQN was assessed in a known murine model, L. amazonensis-chronically infected BALB/c mice. Our results showed that 8-HQN was effective against promastigote and amastigote stages of all tested Leishmania species, presenting a selectivity index of 328.0, 62.0 and 47.0 for L. amazonensis, L. infantum and L. braziliensis, respectively. It was effective in treating infected macrophages, as well as in preventing the infection of these cells using pre-treated parasites. In addition, 8-HQN caused an alteration in the mitochondrial membrane potential of the parasites. When administered at 10mg/kg body weight/day by subcutaneous route, this product was effective in reducing the lesion diameter, as well as the parasite load in evaluated tissues and organs of infected animals. The results showed the in vitro and in vivo efficacy of 8-HQN against three different Leishmania species causing tegumentary and/or visceral leishmaniasis, and it could well be used for future therapeutic optimization studies to treat leishmaniasis. Copyright © 2016 Elsevier B.V. All rights reserved.

Synthesis by combustion in solution of Zn2TiO4+Ag for photocatalytic and photodynamic applications in the visible

NASA Astrophysics Data System (ADS)

Lopera, A. A.; Velásquez, A. M.; Chavarriaga, E. A.; Ocampo, S.; Zaghete, M. A.; Graminha, M. A.; Garcia, C. P.

2017-12-01

Zn2TiO4 + Ag compounds were synthesized by the solution combustion path seeking to enhance their photocatalytic and photodynamic response in the visible. X-ray diffraction tests confirmed the formation of the phase and the presence of metallic silver. Field emission electron microscopy evidenced the formation of aggregates formed by grains lower than 100nm. The diffuse reflectance tests allowed to detect compound absorption in the visible region and activation energy of 2.8eV. The evaluation of the photocatalytic properties was performed by the degradation of methylene blue while the photodynamic response in biological systems was performed by the antilesihmanicidal response of the compounds in promastigotes of Leishmania amazonensis. Indirect measurement of ROS species confirmed the formation of oxygen singlets and OH radicals of the compounds when subjected to the action of visible light.

Antileishmanial and antifungal activity of plants used in traditional medicine in Brazil.

PubMed

Braga, Fernanda G; Bouzada, Maria Lúcia M; Fabri, Rodrigo L; de O Matos, Magnum; Moreira, Francis O; Scio, Elita; Coimbra, Elaine S

2007-05-04

The antileishmanial and antifungal activity of 24 methanol extracts from 20 plants, all of them used in the Brazilian traditional medicine for the treatment of several infectious and inflammatory disorders, were evaluated against promastigotes forms of two species of Leishmania (L. amazonensis and L. chagasi) and two yeasts (Candida albicans and Cryptococcus neoformans). Among the 20 tested methanolic extracts, those of Vernonia polyanthes was the most active against L. amazonensis (IC(50) of 4 microg/ml), those of Ocimum gratissimum exhibited the best activity against L. chagasi (IC(50) of 71 microg/ml). Concerning antifungical activity, Schinus terebintifolius, O. gratissimum, Cajanus cajan, and Piper aduncum extracts were the most active against C. albicans (MIC of 1.25 mg/ml) whereas Bixa orellana, O. gratissimum and Syzygium cumini exhibited the best activity against C. neoformans (MIC of 0.078 mg/ml).

Ultrastructural analysis of Phytomonas species from Euphorbia pinea reveals trans-cytoplasmic filaments 10 nm in diameter.

PubMed

Page, A M; Lagnado, J R

2000-10-01

Phytomonas sp. derived from Euphorbia pinea are digenetic plant trypanosomes that are transmitted by the squashbug Stenocephalus agilis and exist exclusively as promastigotes. The stable sub-pellicular microtubular array, the flagellar axoneme and the paraflagellar rod represent the major cytoskeletal components common to all trypanosomes. The work described in this paper examines in detail the ultrastructural morphology of the organism and highlights a number of novel structural features, and in particular, the presence of some detergent-resistant proteins which take the form of bundles of trans-cytoplasmic filaments of ca. 10 nm in diameter, seen in cells from both log- and stationary-phase cultures. The ultrastructural morphology and immunological cross-reactivity of these filaments are described, and their relationship to filamentous bundles previously reported in stationary-phase cultures of Crithidia fasciculata and to intermediate filaments of animal cells is discussed.

Antileishmanial pharmacomodulation in 8-nitroquinolin-2(1H)-one series.

PubMed

Kieffer, Charline; Cohen, Anita; Verhaeghe, Pierre; Paloque, Lucie; Hutter, Sébastien; Castera-Ducros, Caroline; Laget, Michèle; Rault, Sylvain; Valentin, Alexis; Rathelot, Pascal; Azas, Nadine; Vanelle, Patrice

2015-05-15

An antileishmanial pharmacomodulation at position 4 of 8-nitroquinolin-2(1H)-one was conducted by using the Sonogashira and Suzuki-Miyaura coupling reactions. A series of 25 derivatives was tested in vitro on the promastigote stage of Leishmania donovani along with an in vitro cytotoxicity evaluation on the human HepG2 cell line. Only the derivatives bearing a phenyl moiety at position 4 of the quinoline ring displayed interesting biologic profile, when the phenyl moiety was substituted at the para position by a Br or Cl atom, or by a CF3 group. Among them, molecules 17 and 19 were the most selective and were then tested in vitro on the intracellular amastigote stage of both L. donovani and Leishmania infantum, in parallel with complementary in vitro cytotoxicity assays on the macrophage cell lines THP-1 and J774A.1. Molecule 19 showed no activity on the amastigote stages of the parasites and some cytotoxicity on the J774A.1 cell line while molecule 17, less cytotoxic than 19, showed anti-amastigote activity in L. infantum, being 3 times less active than miltefosine but more active and selective than pentamidine. Nevertheless, hit-molecule 17 did not appear as selective as the parent compound. Copyright © 2015 Elsevier Ltd. All rights reserved.

Differential Subcellular Localization of Leishmania Alba-Domain Proteins throughout the Parasite Development.

PubMed

Dupé, Aurélien; Dumas, Carole; Papadopoulou, Barbara

2015-01-01

Alba-domain proteins are RNA-binding proteins found in archaea and eukaryotes and recently studied in protozoan parasites where they play a role in the regulation of virulence factors and stage-specific proteins. This work describes in silico structural characterization, cellular localization and biochemical analyses of Alba-domain proteins in Leishmania infantum. We show that in contrast to other protozoa, Leishmania have two Alba-domain proteins, LiAlba1 and LiAlba3, representative of the Rpp20- and the Rpp25-like eukaryotic subfamilies, respectively, which share several sequence and structural similarities but also important differences with orthologs in other protozoa, especially in sequences targeted for post-translational modifications. LiAlba1 and LiAlba3 proteins form a complex interacting with other RNA-binding proteins, ribosomal subunits, and translation factors as supported by co-immunoprecipitation and sucrose gradient sedimentation analysis. A higher co-sedimentation of Alba proteins with ribosomal subunits was seen upon conditions of decreased translation, suggesting a role of these proteins in translational repression. The Leishmania Alba-domain proteins display differential cellular localization throughout the parasite development. In the insect promastigote stage, Alba proteins co-localize predominantly to the cytoplasm but they translocate to the nucleolus and the flagellum upon amastigote differentiation in the mammalian host and are found back to the cytoplasm once amastigote differentiation is completed. Heat-shock, a major signal of amastigote differentiation, triggers Alba translocation to the nucleolus and the flagellum. Purification of the Leishmania flagellum confirmed LiAlba3 enrichment in this organelle during amastigote differentiation. Moreover, partial characterization of the Leishmania flagellum proteome of promastigotes and differentiating amastigotes revealed the presence of other RNA-binding proteins, as well as differences in the flagellum composition between these two parasite lifestages. Shuttling of Alba-domain proteins between the cytoplasm and the nucleolus or the flagellum throughout the parasite life cycle suggests that these RNA-binding proteins participate in several distinct regulatory pathways controlling developmental gene expression in Leishmania.

Differential Subcellular Localization of Leishmania Alba-Domain Proteins throughout the Parasite Development

PubMed Central

Dupé, Aurélien; Dumas, Carole; Papadopoulou, Barbara

2015-01-01

Alba-domain proteins are RNA-binding proteins found in archaea and eukaryotes and recently studied in protozoan parasites where they play a role in the regulation of virulence factors and stage-specific proteins. This work describes in silico structural characterization, cellular localization and biochemical analyses of Alba-domain proteins in Leishmania infantum. We show that in contrast to other protozoa, Leishmania have two Alba-domain proteins, LiAlba1 and LiAlba3, representative of the Rpp20- and the Rpp25-like eukaryotic subfamilies, respectively, which share several sequence and structural similarities but also important differences with orthologs in other protozoa, especially in sequences targeted for post-translational modifications. LiAlba1 and LiAlba3 proteins form a complex interacting with other RNA-binding proteins, ribosomal subunits, and translation factors as supported by co-immunoprecipitation and sucrose gradient sedimentation analysis. A higher co-sedimentation of Alba proteins with ribosomal subunits was seen upon conditions of decreased translation, suggesting a role of these proteins in translational repression. The Leishmania Alba-domain proteins display differential cellular localization throughout the parasite development. In the insect promastigote stage, Alba proteins co-localize predominantly to the cytoplasm but they translocate to the nucleolus and the flagellum upon amastigote differentiation in the mammalian host and are found back to the cytoplasm once amastigote differentiation is completed. Heat-shock, a major signal of amastigote differentiation, triggers Alba translocation to the nucleolus and the flagellum. Purification of the Leishmania flagellum confirmed LiAlba3 enrichment in this organelle during amastigote differentiation. Moreover, partial characterization of the Leishmania flagellum proteome of promastigotes and differentiating amastigotes revealed the presence of other RNA-binding proteins, as well as differences in the flagellum composition between these two parasite lifestages. Shuttling of Alba-domain proteins between the cytoplasm and the nucleolus or the flagellum throughout the parasite life cycle suggests that these RNA-binding proteins participate in several distinct regulatory pathways controlling developmental gene expression in Leishmania. PMID:26334886

Molecular Characterization, Expression and in vivo Analysis of LmexCht1: the Chitinase of the Human Pathogen, Leishmania mexicana

PubMed Central

Joshi, Manju B.; Rogers, Matthew E.; Shakarian, Alison M.; Yamage, Mat; Al-Harthi, Saeed A.; Bates, Paul A.; Dwyer, Dennis M.

2010-01-01

SUMMARY Chitinases have been implicated to be of importance in the life cycle development and transmission of a variety of parasitic organisms. Using a molecular approach, we identified and characterized the structure of a single copy LmexCht1-chitinase gene from the primitive trypanosomatid pathogen of humans, Leishmania mexicana. The LmexCht1 encodes an ~50 kDa protein, with well-conserved substrate-binding and catalytic domains characteristic of members of the Chitinase-18 protein family. Further, we showed that LmexCht1 mRNA is constitutively expressed by both the insect vector (i.e. promastigote) and mammalian (i.e. amastigote) life cycle developmental forms of this protozoan parasite. Interestingly, however, amastigotes were found to secrete/release ~ >2-4 fold higher levels of chitinase activity during their growth in vitro than promastigotes. Moreover, a homologous episomal-expression system was devised and used to express an epitope–tagged LmexCht1 chimeric construct in these parasites. Expression of the LmexCht1 chimera was verified in these transfectants by RT-PCR, Western blots and indirect immunofluorescence analyses. Further, results of coupled-immunoprecipitation/ enzyme activity experiments demonstrated that the LmexCht1 chimeric protein was secreted/released by these transfected L. mexicana parasites and that it possessed functional chitinase enzyme activity. Such transfectants were also evaluated for their infectivity both in human macrophages in vitro and in two different strains of mice. Results of those experiments demonstrated that the LmexCht1 transfectants survived significantly better in human macrophages and also produced significantly larger lesions in mice than control parasites. Taken together, our results indicate that the LmexCht1-chimera afforded a definitive survival advantage to the parasite within these mammalian hosts. Thus, the LmexCht1 could potentially represent a new virulence determinant in the mammalian phase of this important human pathogen PMID:15561707

Antiprotozoal and antimicrobial compounds from the plant pathogen Septoria pistaciarum.

PubMed

Kumarihamy, Mallika; Khan, Shabana I; Jacob, Melissa; Tekwani, Babu L; Duke, Stephen O; Ferreira, Daneel; Nanayakkara, N P Dhammika

2012-05-25

Four new 1,4-dihydroxy-5-phenyl-2-pyridinone alkaloids, 17-hydroxy-N-(O-methyl)septoriamycin A (1), 17-acetoxy-N-(O-methyl)septoriamycin A (2), 13-(S)-hydroxy-N-(O-methyl)septoriamycin A (3), and 13-(R)-hydroxy-N-(O-methyl)septoriamycin A (4), together with the known compounds (+)-cercosporin (5), (+)-14-O-acetylcercosporin (6), (+)-di-O-acetylcercosporin (7), lumichrome, and brassicasterol, were isolated from an ethyl acetate extract of a culture medium of Septoria pistaciarum. Methylation of septoriamycin A (8) with diazomethane yielded three di-O-methyl analogues, two of which existed as mixtures of rotamers. We previously reported antimalarial activity of septoriamycin A. This compound also exhibited significant activity against Leishmania donovani promastigotes. Compounds 5-7 showed moderate in vitro activity against L. donovani promastigotes and chloroquine-sensitive (D6) and -resistant (W2) strains of Plasmodium falciparum, whereas compound 5 was fairly active against methicillin-sensitive and methicillin-resistant strains of Staphylococcus aureus. Compounds 5-7 also displayed moderate phytotoxic activity against both a dicot (lettuce, Lactuca sativa) and a monocot (bentgrass, Agrostis stolonifera) and cytotoxicity against a panel of cell lines.

Novel agmatine analogue, {gamma}-guanidinooxypropylamine (GAPA) efficiently inhibits proliferation of Leishmania donovani by depletion of intracellular polyamine levels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Sushma; Jhingran, Anupam; Sharma, Ankur

2008-10-10

The efficacy of {gamma}-guanidinooxypropylamine (GAPA), a novel agmatine analogue against protozoan parasite, Leishmaniadonovani was evaluated. Wild-type and ornithine decarboxylase-overexpressors of L. donovani were used to study the effect and mode of action of this inhibitor. GAPA inhibited the growth of both promastigotes and amastigotes. Ornithine decarboxylase (ODC) activity and polyamine levels were markedly lower in cells treated with GAPA and proliferation was rescued by addition of putrescine or spermidine. GAPA inhibited L. donovani recombinant ODC with K{sub i} value of {approx}60 {mu}M. The ODC-overexpressors showed significant resistance to GAPA. GAPA has pK{sub a} 6.71 and at physiological pH the analoguemore » can mimic protonated state of putrescine and can probably use putrescine transport system. Transport of putrescine in wild-type L. donovani promastigotes was inhibited by GAPA. We for the first time report that GAPA is a potential antileishmanial lead compound and it possibly inhibits L. donovani growth by depletion of intracellular polyamine levels.« less

Altered sterol profile induced in Leishmania amazonensis by a natural dihydroxymethoxylated chalcone

PubMed Central

Torres-Santos, Eduardo Caio; Sampaio-Santos, Maria Isabel; Buckner, Frederick S.; Yokoyama, Kohei; Gelb, Michael; Urbina, Julio A.; Rossi-Bergmann, Bartira

2009-01-01

Objectives The effects of the antileishmanial chalcone 2′,6′-dihydroxy-4′-methoxychalcone (DMC) on Leishmania amazonensis sterol composition and biosynthesis were investigated to obtain information about the mechanism of growth inhibition by DMC on this parasite. Methods The interference of sterol biosynthesis by DMC was studied in drug-treated promastigotes by two different methods. (i) Newly synthesized sterols from parasites grown in the presence of [3H]mevalonate were analysed by thin layer chromatography (TLC)/fluorography. (ii) Total sterols extracted from the parasites grown with or without DMC were characterized by gas chromatography coupled to mass spectroscopy (GC/MS). Results TLC and GC/MS analyses of sterols extracted from DMC-treated promastigotes revealed the accumulation of early precursors and a reduction in the levels of C-14 demethylated and C-24 alkylated sterols, as well as a reduction in exogenous cholesterol uptake. Conclusions This study demonstrates that the natural chalcone DMC alters the sterol composition of L. amazonensis and suggests that the parasite target is different from other known sterol inhibitors. PMID:19176591

Leishmania donovani: expression and characterization of Escherichia coli-expressed recombinant chitinase LdCHT1.

PubMed

Razek-Desouky, A; Specht, C A; Soong, L; Vinetz, J M

2001-12-01

Leishmania parasites produce chitinase activity (EC. 3.2.1.14) thought to be important in parasite-sandfly interactions and transmission of the parasite to the vertebrate host. Previous observations have suggested that parasite chitinases are involved in degradation of the sandfly peritrophic matrix and the chitinous layer of the cardiac valve cuticle. This chitinase activity is thought to produce an incompetent pharyngeal valve sphincter and a route of egress that allow Leishmania promastigotes to be regurgitated into the site of blood feeding. In the studies reported here, enzymatically active L. donovani chitinase LdCHT1 was expressed as a thioredoxin fusion protein in Escherichia coli strain AD494 (DE3). Recombinant LdCHT1 had a predominantly endochitinase activity, in contrast to previous reports of both exo- and endochitinase activities in axenic culture supernatants of diverse Leishmania spp. promastigotes. The predominant endochitinase activity of recombinant LdCHT1 is consistent with the presumed function of the enzyme in disrupting chitinous structures in the sandfly digestive system to allow transmission. Copyright 2001 Elsevier Science (USA).

Intracellular zinc flux causes reactive oxygen species mediated mitochondrial dysfunction leading to cell death in Leishmania donovani.

PubMed

Kumari, Anjali; Singh, Krishn Pratap; Mandal, Abhishek; Paswan, Ranjeet Kumar; Sinha, Preeti; Das, Pradeep; Ali, Vahab; Bimal, Sanjiva; Lal, Chandra Shekhar

2017-01-01

Leishmaniasis caused by Leishmania parasite is a global threat to public health and one of the most neglected tropical diseases. Therefore, the discovery of novel drug targets and effective drug is a major challenge and an important goal. Leishmania is an obligate intracellular parasite that alternates between sand fly and human host. To survive and establish infections, Leishmania parasites scavenge and internalize nutrients from the host. Nevertheless, host cells presents mechanism like nutrient restriction to inhibit microbial growth and control infection. Zinc is crucial for cellular growth and disruption in its homeostasis hinders growth and survival in many cells. However, little is known about the role of zinc in Leishmania growth and survival. In this study, the effect of zinc on the growth and survival of L.donovani was analyzed by both Zinc-depletion and Zinc-supplementation using Zinc-specific chelator N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) and Zinc Sulfate (ZnSO4). Treatment of parasites with TPEN rather than ZnSO4 had significantly affected the growth in a dose- and time-dependent manner. The pre-treatment of promastigotes with TPEN resulted into reduced host-parasite interaction as indicated by decreased association index. Zn depletion resulted into flux in intracellular labile Zn pool and increased in ROS generation correlated with decreased intracellular total thiol and retention of plasma membrane integrity without phosphatidylserine exposure in TPEN treated promastigotes. We also observed that TPEN-induced Zn depletion resulted into collapse of mitochondrial membrane potential which is associated with increase in cytosolic calcium and cytochrome-c. DNA fragmentation analysis showed increased DNA fragments in Zn-depleted cells. In summary, intracellular Zn depletion in the L. donovani promastigotes led to ROS-mediated caspase-independent mitochondrial dysfunction resulting into apoptosis-like cell death. Therefore, cellular zinc homeostasis in Leishmania can be explored for new drug targets and chemotherapeutics to control Leishmanial growth and disease progression.

In vitro 4-Aryloxy-7-chloroquinoline derivatives are effective in mono- and combined therapy against Leishmania donovani and induce mitocondrial membrane potential disruption.

PubMed

Valdivieso, Elizabeth; Mejías, Fabiola; Torrealba, Carlos; Benaim, Gustavo; Kouznetsov, Vladimir V; Sojo, Felipe; Rojas-Ruiz, Fernando A; Arvelo, Francisco; Dagger, Francehuli

2018-07-01

The present study evaluates in vitro the effect of two synthetic compounds of the 7-chloro-4-aryloxyquinoline series, QI (C 17 H 12 ClNO 3 ) and QII (C 18 H 15 ClN 4 O 2 S), on Leishmania donovani parasites. The results obtained demonstrate that these compounds are able to inhibit the proliferation of L. donovani promastigotes in a dose-dependent way (QI IC 50  = 13.03 ± 3.4 and QII IC 50  = 7.90 ± 0.6 μM). Likewise, these compounds significantly reduced the percentage of macrophage infection by amastigotesand the number of amastigotes within macrophage phagolysosomes, the clinical relevant phase of these parasites. Compound QI showed an IC 50 value of 0.66 ± 0.2 μM, while for derivative QII, the corresponding IC 50 was 1.02 ± 0.17 μM. Interestingly, the amastigotes were more susceptible to the drug treatment when compared to promastigotes. Furthermore, no cytotoxic effect of these compounds was observed on the macrophage cell line at the concentrations tested. The combination of these compounds with miltefosine and amphotericin B on both parasite morphotypes was evaluated. The isobolograms showed a synergistic effect for both combinations; with a Fractional Inhibitory Concentration (FIC) Index lower than 1 for promastigotes and less than 0.3 for intracellular amastigotes. The effect of QI and QII on mitochondrial membrane potential was also studied. The combination of quinolone derivatives compounds with miltefosine and amphotericin B showed 5-8-fold stronger depolarization of membrane mitochondrial potential when compared to drugs alone. The present work validates the combination of drugs as an effective alternative to potentiate the action of anti-Leishmania agents and points to the quinoline compounds studied here as possible leishmanicidal drugs. Copyright © 2018 Elsevier B.V. All rights reserved.

Antiparasitic effect of the Psidium guajava L. (guava) and Psidium brownianum MART. EX DC. (araçá-de-veado) extracts.

PubMed

Machado, Antonio J T; Santos, Antonia T L; Martins, Gioconda M A B; Cruz, Rafael P; Costa, Maria do S; Campina, Fábia F; Freitas, Maria A; Bezerra, Camila F; Leal, Antonio L A B; Carneiro, Joara N P; Coronel, Cathia; Rolón, Miriam; Gómez, Celeste V; Coutinho, Henrique D M; Morais-Braga, Maria F B

2018-03-13

In the search for new therapeutic agents against neglected diseases, both aqueous and hydroethanolic extracts from Psidium guajava L. and P. brownianum Mart ex DC leaves were investigated regarding their antiparasitic effect and cytotoxic potential. The extracts were tested at three concentrations (250, 500 and 1000 μg/mL) against Trypanosoma cruzi epimastigote forms (Chagas, 1909), Leishmania braziliensis (Vianna, 1911) and L. infantum promastigotes forms (Nicolle, 1908), as well as against fibroblasts. P. guajava showed no activity against T. cruzi forms, while the hydroethanolic (PBHE), aqueous by decoction (PBAED) and aqueous by infusion (PBAEI) P. browninaum extracts were responsible, respectively, for inhibiting 100, 100 and 92.68% of T. cruzi epimastigote growth at the 1000 μg/mL concentration. The P. brownianum hydroethanolic extract (PBHE) at the highest concentration caused 58.46% death in L. braziliensis, thus demonstrating moderate activity, however when tested against L. infantum, the PBHE inhibited their growth by 37.16%, revealing its low activity. As for the cytotoxicity assays, the P. brownianum aqueous extract by decoction (PBAED) obtained the highest death percentage when compared to the others, causing 90.85% fibroblast mortality at the 1000 μg/mL concentration. Copyright © 2018 Elsevier Ltd. All rights reserved.

Antimony Uptake Systems in the Protozoan Parasite Leishmania and Accumulation Differences in Antimony-Resistant Parasites

PubMed Central

Brochu, Christian; Wang, Jingyu; Roy, Gaétan; Messier, Nadine; Wang, Xiao-Yan; Saravia, Nancy G.; Ouellette, Marc

2003-01-01

The first line drug against leishmaniasis consists of pentavalent antimony [Sb(V)], but there is general belief that the active form of the metal is the trivalent form [Sb(III)]. In this study, we have quantified the accumulation of Sb(V) and Sb(III) in Leishmania by using inductively coupled plasma mass spectrometry. The accumulation was studied in three Leishmania species at various life stages, sensitive or resistant to antimony. Both Sb(III) and Sb(V) are accumulated in promastigote and amastigote parasites, but through competition experiments with arsenite, we found that the routes of entry of Sb(V) and Sb(III) are likely to differ in Leishmania. The level of accumulation of either Sb(III) or Sb(V), however, was not correlated with the susceptibility of wild-type Leishmania cells to antimony. This suggests that other factors may also be implicated in the mode of action of the drugs. In contrast to metal susceptibility, resistance to Sb(III) correlated well with decreased antimony accumulation. This phenotype was energy dependent and highlights the importance of transport systems in drug resistance of this protozoan parasite. PMID:14506011

Pro-apoptotic effect of the landrace Bangla Mahoba of Piper betle on Leishmania donovani may be due to the high content of eugenol.

PubMed

Misra, Pragya; Kumar, Awanish; Khare, Prashant; Gupta, Swati; Kumar, Nikhil; Dube, Anuradha

2009-08-01

In the absence of effective and safe treatment for visceral leishmaniasis or Kala-azar - a devastating parasitic disease caused by Leishmania donovani - the search for anti-leishmanial agents from natural resources in common use is imperative. Recently, the comparative in vitro anti-leishmanial activity of methanolic extracts from two landraces of Piper betle - P. betle landrace Bangla Mahoba (PB-BM) and P. betle landrace Kapoori Vellaikodi (PB-KV) - has been reported. Here, the putative pathway responsible for death induced by the effective extract of PB-BM methanolic extract in promastigotes, as well as the intracellular amastigote form of L. donovani, was assessed using various biochemical approaches. It was found that PB-BM was capable of selectively inhibiting both stages of Leishmania parasites by accelerating apoptotic events by generation of reactive oxygen species targeting the mitochondria without any cytotoxicity towards macrophages. The study was extended to determine the presence or absence of activity of the methanolic extract of PB-BM and PB-KV on the basis of differences in essential oil composition present in the extract assessed by GC and MS. The essential oil from PB-BM was found to be rich in eugenol compared with that from PB-KV. The anti-leishmanial efficacy of PB-BM methanolic extract mediated through apoptosis is probably due to the higher content of eugenol in the active landrace. This observation emphasizes the need to extend studies related to traditional medicines from bioactive plants below the species level to the gender/landrace level for better efficacy and reproducibility.

Biochemistry and Chemotherapy of Malaria and Leishmaniasis

DTIC Science & Technology

1993-12-06

sativum), elephant garlic (Allium scorodoprasum), onion (Allium cepa), and licorice ( Glycyrrhiza glabra). Cells of Leish- mania mexicana 227 and... Glycyrrhiza glabra) inhibited the growth of the leishmanial parasites, but were not toxic to HeLa cells. All of the extracts showed varying inhibitory...root) and Glycyrrhiza glabra (licorice). 10 * Materials & Methods Cultures of Parasitic Protozoa: Promastigotes of Leishmania mexicana Walter Reed

Identification of Leishmania spp. promastigotes in the intestines, ovaries and salivary glands of Rhipicephalus sanguineus actively infesting dogs

USDA-ARS?s Scientific Manuscript database

Sand flies are recognized as the major vector of canine visceral leishmaniasis. However, in some areas of Brazil where sand flies do not occur, this disease is found in humans and dogs. There has been speculation that ticks might play a role in transmission of canine visceral leishmaniasis and the D...

Classical ROS-dependent and early/rapid ROS-independent release of Neutrophil Extracellular Traps triggered by Leishmania parasites.

PubMed

Rochael, Natalia C; Guimarães-Costa, Anderson B; Nascimento, Michelle T C; DeSouza-Vieira, Thiago S; Oliveira, Matheus P; Garcia e Souza, Luiz F; Oliveira, Marcus F; Saraiva, Elvira M

2015-12-17

Neutrophil extracellular traps (NETs) extruded from neutrophils upon activation are composed of chromatin associated with cytosolic and granular proteins, which ensnare and kill microorganisms. This microbicidal mechanism named classical netosis has been shown to dependent on reactive oxygen species (ROS) generation by NADPH oxidase and also chromatin decondensation dependent upon the enzymes (PAD4), neutrophil elastase (NE) and myeloperoxidase (MPO). NET release also occurs through an early/rapid ROS-independent mechanism, named early/rapid vital netosis. Here we analyze the role of ROS, NE, MPO and PAD4 in the netosis stimulated by Leishmania amazonensis promastigotes in human neutrophils. We demonstrate that promastigotes induce a classical netosis, dependent on the cellular redox imbalance, as well as by a chloroamidine sensitive and elastase activity mechanism. Additionally, Leishmania also induces the early/rapid NET release occurring only 10 minutes after neutrophil-parasite interaction. We demonstrate here, that this early/rapid mechanism is dependent on elastase activity, but independent of ROS generation and chloroamidine. A better understanding of both mechanisms of NET release, and the NETs effects on the host immune system modulation, could support the development of new potential therapeutic strategies for leishmaniasis.

Leishmanicidal activity of polyphenolic-rich extract from husk fiber of Cocos nucifera Linn. (Palmae).

PubMed

Mendonça-Filho, Ricardo R; Rodrigues, Igor A; Alviano, Daniela S; Santos, André L S; Soares, Rosangela M A; Alviano, Celuta S; Lopes, Angela H C S; Rosa, Maria do Socorro S

2004-04-01

The available therapy for leishmaniasis, which affects 2 million people per annum, still causes serious side effects. The polyphenolic-rich extract from the husk fiber of Cocos nucifera Linn. (Palmae) presents antibacterial and antiviral activities, also inhibiting lymphocyte proliferation, as shown by our group in previous works. In the present study, the in vitro leishmanicidal effects of C. nucifera on Leishmania amazonensis were evaluated. The minimal inhibitory concentration of the polyphenolic-rich extract from C. nucifera to completely abrogate parasite growth was 10 microg/ml. Pretreatment of peritoneal mouse macrophages with 10 microg/ml of C. nucifera polyphenolic-rich extract reduced approximately 44% the association index between these macrophages and L. amazonensis promastigotes, with a concomitant increase of 182% in nitric oxide production by the infected macrophage in comparison to nontreated macrophages. These results provide new perspectives on drug development against leishmaniasis, since the extract of C. nucifera at 10 microg/ml is a strikingly potent leishmanicidal substance which inhibited the growth of both promastigote and amastigote developmental stages of L. amazonensis after 60 min, presenting no in vivo allergenic reactions or in vitro cytotoxic effects in mammalian systems.

Nerolidol, the main constituent of Piper aduncum essential oil, has anti-Leishmania braziliensis activity.

PubMed

Ceole, Ligia Fernanda; Cardoso, Maria DAS Graças; Soares, Maurilio José

2017-08-01

Leishmania (Viannia) braziliensis is a protozoan that causes mucocutaneous leishmaniasis, which is an infectious disease that affects more than 12 million people worldwide. The available treatment is limited, has side-effects or is inefficient. In a search for alternative compounds of natural origin, we tested the microbicidal activity of Piper aduncum essential oil (PaEO) on this parasite. Our data showed that PaEO had an inhibitory effect on the growth of L. braziliensis promastigotes with an IC50/24 h=77·9 µg mL-1. The main constituent (nerolidol: 25·22%) presented a similar inhibitory effect (IC50/24 h = 74·3 µg mL-1). Ultrastructural observation of nerolidol-treated parasites by scanning and transmission electron microscopies revealed cell shrinkage and morphological alterations in the mitochondrion, nuclear chromatin and flagellar pocket. Flow cytometry analysis showed a reduction in the cell size, loss of mitochondrial membrane potential, phosphatidylserine exposure and DNA degradation, which when associated with the morphological changes indicated that nerolidol induced incidental cell death in the L. braziliensis promastigotes. The results presented here indicate that nerolidol derivatives are promising compounds for further evaluation against Leishmania parasites.

Leishmania donovani Resistance to Miltefosine Involves a Defective Inward Translocation of the Drug

PubMed Central

Pérez-Victoria, F. Javier; Castanys, Santiago; Gamarro, Francisco

2003-01-01

Miltefosine (hexadecylphosphocholine [HePC]) is the first drug approved for the oral treatment of visceral leishmaniasis. As part of a study on the mechanisms of action of this drug and on the rates of resistance to this drug, we have been working in vitro with an Leishmania donovani line that was previously shown to be 15-fold more resistant to HePC. We have studied the accumulation of [14C]HePC by L. donovani promastigotes and have found a drastic reduction (>95%) in the ability of the resistant line to internalize the drug. Binding of HePC to the plasma membrane and drug efflux from preloaded cells were similar in both drug-sensitive and -resistant lines, and no [14C]HePC metabolism was evident in either line. Resistant parasites were also unable to take up other short-chain phospholipid analogs, independently of their polar head group, even though endocytosis remained unaltered. Finally, HePC uptake was temperature and energy dependent and sensitive to the thiol-reactive agent N-ethylmaleimide. We propose that inward translocation of a short-chain phospholipid across the plasma membrane may exist in Leishmania promastigotes and that such activity is defective in the resistant line. PMID:12878496

In vivo Antimalarial and Antitrypanosomal Activity of Strychnogucine B, a Bisindole Alkaloid from Strychnos icaja.

PubMed

Beaufay, Claire; Ledoux, Allison; Jansen, Olivia; Bordignon, Annélise; Zhao, Senzhi; Teijaro, Christiana N; Andrade, Rodrigo B; Quetin-Leclercq, Joëlle; Frédérich, Michel

2018-06-21

Strychnogucine B is a bisindole alkaloid previously isolated from Strychnos icaja that possesses promising in vitro antiplasmodial properties. This compound was synthesized in four steps from (-)-strychnine. As no acute toxicity was observed at the highest tested cumulative dose of 60 mg/kg, its in vivo antimalarial activity was determined intraperitoneally at 30 mg/kg/d in a Plasmodium berghei murine model. In the Peters's 4-d suppressive test, this alkaloid suppressed the parasitaemia by almost 36% on day 5 and 60% on day 7 compared to vehicle-treated mice. In addition to this interesting antimalarial activity, it showed moderate in vitro antitrypanosomal activity but no in vivo activity in an acute Trypanosoma brucei model. It was also inactive in vitro on Leishmania mexicana promastigotes. This highlights its selective antimalarial efficacy and leads to further investigation to assess its potential as new antimalarial lead compound. Georg Thieme Verlag KG Stuttgart · New York.

Augmented Oxygen-Dependent Killing of Leishmania.

DTIC Science & Technology

1992-06-30

reduction-oxidation cycling drugs: amphotericin B, menadione , and phenazine methosulfate. Promastigotes were exposed to the above drugs under...P02 = 2]..1 kPa) or hyperoxic conditions(P02 - 91.7 kPa). High oxygen tensions did not alter the lethal effects of either menadione or phenazine...effects of high oxygen tensions on the lethal effects of three reduction-oxidation cycling drugs: amphotericin B, menadione , and phenazine

Investigations of Cross Immunity between Leishmania tropica (Jericho) and Leishmania braziliensis in Experimentally Infected Mystromys albacaudatus.

DTIC Science & Technology

1979-09-01

would be the utilization of irradiated killed promastigotes of L. braziliensis. Precedence of this exists in malaria with the radiation of attenuated...34 "’Vaccination with a 5-year-old IHurman Strain of I. tropica from the Negev ," transcript of the Royal S:ociety of T7ropical Medicine and Hy,-iene; 66 - 1972

Defeating Leishmania resistance to miltefosine (hexadecylphosphocholine) by peptide-mediated drug smuggling: a proof of mechanism for trypanosomatid chemotherapy.

PubMed

Luque-Ortega, Juan Román; de la Torre, Beatriz G; Hornillos, Valentín; Bart, Jean-Mathieu; Rueda, Cristina; Navarro, Miguel; Amat-Guerri, Francisco; Acuña, A Ulises; Andreu, David; Rivas, Luis

2012-08-10

Miltefosine (hexadecylphosphocholine, HePC), the first orally active drug successful against leishmaniasis, is especially active on the visceral form of the disease. Resistance mechanisms are almost exclusively associated to dysfunction in HePC uptake systems. In order to evade the requirements of its cognate receptor/translocator, HePC-resistant Leishmania donovani parasites (R40 strain) were challenged with constructs consisting of an ω-thiol-functionalized HePC analogue conjugated to the cell-penetrating peptide (CPP) Tat(48-60), either through a disulfide or a thioether bond. The conjugates enter and kill both promastigote and intracellular amastigote forms of the R40 strain. Intracellular release of HePC by reduction of the disulfide-based conjugate was confirmed by means of double tagging at both the CPP (Quasar 670) and HePC (BODIPY) moieties. Scission of the conjugate, however, is not mandatory, as the metabolically more stable thioether conjugate retained substantial activity. The disulfide conjugate is highly active on the bloodstream form of Trypanosoma b. brucei, naturally resistant to HePC. Our results provide proof-of-mechanism for the use of CPP conjugates to avert drug resistance by faulty drug accumulation in parasites, as well as the possibility to extend chemotherapy into other parasites intrinsically devoid of membrane translocation systems. Copyright © 2012 Elsevier B.V. All rights reserved.

Development of a LAMP assay for detection of Leishmania infantum infection in dogs using conjunctival swab samples.

PubMed

Gao, Chun-hua; Ding, Dan; Wang, Jun-yun; Steverding, Dietmar; Wang, Xia; Yang, Yue-tao; Shi, Feng

2015-07-15

Leishmania infantum infections in dogs play a crucial role in the transmission of pathogens causing visceral leishmaniasis to humans in the Gansu province, northwest China. To be able to control zoonotic transmission of the parasite to humans, a non-invasive loop-mediated isothermal amplification (LAMP) assay to specifically detect L. infantum infections in dogs was developed. The primers used in the LAMP assay were designed to target kinetoplast DNA minicircle sequences of the L. infantum isolate MCAN/CN/90/SC and tested using DNA isolated from promastigotes of different Leishmania species. The LAMP assay was evaluated with conjunctional swab samples obtained from 111 and 33 dogs living in an endemic and a non-endemic region of zoonotic visceral leishmaniasis in the Gansu province, respectively. The LAMP assay was also compared with conventional PCR, ELISA and microscopy using conjunctional swab, serum and bone marrow samples from the dogs, respectively. The LAMP assay detected 1 fg of L. infantum DNA purified from cultured promastigotes which was 10-fold more sensitive than a conventional PCR test using Leishmania genus-specific primers. No cross reaction was observed with DNA isolated from promastigotes of L. donovani, L. major, L. tropica, and L. braziliensis, and the L. infantum reference strain MHOM/TN/80/IPT1. The L. infantum-positive rates obtained for field-collected samples were 61.3%, 58.6%, 40.5% and 10.8% by LAMP, PCR, ELISA and microscopy, respectively. As only one out of the 33 samples from control dogs from the non-endemic region of zoonotic visceral leishmaniasis was positive by the LAMP assay and the PCR test, the observed true negative rate (specificity) was 97% for both methods. This study has shown that the non-invasive, conjunctional swab-based LAMP assay developed was more sensitive in the detection of leishmaniasis in dogs than PCR, ELISA and microscopy. The findings indicate that the LAMP assay is a sensitive and specific method for the field surveillance of domestic dogs, particularly of asymptomatic canines, in ZVL-endemic areas in western China.

Development of Serologic Assays for the Diagnosis of New World Leishmaniasis

DTIC Science & Technology

1986-02-20

the skin by the bite of the phlebotomine sandfly vector , and is maintained by subsequent parasite invasion of and multiplication within cells of the...distribution within the lipid bilayer may play a significant role in the behavior of the parasite within the insect vector and the mammalian host...by the phlebotomine sandfly vector . Promastigotes are then phagocytized by dermal macrophages, where they differentiate into the intracellular, or

Arrabidaea chica hexanic extract induces mitochondrion damage and peptidase inhibition on Leishmania spp.

PubMed

Rodrigues, Igor A; Azevedo, Mariana M B; Chaves, Francisco C M; Alviano, Celuta S; Alviano, Daniela S; Vermelho, Alane B

2014-01-01

Currently available leishmaniasis treatments are limited due to severe side effects. Arrabidaea chica is a medicinal plant used in Brazil against several diseases. In this study, we investigated the effects of 5 fractions obtained from the crude hexanic extract of A. chica against Leishmania amazonensis and L. infantum, as well as on the interaction of these parasites with host cells. Promastigotes were treated with several concentrations of the fractions obtained from A. chica for determination of their minimum inhibitory concentration (MIC). In addition, the effect of the most active fraction (B2) on parasite's ultrastructure was analyzed by transmission electron microscopy. To evaluate the inhibitory activity of B2 fraction on Leishmania peptidases, parasites lysates were treated with the inhibitory and subinhibitory concentrations of the B2 fraction. The minimum inhibitory concentration of B2 fraction was 37.2 and 18.6 μg/mL for L. amazonensis and L. infantum, respectively. Important ultrastructural alterations as mitochondrial swelling with loss of matrix content and the presence of vesicles inside this organelle were observed in treated parasites. Moreover, B2 fraction was able to completely inhibit the peptidase activity of promastigotes at pH 5.5. The results presented here further support the use of A. chica as an interesting source of antileishmanial agents.

Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species

PubMed Central

Raymond, Frédéric; Boisvert, Sébastien; Roy, Gaétan; Ritt, Jean-François; Légaré, Danielle; Isnard, Amandine; Stanke, Mario; Olivier, Martin; Tremblay, Michel J.; Papadopoulou, Barbara; Ouellette, Marc; Corbeil, Jacques

2012-01-01

The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved to an average 16-fold mean coverage by next-generation DNA sequencing technologies. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described thus providing an opportunity for comparison with the completed genomes of pathogenic Leishmania species. A high synteny was observed between all sequenced Leishmania species. A limited number of chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic to L. major. Globally, >90% of the L. tarentolae gene content was shared with the other Leishmania species. We identified 95 predicted coding sequences unique to L. tarentolae and 250 genes that were absent from L. tarentolae. Interestingly, many of the latter genes were expressed in the intracellular amastigote stage of pathogenic species. In addition, genes coding for products involved in antioxidant defence or participating in vesicular-mediated protein transport were underrepresented in L. tarentolae. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the zinc metallo-peptidase surface glycoprotein GP63 and the promastigote surface antigen PSA31C. Overall, L. tarentolae's gene content appears better adapted to the promastigote insect stage rather than the amastigote mammalian stage. PMID:21998295

Enterocin AS-48 as Evidence for the Use of Bacteriocins as New Leishmanicidal Agents.

PubMed

Abengózar, María Ángeles; Cebrián, Rubén; Saugar, José María; Gárate, Teresa; Valdivia, Eva; Martínez-Bueno, Manuel; Maqueda, Mercedes; Rivas, Luis

2017-04-01

We report the feasibility of enterocin AS-48, a circular cationic peptide produced by Enterococcus faecalis , as a new leishmanicidal agent. AS-48 is lethal to Leishmania promastigotes as well as to axenic and intracellular amastigotes at low micromolar concentrations, with scarce cytotoxicity to macrophages. AS-48 induced a fast bioenergetic collapse of L. donovani promastigotes but only a partial permeation of their plasma membrane with limited entrance of vital dyes, even at concentrations beyond its full lethality. Fluoresceinated AS-48 was visualized inside parasites by confocal microscopy and seen to cause mitochondrial depolarization and reactive oxygen species production. Altogether, AS-48 appeared to have a mixed leishmanicidal mechanism that includes both plasma membrane permeabilization and additional intracellular targets, with mitochondrial dysfunctionality being of special relevance. This complex leishmanicidal mechanism of AS-48 persisted even for the killing of intracellular amastigotes, as evidenced by transmission electron microscopy. We demonstrated the potentiality of AS-48 as a new and safe leishmanicidal agent, expanding the growing repertoire of eukaryotic targets for bacteriocins, and our results provide a proof of mechanism for the search of new leishmanicidal bacteriocins, whose diversity constitutes an almost endless source for new structures at moderate production cost and whose safe use on food preservation is well established. Copyright © 2017 American Society for Microbiology.

Resveratrol Is Active against Leishmania amazonensis: In Vitro Effect of Its Association with Amphotericin B

PubMed Central

Ferreira, Christian; Soares, Deivid Costa; do Nascimento, Michelle Tanny Cunha; Pinto-da-Silva, Lucia Helena; Sarzedas, Carolina Galvão; Tinoco, Luzineide Wanderley

2014-01-01

Resveratrol is a polyphenol found in black grapes and red wine and has many biological activities. In this study, we evaluated the effect of resveratrol alone and in association with amphotericin B (AMB) against Leishmania amazonensis. Our results demonstrate that resveratrol possesses both antipromastigote and antiamastigote effects, with 50% inhibitory concentrations (IC50s) of 27 and 42 μM, respectively. The association of resveratrol with AMB showed synergy for L. amazonensis amastigotes, as demonstrated by the mean sums of fractional inhibitory index concentration (mean ΣFIC) of 0.483, although for promastigotes, this association was indifferent. Treatment with resveratrol increased the percentage of promastigotes in the sub-G0/G1 phase of the cell cycle, reduced the mitochondrial potential, and showed an elevated choline peak and CH2-to-CH3 ratio in the nuclear magnetic resonance (NMR) spectroscopy analysis; all these features indicate parasite death. Resveratrol also decreased the activity of the enzyme arginase in uninfected and infected macrophages with and without stimulation with interleukin-4 (IL-4), also implicating arginase inhibition in parasite death. The anti-Leishmania effect of resveratrol and its potential synergistic association with AMB indicate that these compounds should be subjected to further studies of drug association therapy in vivo. PMID:25114129

Activity of cycloartane-type triterpenes and sterols isolated from Musa paradisiaca fruit peel against Leishmania infantum chagasi.

PubMed

Silva, A A S; Morais, S M; Falcão, M J C; Vieira, I G P; Ribeiro, L M; Viana, S M; Teixeira, M J; Barreto, F S; Carvalho, C A; Cardoso, R P A; Andrade-Junior, H F

2014-09-25

The aim of the study was to evaluate in vitro the antileishmanial activity of triterpenes and sterols isolated from Musa paradisiaca (banana) fruit peel used traditionally to treat leishmaniasis. The compounds were isolated from the ethanolic extract of the peel of the banana fruit by column chromatography. The chemical structure of compounds was determined by (1)H and (13)C - nuclear magnetic resonance spectroscopy. The cytotoxicity was measured in RAW 264.7 cells and LLC-MK2. Leishmanicidal activity against L. infantum chagasi promastigotes was performed by the MTT colorimetric method and activity against amastigotes was assayed in mammalian cells using in situ ELISA method. Five compounds were identified, consisting of three triterpenes: cycloeucalenone, 31-norcyclolaudenone and 24-methylene-cicloartanol and a mixture of two sterols: beta-sitosterol and stigmasterol. With the exception of cycloeucalenone, all compounds showed statistically similar activity against promastigote to pentamidine. While, acting against amastigotes, excluding 31-norcyclolaudenone, other compounds showed activity similar to amphotericin B. All compounds showed low cytotoxicity in mammalian cells. This study partially confirms the use of Musa paradisiaca in folk medicine against leishmaniasis. Further in vivo studies are necessary to evaluate the efficacy. Copyright © 2014 Elsevier GmbH. All rights reserved.

Enterocin AS-48 as Evidence for the Use of Bacteriocins as New Leishmanicidal Agents

PubMed Central

Abengózar, María Ángeles; Cebrián, Rubén; Saugar, José María; Gárate, Teresa; Valdivia, Eva; Martínez-Bueno, Manuel; Maqueda, Mercedes

2017-01-01

ABSTRACT We report the feasibility of enterocin AS-48, a circular cationic peptide produced by Enterococcus faecalis, as a new leishmanicidal agent. AS-48 is lethal to Leishmania promastigotes as well as to axenic and intracellular amastigotes at low micromolar concentrations, with scarce cytotoxicity to macrophages. AS-48 induced a fast bioenergetic collapse of L. donovani promastigotes but only a partial permeation of their plasma membrane with limited entrance of vital dyes, even at concentrations beyond its full lethality. Fluoresceinated AS-48 was visualized inside parasites by confocal microscopy and seen to cause mitochondrial depolarization and reactive oxygen species production. Altogether, AS-48 appeared to have a mixed leishmanicidal mechanism that includes both plasma membrane permeabilization and additional intracellular targets, with mitochondrial dysfunctionality being of special relevance. This complex leishmanicidal mechanism of AS-48 persisted even for the killing of intracellular amastigotes, as evidenced by transmission electron microscopy. We demonstrated the potentiality of AS-48 as a new and safe leishmanicidal agent, expanding the growing repertoire of eukaryotic targets for bacteriocins, and our results provide a proof of mechanism for the search of new leishmanicidal bacteriocins, whose diversity constitutes an almost endless source for new structures at moderate production cost and whose safe use on food preservation is well established. PMID:28167557

Assessment of India’s Research Literature

DTIC Science & Technology

2006-01-01

promastigotes. • (19) [inhibitor 16.0%, acid 6.4%, amino 3.5%, activ 3.5%, antibacteri 3.1%, trypsin 2.9%, amino.acid 2.0%, chymotrypsin...substitut 2.1%] – Antibacterial and antifungal activity in compounds, particularly screening for such activity • (46) [compound 44.0%, activ 2.9...studies, and its presence in the midst of the high tech top tier countries stands out. The presence of Philippines in this list has strong reasons

Field Evaluation of a Fluorogenic Probe-Based PCR Assay for Identification of a Visceral Leishmaniasis Gene Target

DTIC Science & Technology

2004-06-01

encodes protein required for amastigote development, which can ultimately be expressed in humans as VL (3, 4, 5). The leishmaniasises are also expressed ...Leishmania surveillance at Tallil Air Base, south central Iraq, expressed concern of a potential leishmaniasis outbreak situation. In response, we...site. That L. donovani promastigote-to-amastigote development, and VL pathogenesis, requires an A2 gene family encoded factor defines this protein

Evaluation of the Leishmanicidal Activity of Rutaceae and Lauraceae Ethanol Extracts on Golden Syrian Hamster (Mesocricetus auratus) Peritoneal Macrophages.

PubMed

Chávez Enciso, N A; Coy-Barrera, E D; Patiño, O J; Cuca, L E; Delgado, Gabriela

2014-05-01

Traditional medicine has provided a number of therapeutic solutions for the control of infectious agents, cancers, and other diseases. After screening a wide variety of Colombian plant extracts, we have identified promising antileishmanial activity in ethanol extracts from Ocotea macrophylla (Lauraceae) and Zanthoxyllum monophyllum (Rutaceae). In this study, we evaluated the in vitro activity of two ethanol extracts, one from Ocotea macrophylla and the other from Zanthoxyllum monophyllum and one alkaloid fraction of ethanol extract of Zanthoxyllum monophyllum, on peritoneal macrophages isolated from golden Syrian hamsters (Mesocricetus auratus) infected with Leishmania panamensis and Leishmania major promastigotes. All of the extracts studied displayed promising (≥2) selectivity indices (S/I), the most significant of which were for ethanol extract of Zanthoxyllum monophyllum against Leishmania panamensis (S/I=12) and alkaloid fraction of ethanol extract of Zanthoxyllum monophyllum against Leishmania major (S/I=11). These results support the use of ethanol extracts and alkaloid fractions isolated from Ocotea macrophylla and Zanthoxyllum monophyllum, respectively; as therapeutic options for cutaneous leishmaniasis.

Fragment-Based Phenotypic Lead Discovery: Cell-Based Assay to Target Leishmaniasis.

PubMed

Ayotte, Yann; Bilodeau, François; Descoteaux, Albert; LaPlante, Steven R

2018-05-02

A rapid and practical approach for the discovery of new chemical matter for targeting pathogens and diseases is described. Fragment-based phenotypic lead discovery (FPLD) combines aspects of traditional fragment-based lead discovery (FBLD), which involves the screening of small-molecule fragment libraries to target specific proteins, with phenotypic lead discovery (PLD), which typically involves the screening of drug-like compounds in cell-based assays. To enable FPLD, a diverse library of fragments was first designed, assembled, and curated. This library of soluble, low-molecular-weight compounds was then pooled to expedite screening. Axenic cultures of Leishmania promastigotes were screened, and single hits were then tested for leishmanicidal activity against intracellular amastigote forms in infected murine bone-marrow-derived macrophages without evidence of toxicity toward mammalian cells. These studies demonstrate that FPLD can be a rapid and effective means to discover hits that can serve as leads for further medicinal chemistry purposes or as tool compounds for identifying known or novel targets. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-Affinity Binding of Silybin Derivatives to the Nucleotide-Binding Domain of a Leishmania tropica P-Glycoprotein-Like Transporter and Chemosensitization of a Multidrug-Resistant Parasite to Daunomycin

PubMed Central

Pérez-Victoria, José M.; Pérez-Victoria, F. Javier; Conseil, Gwenaëlle; Maitrejean, Mathias; Comte, Gilles; Barron, Denis; Di Pietro, Attilio; Castanys, Santiago; Gamarro, Francisco

2001-01-01

In order to overcome the multidrug resistance mediated by P-glycoprotein-like transporters in Leishmania spp., we have studied the effects produced by derivatives of the flavanolignan silybin and related compounds lacking the monolignol unit on (i) the affinity of binding to a recombinant C-terminal nucleotide-binding domain of the L. tropica P-glycoprotein-like transporter and (ii) the sensitization to daunomycin on promastigote forms of a multidrug-resistant L. tropica line overexpressing the transporter. Oxidation of the flavanonol silybin to the corresponding flavonol dehydrosilybin, the presence of the monolignol unit, and the addition of a hydrophobic substituent such as dimethylallyl, especially at position 8 of ring A, considerably increased the binding affinity. The in vitro binding affinity of these compounds for the recombinant cytosolic domain correlated with their modulation of drug resistance phenotype. In particular, 8-(3,3-dimethylallyl)-dehydrosilybin effectively sensitized multidrug-resistant Leishmania spp. to daunomycin. The cytosolic domains are therefore attractive targets for the rational design of inhibitors against P-glycoprotein-like transporters. PMID:11158738

Leishmanicidal evaluation of extracts from native plants of the Yucatan peninsula.

PubMed

Peraza-Sánchez, S R; Cen-Pacheco, F; Noh-Chimal, A; May-Pat, F; Simá-Polanco, P; Dumonteil, E; García-Miss, M R; Mut-Martín, M

2007-06-01

Methanol extracts were prepared from different parts of 18 plants collected in the Yucatan peninsula and evaluated in an in vitro bioassay for leishmanicidal activity against Leishmania mexicana promastigotes. The ten most potent plant extracts (IC(50)<50 microg/ml) were Aphelandra scabra leaves, Byrsonima bucidaefolia bark, Byrsonima crassifolia bark, Clusia flava leaves, Cupania dentata bark, Diphysa carthagenensis leaves, Dorstenia contrajerva whole plant, Milleria quinqueflora roots, Tridax procumbens whole plant, and Vitex gaumeri bark.

Antiprotozoal activity of Neurolaena lobata.

PubMed

Berger, I; Passreiter, C M; Cáceres, A; Kubelka, W

2001-06-01

Extracts, fractions and sesquiterpene lactones from Neurolaena lobata (L.) R. Br. (Asteraceae), a traditional medicinal plant from Guatemala, were tested in vitro against Leishmania spp. promastigotes, Trypanosoma cruzi trypomastigotes and epimastigotes and Trichomonas vaginalis trophozoites. The ethanol extract inhibited the parasite growth of L. mexicana, T. cruzi and T. vaginalis significantly. The pure germacranolides 1 and a mixture of 2 and 3, isolated from the ethonal extract, were highly active against L. mexicana and T. cruzi. Copyright 2001 John Wiley & Sons, Ltd.

Integrity of the actin cytoskeleton of host macrophages is essential for Leishmania donovani infection.

PubMed

Roy, Saptarshi; Kumar, G Aditya; Jafurulla, Md; Mandal, Chitra; Chattopadhyay, Amitabha

2014-08-01

Visceral leishmaniasis is a vector-borne disease caused by an obligate intracellular protozoan parasite Leishmania donovani. The molecular mechanism involved in internalization of Leishmania is poorly understood. The entry of Leishmania involves interaction with the plasma membrane of host cells. We have previously demonstrated the requirement of host membrane cholesterol in the binding and internalization of L. donovani into macrophages. In the present work, we explored the role of the host actin cytoskeleton in leishmanial infection. We observed a dose-dependent reduction in the attachment of Leishmania promastigotes to host macrophages upon destabilization of the actin cytoskeleton by cytochalasin D. This is accompanied by a concomitant reduction in the intracellular amastigote load. We utilized a recently developed high resolution microscopy-based method to quantitate cellular F-actin content upon treatment with cytochalasin D. A striking feature of our results is that binding of Leishmania promastigotes and intracellular amastigote load show close correlation with cellular F-actin level. Importantly, the binding of Escherichia coli remained invariant upon actin destabilization of host cells, thereby implying specific involvement of the actin cytoskeleton in Leishmania infection. To the best of our knowledge, these novel results constitute the first comprehensive demonstration on the specific role of the host actin cytoskeleton in Leishmania infection. Our results could be significant in developing future therapeutic strategies to tackle leishmaniasis. Copyright © 2014 Elsevier B.V. All rights reserved.

Antifungal, Antileishmanial, and Cytotoxicity Activities of Various Extracts of Berberis vulgaris (Berberidaceae) and Its Active Principle Berberine

PubMed Central

Mahmoudvand, Hossein; Ayatollahi Mousavi, Seyyed Amin; Sepahvand, Asghar; Sharififar, Fariba; Ezatpour, Behrouz; Gorohi, Fatemeh; Saedi Dezaki, Ebrahim; Jahanbakhsh, Sareh

2014-01-01

In this study, in vitro antidermatophytic activity against Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis, and Microsporum gypseum was studied by disk diffusion test and assessment of minimum inhibitory concentration (MIC) using CLSI broth macrodilution method (M38-A2). Moreover, antileishmanial and cytotoxicity activity of B. vulgaris and berberine against promastigotes of Leishmania major and Leishmania tropica were evaluated by colorimetric MTT assay. The findings indicated that the various extracts of B. vulgaris particularly berberine showed high potential antidermatophytic against pathogenic dermatophytes tested with MIC values varying from 0.125 to >4 mg/mL. The results revealed that B. vulgaris extracts as well as berberine were effective in inhibiting L. major and L. tropica promastigotes growth in a dose-dependent manner with IC50 (50% inhibitory concentration) values varying from 2.1 to 26.6 μg/mL. Moreover, it could be observed that berberine as compared with B. vulgaris exhibited more cytotoxicity against murine macrophages with CC50 (cytotoxicity concentration for 50% of cells) values varying from 27.3 to 362.6 μg/mL. Results of this investigation were the first step in the search for new antidermatophytic and antileishmanial drugs. However, further works are required to evaluate exact effect of these extracts in animal models as well as volunteer human subjects. PMID:24977052

Antifungal, Antileishmanial, and Cytotoxicity Activities of Various Extracts of Berberis vulgaris (Berberidaceae) and Its Active Principle Berberine.

PubMed

Mahmoudvand, Hossein; Ayatollahi Mousavi, Seyyed Amin; Sepahvand, Asghar; Sharififar, Fariba; Ezatpour, Behrouz; Gorohi, Fatemeh; Saedi Dezaki, Ebrahim; Jahanbakhsh, Sareh

2014-01-01

In this study, in vitro antidermatophytic activity against Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis, and Microsporum gypseum was studied by disk diffusion test and assessment of minimum inhibitory concentration (MIC) using CLSI broth macrodilution method (M38-A2). Moreover, antileishmanial and cytotoxicity activity of B. vulgaris and berberine against promastigotes of Leishmania major and Leishmania tropica were evaluated by colorimetric MTT assay. The findings indicated that the various extracts of B. vulgaris particularly berberine showed high potential antidermatophytic against pathogenic dermatophytes tested with MIC values varying from 0.125 to >4 mg/mL. The results revealed that B. vulgaris extracts as well as berberine were effective in inhibiting L. major and L. tropica promastigotes growth in a dose-dependent manner with IC50 (50% inhibitory concentration) values varying from 2.1 to 26.6  μ g/mL. Moreover, it could be observed that berberine as compared with B. vulgaris exhibited more cytotoxicity against murine macrophages with CC50 (cytotoxicity concentration for 50% of cells) values varying from 27.3 to 362.6  μ g/mL. Results of this investigation were the first step in the search for new antidermatophytic and antileishmanial drugs. However, further works are required to evaluate exact effect of these extracts in animal models as well as volunteer human subjects.

Phototoxic effects of silicon bis (dimetilaminoetanoxi)-phthalocyanine (SiPc) on the viability of Leishmania major and Leishmania braziliensis promastigotes

NASA Astrophysics Data System (ADS)

Guerra Pinto, Juliana; Ferreira-Strixino, Juliana; Mittmann, Josane

2016-06-01

American cutaneous leishmaniasis (ACL) is an infectious disease caused by protozoans of the genus Leishmania. The treatment may consist of pentavalent antimonials or pentamidine and amphotericin. However, these treatments are extremely aggressive. Photodynamic antimicrobial chemotherapy (PACT) involves the same mechanism of photodynamic therapy which associates a photosensitizer with oxygen and a light source generating a photochemical reaction leading to cell death. The aim of this study was to verify the potential use of silicon bis (dimetilaminoetanoxi)-phthalocyanine (SiPc) compound in photodynamic treatment through evaluation of its phototoxic effect in promastigotes of the genus Leishmania braziliensis and Leishmania major. Treatment with SiPc was able to drastically affect the viability of the parasites as well as affect their growth and morphology, after PACT treatment. The data shown in this study allows us to conclude that SiPc is a promising photosensitizer (PS) since it does not affect parasite growth and viability in the dark. After PACT with this phthalocyanine, over 99% of parasites were killed with the higher concentration and a light dose used. These results suggest that SiPc can be used in future to treat CL, however, further studies are necessary to determine whether the PS are toxic to mononuclear phagocytic cells and epithelial cells which will also be affected by therapy when applied topically.

Coiling Phagocytosis of Trypanosomatids and Fungal Cells

PubMed Central

Rittig, M. G.; Schröppel, K.; Seack, K.-H.; Sander, U.; N’Diaye, E.-N.; Maridonneau-Parini, I.; Solbach, W.; Bogdan, C.

1998-01-01

Coiling phagocytosis has previously been studied only with the bacteria Legionella pneumophila and Borrelia burgdorferi, and the results were inconsistent. To learn more about this unconventional phagocytic mechanism, the uptake of various eukaryotic microorganisms by human monocytes, murine macrophages, and murine dendritic cells was investigated in vitro by video and electron microscopy. Unconventional phagocytosis of Leishmania spp. promastigotes, Trypanosoma cruzi trypomastigotes, Candida albicans hyphae, and zymosan particles from Saccharomyces cerevisiae differed in (i) morphology (rotating unilateral pseudopods with the trypanosomatids, overlapping bilateral pseudopods with the fungi), (ii) frequency (high with Leishmania; occasional with the fungi; rare with T. cruzi), (iii) duration (rapid with zymosan; moderate with the trypanosomatids; slow with C. albicans), (iv) localization along the promastigotes (flagellum of Leishmania major and L. aethiopica; flagellum or posterior pole of L. donovani), and (v) dependence on complement (strong with L. major and L. donovani; moderate with the fungi; none with L. aethiopica). All of these various types of unconventional phagocytosis gave rise to similar pseudopod stacks which eventually transformed to a regular phagosome. Further video microscopic studies with L. major provided evidence for a cytosolic localization, synchronized replication, and exocytic release of the parasites, extending traditional concepts about leishmanial infection of host cells. It is concluded that coiling phagocytosis comprises phenotypically similar consequences of various disturbances in conventional phagocytosis rather than representing a single separate mechanism. PMID:9712785

Passive transfer of leishmania lipopolysaccharide confers parasite survival in macrophages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Handman, E.; Schnur, L.F.; Spithill, T.W.

1986-12-01

Infection of macrophages by the intracellular protozoan parasite Leishmania involves specific attachment to the host membrane, followed by phagocytosis and intracellular survival and growth. Two parasite molecules have been implicated in the attachment event: Leishmania lipopolysaccharide (L-LPS) and a glycoprotein (gp63). This study was designed to clarify the role of L-LPS in infection and the stage in the process of infection at which it operates. The authors have recently identified a Leishmania major strain (LRC-L119) which lacks the L-LPS molecule and is not infective for hamsters or mice. This parasite was isolated from a gerbil in Kenya and was identifiedmore » phenotypically as L. major by isoenzyme and fatty acid analysis. In this study they have confirmed at the genotype level that LRC-L119 is L. major by analyzing and comparing the organization of cloned DNA sequences in the genome of different strains of L. major. Here they show that LRC-L119 promastigotes are phagocytosed rapidly by macrophages in vitro, but in contrast to virulent strains of L. major, they are then killed over a period of 18 hr. In addition, they show that transfer of purified L-LPS from a virulent clone of L. major (V121) into LRC-L119 promastigotes confers on them the ability to survive in macrophages in vitro.« less

In vitro evaluation of photodynamic therapy using curcumin on Leishmania major and Leishmania braziliensis.

PubMed

Pinto, Juliana Guerra; Fontana, Letícia Correa; de Oliveira, Marco Antonio; Kurachi, Cristina; Raniero, Leandro José; Ferreira-Strixino, Juliana

2016-07-01

Cutaneous leishmaniasis is an infectious disease caused by the Leishmania protozoan. The conventional treatment is long-lasting and aggressive, in addition to causing harmful effect. Photodynamic therapy has emerged as a promising alternative treatment, which allows local administration with fewer side effects. This study investigated the photodynamic activity of curcumin on Leishmania major and Leishmania braziliensis promastigote. Both species were submitted to incubation with curcumin in serial dilutions from 500 μg/ml up to 7.8 μg/ml. Control groups were kept in the dark while PDT groups received a fluency of 10 J/cm(2) at 450 nm. Mitochondrial activity was assessed by MTT assay 18 h after light treatment, and viability was measured by Trypan blue dye exclusion test. Morphological alterations were observed by Giemsa staining. Confocal microscopy showed the uptake of curcumin by both tested Leishmania species. Mitochondrial activity was inconclusive to determine viability; however, Trypan blue test was able to show that curcumin photodynamic treatment had a significant effect on viability of parasites. The morphology of promastigotes was highly affected by the photodynamic therapy. These results indicated that curcumin may be a promising alternative photosensitizer, because it presents no toxicity in the dark; however, further tests in co-culture with macrophages and other species of Leishmania should be conducted to determine better conditions before in vivo tests are performed.

Evaluation of Enzyme-Linked Immunosorbent Assay for Diagnosis of Post-Kala-Azar Dermal Leishmaniasis with Crude or Recombinant k39 Antigen

PubMed Central

Salotra, P.; Sreenivas, G.; Nasim, A. A.; Subba Raju, B. V.; Ramesh, V.

2002-01-01

The diagnosis of post-kala-azar dermal leishmaniasis (PKDL), a dermatosis that provides the only known reservoir for the parasite Leishmania donovani in India, remains a problem. Timely recognition and treatment of PKDL would contribute significantly to the control of kala-azar. We evaluated here the potential of the enzyme-linked immunosorbent assay (ELISA) as a diagnostic tool for PKDL. Antigen prepared from promastigotes and axenic amastigotes with parasite isolates that were derived from skin lesions of a PKDL patient gave sensitivities of 86.36 and 92%, respectively, in the 88 PKDL cases examined. The specificity of the ELISA test was examined by testing groups of patients with other skin disorders (leprosy and vitiligo) or coendemic infections (malaria and tuberculosis), as well as healthy controls from areas where this disease is endemic or is not endemic. A false-positive reaction was obtained in 14 of 144 (9.8%) of the controls with the promastigote antigen and in 14 of 145 (9.7%) of the controls with the amastigote antigen. Evaluation of the serodiagnostic potential of recombinant k39 by ELISA revealed a higher sensitivity (94.5%) and specificity (93.7%) compared to the other two antigens used. The data demonstrate that ELISA with crude or recombinant antigen k39 provides a relatively simple and less-invasive test for the reliable diagnosis of PKDL. PMID:11874880

Identification of Potent Chemotypes Targeting Leishmania major Using a High-Throughput, Low-Stringency, Computationally Enhanced, Small Molecule Screen

PubMed Central

Sharlow, Elizabeth R.; Close, David; Shun, Tongying; Leimgruber, Stephanie; Reed, Robyn; Mustata, Gabriela; Wipf, Peter; Johnson, Jacob; O'Neil, Michael; Grögl, Max; Magill, Alan J.; Lazo, John S.

2009-01-01

Patients with clinical manifestations of leishmaniasis, including cutaneous leishmaniasis, have limited treatment options, and existing therapies frequently have significant untoward liabilities. Rapid expansion in the diversity of available cutaneous leishmanicidal chemotypes is the initial step in finding alternative efficacious treatments. To this end, we combined a low-stringency Leishmania major promastigote growth inhibition assay with a structural computational filtering algorithm. After a rigorous assay validation process, we interrogated ∼200,000 unique compounds for L. major promastigote growth inhibition. Using iterative computational filtering of the compounds exhibiting >50% inhibition, we identified 553 structural clusters and 640 compound singletons. Secondary confirmation assays yielded 93 compounds with EC50s ≤ 1 µM, with none of the identified chemotypes being structurally similar to known leishmanicidals and most having favorable in silico predicted bioavailability characteristics. The leishmanicidal activity of a representative subset of 15 chemotypes was confirmed in two independent assay formats, and L. major parasite specificity was demonstrated by assaying against a panel of human cell lines. Thirteen chemotypes inhibited the growth of a L. major axenic amastigote-like population. Murine in vivo efficacy studies using one of the new chemotypes document inhibition of footpad lesion development. These results authenticate that low stringency, large-scale compound screening combined with computational structure filtering can rapidly expand the chemotypes targeting in vitro and in vivo Leishmania growth and viability. PMID:19888337

Antileishmanial and antitrypanosomal activity of bufadienolides isolated from the toad Rhinella jimi parotoid macrogland secretion.

PubMed

Tempone, André Gustavo; Pimenta, Daniel Carvalho; Lebrun, Ivo; Sartorelli, Patrícia; Taniwaki, Noemi N; de Andrade, Heitor Franco; Antoniazzi, Marta Maria; Jared, Carlos

2008-07-01

Amphibian skin secretions are considered a rich source of biologically active compounds and are known to be rich in peptides, bufadienolides and alkaloids. Bufadienolides are cardioactive steroids from animals and plants that have also been reported to possess antimicrobial activities. Leishmaniasis and American Trypanosomiasis are parasitic diseases found in tropical and subtropical regions. The efforts toward the discovery of new treatments for these diseases have been largely neglected, despite the fact that the only available treatments are highly toxic drugs. In this work, we have isolated, through bioguided assays, the major antileishmanial compounds of the toad Rhinella jimi parotoid macrogland secretion. Mass spectrometry and (1)H and (13)C NMR spectroscopic analyses were able to demonstrate that the active molecules are telocinobufagin and hellebrigenin. Both steroids demonstrated activity against Leishmania (L.) chagasi promastigotes, but only hellebrigenin was active against Trypanosoma cruzi trypomastigotes. These steroids were active against the intracellular amastigotes of Leishmania, with no activation of nitric oxide production by macrophages. Neither cytotoxicity against mouse macrophages nor hemolytic activities were observed. The ultrastructural studies with promastigotes revealed the induction of mitochondrial damage and plasma membrane disturbances by telocinobufagin, resulting in cellular death. This novel biological effect of R. jimi steroids could be used as a template for the design of new therapeutics against Leishmaniasis and American Trypanosomiasis.

Combining epidemiology with basic biology of sand flies, parasites, and hosts to inform leishmaniasis transmission dynamics and control.

PubMed

Courtenay, Orin; Peters, Nathan C; Rogers, Matthew E; Bern, Caryn

2017-10-01

Quantitation of the nonlinear heterogeneities in Leishmania parasites, sand fly vectors, and mammalian host relationships provides insights to better understand leishmanial transmission epidemiology towards improving its control. The parasite manipulates the sand fly via production of promastigote secretory gel (PSG), leading to the "blocked sand fly" phenotype, persistent feeding attempts, and feeding on multiple hosts. PSG is injected into the mammalian host with the parasite and promotes the establishment of infection. Animal models demonstrate that sand flies with the highest parasite loads and percent metacyclic promastigotes transmit more parasites with greater frequency, resulting in higher load infections that are more likely to be both symptomatic and efficient reservoirs. The existence of mammalian and sand fly "super-spreaders" provides a biological basis for the spatial and temporal clustering of clinical leishmanial disease. Sand fly blood-feeding behavior will determine the efficacies of indoor residual spraying, topical insecticides, and bed nets. Interventions need to have sufficient coverage to include transmission hot spots, especially in the absence of field tools to assess infectiousness. Interventions that reduce sand fly densities in the absence of elimination could have negative consequences, for example, by interfering with partial immunity conferred by exposure to sand fly saliva. A deeper understanding of both sand fly and host biology and behavior is essential to ensuring effectiveness of vector interventions.

The Limits on Trypanosomatid Morphological Diversity

PubMed Central

Wheeler, Richard John; Gluenz, Eva; Gull, Keith

2013-01-01

Cell shape is one, often overlooked, way in which protozoan parasites have adapted to a variety of host and vector environments and directional transmissions between these environments. Consequently, different parasite life cycle stages have characteristic morphologies. Trypanosomatid parasites are an excellent example of this in which large morphological variations between species and life cycle stage occur, despite sharing well-conserved cytoskeletal and membranous structures. Here, using previously published reports in the literature of the morphology of 248 isolates of trypanosomatid species from different hosts, we perform a meta-analysis of the occurrence and limits on morphological diversity of different classes of trypanosomatid morphology (trypomastigote, promastigote, etc.) in the vertebrate bloodstream and invertebrate gut environments. We identified several limits on cell body length, cell body width and flagellum length diversity which can be interpreted as biomechanical limits on the capacity of the cell to attain particular dimensions. These limits differed for morphologies with and without a laterally attached flagellum which we suggest represent two morphological superclasses, the ‘juxtaform’ and ‘liberform’ superclasses. Further limits were identified consistent with a selective pressure from the mechanical properties of the vertebrate bloodstream environment; trypanosomatid size showed limits relative to host erythrocyte dimensions. This is the first comprehensive analysis of the limits of morphological diversity in any protozoan parasite, revealing the morphogenetic constraints and extrinsic selection pressures associated with the full diversity of trypanosomatid morphology. PMID:24260255

Antimony susceptibility of Leishmania isolates collected over a 30-year period in Algeria

PubMed Central

Eddaikra, Naouel; Ait-Oudhia, Khatima; Kherrachi, Ihcen; Oury, Bruno; Moulti-Mati, Farida; Benikhlef, Razika; Harrat, Zoubir

2018-01-01

Background In Algeria, the treatment of visceral and cutaneous leishmanioses (VL and CL) has been and continues to be based on antimony-containing drugs. It is suspected that high drug selective pressure might favor the emergence of chemoresistant parasites. Although treatment failure is frequently reported during antimonial therapy of both CL and VL, antimonial resistance has never been thoroughly investigated in Algeria. Determining the level of antimonial susceptibility, amongst Leishmania transmitted in Algeria, is of great importance for the development of public health policies. Methodology/Principal findings Within the framework of the knowledge about the epidemiology of VL and CL amassed during the last 30 years, we sampled Leishmania isolates to determine their susceptibility to antimony. We analyzed a total of 106 isolates including 88 isolates collected between 1976 and 2013 in Algeria from humans, dogs, rodents, and phlebotomines and 18 collected from dogs in France. All the Algerian isolates were collected in 14 localities where leishmaniasis is endemic. The 50% inhibitory concentrations (IC50) of potassium antimony tartrate (the trivalent form of antimony, Sb(III)) and sodium stibogluconate (the pentavalent form of antimony, Sb(V)) were determined in promastigotes and intramacrophage amastigotes, respectively. The epidemiological cutoff (ECOFF) that allowed us to differentiate between Leishmania species causing cutaneous or visceral leishmaniases that were susceptible (S+) or insusceptible (S-) to the trivalent form of antimony was determined. The computed IC50 cutoff values were 23.83 μg/mL and 15.91 μg/mL for VL and CL, respectively. We report a trend of increasing antimony susceptibility in VL isolates during the 30-year period. In contrast, an increase in the frequency of S- phenotypes in isolates causing CL was observed during the same period. In our study, the emergence of S- phenotypes correlates with the inclusion of L. killicki (syn: L. tropica) isolates that cause cutaneous leishmaniasis and that have emerged in Algeria during the last decade. Conclusion/Significance Our results provide insight into the spatiotemporal dynamics of Leishmania antimony susceptibility in Algeria. We highlight the need for the future implementation of an effective methodology to determine the antimony susceptibility status of Leishmania isolates to detect the emergence of and prevent the dissemination of drug-resistant strains. PMID:29561842

Carboxylate derivatives of tributyltin (IV) complexes as anticancer and antileishmanial agents.

PubMed

Waseem, Durdana; Butt, Arshad Farooq; Haq, Ihsan-Ul; Bhatti, Moazzam Hussain; Khan, Gul Majid

2017-04-04

Tributyltin (IV) compounds are promising candidates for drug development. In the current study, we evaluated in-vitro and in-silico profile of carboxylate derivatives of tributyltin (IV) complexes. ADMET and drug-likeliness properties were predicted using MetaPrint2D React, preADMET, SwissADME and Molsoft tools. SwissTargetPrediction predicted molecular targets for compounds. In-vitro bioactivity was evaluated by quantifying cytotoxicity against HepG2, THP-1 cell lines, isolated lymphocytes and leishmania promastigotes as well as measuring protein kinase (PK) inhibition activity. Results indicate partial compliance of compounds with drug-likeliness rules. Ch-409 complies with WDI and Lipinski rules. ADMET profile prediction shows strong plasma protein binding except for Ch-409, low to high GI absorption and BBB penetration (C brain /C blood  = 0.942-11; caco-2 cells permeability 20.13-26.75 nm/sec), potential efflux by P-glycoprotein, metabolism by CYP3A4, medium inhibition of hERG, mutagenicity and capacity to be detoxified by glutathionation and glucuronidation. Molecular targets include proteases, enzymes, membrane receptors, transporters and ion channels where Ch-409 targets membrane receptors only. Compounds are significantly (p < 0.05) cytotoxic against HepG2 cell line and leishmania as compared with normal isolated lymphocytes. Ch-459 indicates highest toxicity against leishmania (mortality 97.9 ± 3.99%; LC50 0.323 ± 0.002 μg/mL) whereas Ch-409 possesses maximum cytotoxicity against HepG2 cell line (IC50 0.08 ± 0.001 μg/mL) as well as 97.5 ± 1.98% (LC50 0.954 ± 0.158 μg/mL) mortality of leishmania promastigotes. It was observed that antileishmanial effect was reduced by 16.38%-34.38% and 15-38.2% in the presence of NaN 3 and mannitol respectively. PK inhibition and reactive oxygen species production are possible mechanisms for cytotoxicity. Selected carboxylate derivatives of tributyltin (IV) complexes possess significant antileishmanial and cytotoxic potential. These are promising compounds for the development of antileishmanial and anticancer drugs. Graphical Abstract Carboxylate derivatives of tributyltin (IV) complexes as anticancer and antileishmanial agents.

A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

PubMed Central

Beneke, Tom; Makin, Laura; Valli, Jessica; Sunter, Jack

2017-01-01

Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource (LeishGEdit.net) for automated primer design. We produced a set of plasmids that allows easy and scalable generation of DNA constructs for transfections in just a few hours. We show how these tools allow knock-in of fluorescent protein tags, modified biotin ligase BirA*, luciferase, HaloTag and small epitope tags, which can be fused to proteins at the N- or C-terminus, for functional studies of proteins and localization screening. These tools enabled generation of null mutants in a single round of transfection in promastigote form Leishmania major, Leishmania mexicana and bloodstream form Trypanosoma brucei; deleted genes were undetectable in non-clonal populations, enabling for the first time rapid and large-scale knockout screens. PMID:28573017

N-cinnamoylated aminoquinolines as promising antileishmanial agents.

PubMed

Vale-Costa, S; Costa-Gouveia, J; Pérez, B; Silva, T; Teixeira, C; Gomes, P; Gomes, M S

2013-10-01

A series of cinnamic acid conjugates of primaquine and chloroquine were evaluated for their in vitro antileishmanial activities. Although primaquine derivatives had modest activity, chloroquine conjugates exhibited potent activity against both promastigotes (50% inhibitory concentration [IC50] = 2.6 to 21.8 μM) and intramacrophagic amastigotes (IC50 = 1.2 to 9.3 μM) of Leishmania infantum. Both the high activity of these chloroquine analogues and their mild-to-low toxicity toward host cells make them promising leads for the discovery of new antileishmanial agents.

First Report on Natural Infection of Phlebotomus sergenti with Leishmania Promastigotes in the Cutaneous Leishmaniasis Focus in Southeastern Tunisia

PubMed Central

Tabbabi, Ahmed; Bousslimi, Nadia; Rhim, Adel; Aoun, Karim; Bouratbine, Aïda

2011-01-01

During September 2010, 133 female sand flies were caught inside houses of patients with cutaneous leishmaniasis in the focus for this disease in southeastern Tunisia and subsequently dissected. One specimen was positive for Leishmania protozoa. This sand fly species was identified as Phlebotomus sergenti, and the parasite was identified as L. tropica. This is the first report of P. sergenti involvement in transmission of L. tropica in Tunisia. PMID:21976566

Chemotherapy and Drug Targeting in the Treatment of Leishmaniasis

DTIC Science & Technology

1990-05-30

extremely inhibitory. ED50 for growth of promastigotes of L. mexicana sp. was as low as 5nM and up to 1O0uM. Sinefungi a natural nucleoside isclated from...synthesis increases and polyamine levels rise when the growth rate is maximal. Growth apnears to be related to and dependent upon polyamine biosynthesis...the growth of Hemophilus parainfluenzae. Since that time, pol, imines have been shown to be stimulatory to (or essential for) the grc~th of various

Leishmanicidal activity of Yucatecan medicinal plants on Leishmania species responsible for cutaneous leishmaniasis.

PubMed

Getti, Giulia; Durgadoss, Priyanka; Domínguez-Carmona, Dafne; Martin-Quintal, Zhelmy; Peraza-Sánchez, Sergio; Peña-Rodriguez, Luis Manuel; Humber, David

2009-04-01

The leishmanicidal activity of 15 extracts and 4 pure metabolites obtained from Urechites andrieuxii, Colubrina greggii, Dorstenia contrajerva, and Tridax procumbens was evaluated using the newly developed MTS ({3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay, optimized for promastigotes of Leishmania major, Leishmania tropica, and Leishmania aethiopica, as well as for L. aethiopica axenic amastigotes. The assay was then used for calculating the percentage of viable stationary phase parasites after a 24-hr treatment with each plant extract or pure metabolite. The 3 most active samples, 2 from C. greggii (NCG-5C and DCG-3A) and 1 from T. procumbens (TPZ-2A), showed LD50 values of 62.4, 7.2, and 18.5 microg/ml, respectively, on stationary promastigotes, and of 94.2, 27.1, and 95.2 microg/ml, on amastigotes of L. aethiopica. Moreover, TPZ-2A and DCG-3A significantly reduced the percentage of infected monocyte-derived macrophages (THP-I). The percentage of infected cells decreased from 69.9% +/- 2.5% to 20.8% +/- 2% when the cells were treated with the DCG-3A fraction and to 14.9% +/- 0.5% when treated with TPZ-2A, without significantly decreasing the number of human cells. These findings indicate the presence of potentially bioactive metabolites in the roots of C. greggii and in T. procumbens and reflect the importance of pursuing the bioassay-guided purification of these metabolites.

In Vitro Inhibitory Effect of Berberis vulgaris (Berberidaceae) and Its Main Component, Berberine against Different Leishmania Species.

PubMed

Mahmoudvand, Hossein; Sharififar, Fariba; Sharifi, Iraj; Ezatpour, Behrouz; Fasihi Harandi, Majid; Makki, Mahsa Sadat; Zia-Ali, Naser; Jahanbakhsh, Sareh

2014-03-01

Leishmaniasis has been identified as a major public health problem in tropical and sub-tropical countries. The present study was aimed to investigate antileishmanial effects of various extracts of Berberis vulgaris also its active compoenent, berberine against Leishmania tropica and L. infantum species on in vitro experiments. In this study in vitro antileishmanial activity of various extracts of B. vulgaris also its active compoenent, berberine against promastigote and amastigote stages of L. tropica and L. infantum was evaluated, using MTT assay and in a macrophage model, respectively. Furthermore, infectivity rate and cytotoxicity effects of B. vulgaris and berberine in murine macrophage cells were investigated. The findings of optical density (OD) and IC50 indicated that B. vulgaris particulary berberine significantly (P<0.05) inhibited the growth rate of promastigote stage of L.tropica and L.infantum in comparison to meglumine antimoniate (MA). In addition, B. vulgaris and berberine significantly (P<0.05) decreased the mean number of amastigotes in each macrophage as compared with positive control. In the evaluation of cytotoxicity effects, it could be observed that berberine as compared with B. vulgaris exhibited more cytotoxicity against murine macrophages. Results also showed that when parasites were pre-incubated with B. vulgaris their ability to infect murine macrophages was significantly decreased. B.vulgaris particularly berberine exhibited potent in vitro leishmanicidal effects against L. tropica and L.infantum. Further works are required to evaluate the antileishmanial effects of B.vulgaris on Leishmania species using clinical settings.

IN VIVO AND IN VITRO ANTILEISHMANIAL EFFECTS OF METHANOLIC EXTRACT FROM BARK OF BURSERA APTERA.

PubMed

Nieto-Yañez, O J; Resendiz-Albor, A A; Ruiz-Hurtado, P A; Rivera-Yañez, N; Rodriguez-Canales, M; Rodriguez-Sosa, M; Juarez-Avelar, I; Rodriguez-Lopez, M G; Canales-Martinez, M M; Rodriguez-Monroy, M A

2017-01-01

Cutaneous leishmaniasis lacks effective and well-tolerated treatments. The current therapies mainly rely on antimonial drugs that are inadequate because of their poor efficacy. Traditional medicine offers a complementary alternative for the treatment of various diseases. Additionally, several plants have shown success as anti-leishmanial agents. Therefore, we sought to evaluate the in vitro and in vivo activity of MEBA against Leishmania mexicana . Methanolic extract of B. aptera was obtained by macetration, after we determined in vitro anti-leishmanial activity of MEBA by MTT assay and the induced apoptosis in promastigotes by flow cytometry. To analyze the in vivo anti-leishmanial activity, we used infected mice that were treated and not treated with MEBA and we determined the levels of cytokines using ELISA. The phytochemical properties were determined by CG-MS and DPPH assay. We determined of LC 50 of 0.408 mg/mL of MEBA for in vitro anti-leishmanial activity. MEBA induced apoptosis in promastigotes (15.3% ± 0.86). Treated mice exhibited smaller lesions and contained significantly fewer parasites than did untreated mice; in addition, we found that IFN-γ and TNF-α increased in the sera of MEBA-treated mice. GC-MS analysis showed that podophyllotoxin was the most abundant compound. Evaluation of the activity by DPPH assay demonstrated an SC 50 of 11.72 μg/mL. Based on the above data, it was concluded that MEBA is a good candidate in the search for new anti-leishmanial agents.

Chlorin E6 phototoxicity in L. major and L. braziliensis promastigotes-In vitro study.

PubMed

Pinto, Juliana Guerra; Pereira, André Henrique Correia; de Oliveira, Marco Antonio; Kurachi, Cristina; Raniero, Leandro José; Ferreira-Strixino, Juliana

2016-09-01

Cutaneous leishmaniasis is a zoonosis caused by protozoa of the genus Leishmania. Conventional treatments are long and aggressive, and they trigger a diversity of side effects. Photodynamic Therapy was originally proposed as a treatment for cancer, and it now appears to be a promising therapy for local treatment with fewer side effects of infectious diseases. This study aimed to evaluate Chlorin e6 internalization by Leishmania major and Leishmania braziliensis promastigotes and its viability and effects on mitochondrial activity. Control groups were kept in the dark, while PDT groups received fluence of 10J/cm(2) (660nm). Chlorin internalization was evaluated using confocal microscopy after one hour of incubation for both species. The mitochondrial activity was evaluated by MTT assay, and viability was measured by the Trypan blue exclusion test. Giemsa staining was used to observe morphological changes. PS was internalized in both species and mitochondrial activity changed in all groups. However, the obtained MTT and Trypan results indicated that despite the change in mitochondrial activity in the dark groups, their viability was not affected, whereas the PDT treated groups had significantly reduced viability. Morphology was drastically altered in PDT treated groups, while groups kept in the dark exhibited the standard morphology. This study demonstrates that Chlorin has great potential for being used in PDT as a treatment for cutaneous leishmaniasis, although more studies are needed to determine in vivo application protocols. Copyright © 2016 Elsevier B.V. All rights reserved.

Trypanosomatidae produce acetate via a mitochondrial acetate:succinate CoA transferase

PubMed Central

Van Hellemond, Jaap J.; Opperdoes, Fred R.; Tielens, Aloysius G. M.

1998-01-01

Hydrogenosome-containing anaerobic protists, such as the trichomonads, produce large amounts of acetate by an acetate:succinate CoA transferase (ASCT)/succinyl CoA synthetase cycle. The notion that mitochondria and hydrogenosomes may have originated from the same α-proteobacterial endosymbiont has led us to look for the presence of a similar metabolic pathway in trypanosomatids because these are the earliest-branching mitochondriate eukaryotes and because they also are known to produce acetate. The mechanism of acetate production in these organisms, however, has remained unknown. Four different members of the trypanosomatid family: promastigotes of Leishmania mexicana mexicana, L. infantum and Phytomonas sp., and procyclics of Trypanosoma brucei were analyzed as well as the parasitic helminth Fasciola hepatica. They all use a mitochondrial ASCT for the production of acetate from acetyl CoA. The succinyl CoA that is produced during acetate formation by ASCT is recycled presumably to succinate by a mitochondrial succinyl CoA synthetase, concomitantly producing ATP from ADP. The ASCT of L. mexicana mexicana promastigotes was further characterized after partial purification of the enzyme. It has a high affinity for acetyl CoA (Km 0.26 mM) and a low affinity for succinate (Km 6.9 mM), which shows that significant acetate production can occur only when high mitochondrial succinate concentrations prevail. This study identifies a metabolic pathway common to mitochondria and hydrogenosomes, which strongly supports a common origin for these two organelles. PMID:9501211

Identification of novel inhibitors against UDP-galactopyranose mutase to combat leishmaniasis.

PubMed

Kashif, Mohammad; Tabrez, Shams; Husein, Atahar; Arish, Mohd; Kalaiarasan, Ponnusamy; Manna, Partha P; Subbarao, Naidu; Akhter, Yusuf; Rub, Abdur

2018-03-01

Leishmania, a protozoan parasite that causes leishmaniasis, affects 1-2 million people every year worldwide. Leishmaniasis is a vector born disease and characterized by a diverse group of clinical syndromes. Current treatment is limited because of drug resistance, high cost, poor safety, and low efficacy. The urgent need for potent agents against Leishmania has led to significant advances in the development of novel antileishmanial drugs. β-galactofuranose (β-Galf) is an important component of Leishmanial cell surface matrix and plays a critical role in the pathogenesis of parasite. UDP-galactopyranose mutase (UGM) converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf) which acts as the precursor for β-Galf synthesis. Due to its absence in human, this enzyme is selected as the potential target in search of new antileishmanial drugs. Three dimensional protein structure model of Leishmania major UGM (LmUGM) has been homology modeled using Trypanosoma cruzi UGM (TcUGM) as a template. The stereochemistry was validated further. We selected already reported active compounds from PubChem database to target the LmUGM. Three compounds (6064500, 44570814, and 6158954) among the top hit occupied the UDP binding site of UGM suggested to work as a possible inhibitor for it. In vitro antileishmanial activity assay was performed with the top ranked inhibitor, 6064500. The 6064500 molecule has inhibited the growth of Leishmania donovani promastigotes significantly. Further, at similar concentrations it has exhibited significantly lesser toxicity than standard drug miltefosine hydrate in mammalian cells. © 2017 Wiley Periodicals, Inc.

Lipophosphoglycan polymorphisms do not affect Leishmania amazonensis development in the permissive vectors Lutzomyia migonei and Lutzomyia longipalpis.

PubMed

Nogueira, Paula M; Guimarães, Agna C; Assis, Rafael R; Sadlova, Jovana; Myskova, Jitka; Pruzinova, Katerina; Hlavackova, Jana; Turco, Salvatore J; Torrecilhas, Ana C; Volf, Petr; Soares, Rodrigo P

2017-12-16

Lipophosphoglycan (LPG) is a dominant surface molecule of Leishmania promastigotes. Its species-specific polymorphisms are found mainly in the sugars that branch off the conserved Gal(β1,4)Man(α1)-PO 4 backbone of repeat units. Leishmania amazonensis is one of the most important species causing human cutaneous leishmaniasis in the New World. Here, we describe LPG intraspecific polymorphisms in two Le. amazonensis reference strains and their role during the development in three sand fly species. Strains isolated from Lutzomyia flaviscutellata (PH8) and from a human patient (Josefa) displayed structural polymorphism in the LPG repeat units, possessing side chains with 1 and 2 β-glucose or 1 to 3 β-galactose, respectively. Both strains successfully infected permissive vectors Lutzomyia longipalpis and Lutzomyia migonei and could colonize their stomodeal valve and differentiate into metacyclic forms. Despite bearing terminal galactose residues on LPG, Josefa could not sustain infection in the restrictive vector Phlebotomus papatasi. LPG polymorphisms did not affect the ability of Le. amazonensis to develop late-stage infections in permissive vectors. However, the non-establishment of infection in Ph. papatasi by Josefa strain suggested other LPG-independent factors in this restrictive vector.

N-Cinnamoylated Aminoquinolines as Promising Antileishmanial Agents

PubMed Central

Vale-Costa, S.; Costa-Gouveia, J.; Pérez, B.; Silva, T.; Teixeira, C.; Gomes, P.

2013-01-01

A series of cinnamic acid conjugates of primaquine and chloroquine were evaluated for their in vitro antileishmanial activities. Although primaquine derivatives had modest activity, chloroquine conjugates exhibited potent activity against both promastigotes (50% inhibitory concentration [IC50] = 2.6 to 21.8 μM) and intramacrophagic amastigotes (IC50 = 1.2 to 9.3 μM) of Leishmania infantum. Both the high activity of these chloroquine analogues and their mild-to-low toxicity toward host cells make them promising leads for the discovery of new antileishmanial agents. PMID:23917315

Fluorogenic Substrate Detection of Viable Intracellular and Extracellular Pathogenic Protozoa

NASA Astrophysics Data System (ADS)

Jackson, Peter R.; Pappas, Michael G.; Hansen, Brian D.

1985-01-01

Viable Leishmania promastigotes and amastigotes were detected by epifluorescence microscopy with fluorescein diacetate being used to mark living parasites and the nucleic acid-binding compound ethidium bromide to stain dead cells. This procedure is superior to other assays because it is faster and detects viable intracellular as well as extracellular Leishmania. Furthermore, destruction of intracellular pathogens by macrophages is more accurately determined with fluorescein diacetate than with other stains. The procedure may have applications in programs to develop drugs and vaccines against protozoa responsible for human and animal disease.

Characterization of a ricin-resistant mutant of Leishmania donovani that expresses lipophosphoglycan.

PubMed

Phillips, Megan R; Turco, Salvatore J

2015-04-01

The abundant cell-surface lipophosphoglycan (LPG) of Leishmania parasites plays a central role throughout the eukaryote's life cycle. A number of LPG-defective mutants and their complementing genes have been isolated and have proven invaluable in assessing the importance of LPG and related glycoconjugates in parasite virulence. While ricin agglutination selection protocols frequently result in lpg- mutants, one  Leishmania donovani variant we isolated, named JABBA, was found to be lpg+. Procyclic (logarithmic) JABBA expresses significant amounts of a large-sized LPG, larger than observed from procyclic wild type but similar in size to LPG from wild type from metacyclic (stationary) phase. Structural analysis of the LPG from logarithmically grown JABBA by capillary electrophoresis protocols revealed that it averaged 30 repeat units composed of the unsubstituted Gal(β1,4)Man(α1)-PO4 typical of wild-type L. donovani. Analysis of JABBA LPG caps indicated that 20% is branched trisaccharide Gal(β1,4)[Glc(β1,2)]Man and tetrasaccharide Gal(β1,4)[Glc(β1,2)Man(α1,2)]Man instead of the usual Gal(β1,4)Man and Man(α1,2)Man terminating caps. Consistent with these structural observations, analyses of the relevant glycosyltransferases in JABBA microsomes involved in LPG biosynthesis showed a 2-fold increase in elongating mannosylphosphoryltransferase activity and up-regulation of a β-glucosyltransferase activity. Furthermore, the caps of JABBA LPG are cryptic in presentation as shown by the loss of binding by the lectins, ricin, peanut agglutinin and concanavalin A and reduced accessibility of the terminal galactose residues to oxidation by galactose oxidase. These results indicate that LPG from JABBA is intriguingly similar to the larger LPG in wild-type parasites that arises following the differentiation of the non-infectious procyclic promastigotes to infectious, metacyclic forms. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Characterization of a ricin-resistant mutant of Leishmania donovani that expresses lipophosphoglycan

PubMed Central

Phillips, Megan R; Turco, Salvatore J

2015-01-01

The abundant cell-surface lipophosphoglycan (LPG) of Leishmania parasites plays a central role throughout the eukaryote's life cycle. A number of LPG-defective mutants and their complementing genes have been isolated and have proven invaluable in assessing the importance of LPG and related glycoconjugates in parasite virulence. While ricin agglutination selection protocols frequently result in lpg− mutants, one  Leishmania donovani variant we isolated, named JABBA, was found to be lpg+. Procyclic (logarithmic) JABBA expresses significant amounts of a large-sized LPG, larger than observed from procyclic wild type but similar in size to LPG from wild type from metacyclic (stationary) phase. Structural analysis of the LPG from logarithmically grown JABBA by capillary electrophoresis protocols revealed that it averaged 30 repeat units composed of the unsubstituted Gal(β1,4)Man(α1)-PO4 typical of wild-type L. donovani. Analysis of JABBA LPG caps indicated that 20% is branched trisaccharide Gal(β1,4)[Glc(β1,2)]Man and tetrasaccharide Gal(β1,4)[Glc(β1,2)Man(α1,2)]Man instead of the usual Gal(β1,4)Man and Man(α1,2)Man terminating caps. Consistent with these structural observations, analyses of the relevant glycosyltransferases in JABBA microsomes involved in LPG biosynthesis showed a 2-fold increase in elongating mannosylphosphoryltransferase activity and up-regulation of a β-glucosyltransferase activity. Furthermore, the caps of JABBA LPG are cryptic in presentation as shown by the loss of binding by the lectins, ricin, peanut agglutinin and concanavalin A and reduced accessibility of the terminal galactose residues to oxidation by galactose oxidase. These results indicate that LPG from JABBA is intriguingly similar to the larger LPG in wild-type parasites that arises following the differentiation of the non-infectious procyclic promastigotes to infectious, metacyclic forms. PMID:25472443

Inhibition of fumarate reductase in Leishmania major and L. donovani by chalcones.

PubMed

Chen, M; Zhai, L; Christensen, S B; Theander, T G; Kharazmi, A

2001-07-01

Our previous studies have shown that chalcones exhibit potent antileishmanial and antimalarial activities in vitro and in vivo. Preliminary studies showed that these compounds destroyed the ultrastructure of Leishmania parasite mitochondria and inhibited the respiration and the activity of mitochondrial dehydrogenases of Leishmania parasites. The present study was designed to further investigate the mechanism of action of chalcones, focusing on the parasite respiratory chain. The data show that licochalcone A inhibited the activity of fumarate reductase (FRD) in the permeabilized Leishmania major promastigote and in the parasite mitochondria, and it also inhibited solubilized FRD and a purified FRD from L. donovani. Two other chalcones, 2,4-dimethoxy-4'-allyloxychalcone (24m4ac) and 2,4-dimethoxy-4'-butoxychalcone (24mbc), also exhibited inhibitory effects on the activity of solubilized FRD in L. major promastigotes. Although licochalcone A inhibited the activities of succinate dehydrogenase (SDH), NADH dehydrogenase (NDH), and succinate- and NADH-cytochrome c reductases in the parasite mitochondria, the 50% inhibitory concentrations (IC(50)) of licochalcone A for these enzymes were at least 20 times higher than that for FRD. The IC(50) of licochalcone A for SDH and NDH in human peripheral blood mononuclear cells were at least 70 times higher than that for FRD. These findings indicate that FRD, one of the enzymes of the parasite respiratory chain, might be the specific target for the chalcones tested. Since FRD exists in the Leishmania parasite and does not exist in mammalian cells, it could be an excellent target for antiprotozoal drugs.

IN VIVO AND IN VITRO ANTILEISHMANIAL EFFECTS OF METHANOLIC EXTRACT FROM BARK OF BURSERA APTERA

PubMed Central

Nieto-Yañez, O. J.; Resendiz-Albor, A. A.; Ruiz-Hurtado, P. A.; Rivera-Yañez, N.; Rodriguez-Canales, M.; Rodriguez-Sosa, M.; Juarez-Avelar, I.; Rodriguez-Lopez, M. G.; Canales-Martinez, M. M.; Rodriguez-Monroy, M. A.

2017-01-01

Background: Cutaneous leishmaniasis lacks effective and well-tolerated treatments. The current therapies mainly rely on antimonial drugs that are inadequate because of their poor efficacy. Traditional medicine offers a complementary alternative for the treatment of various diseases. Additionally, several plants have shown success as anti-leishmanial agents. Therefore, we sought to evaluate the in vitro and in vivo activity of MEBA against Leishmania mexicana. Materials and Methods: Methanolic extract of B. aptera was obtained by macetration, after we determined in vitro anti-leishmanial activity of MEBA by MTT assay and the induced apoptosis in promastigotes by flow cytometry. To analyze the in vivo anti-leishmanial activity, we used infected mice that were treated and not treated with MEBA and we determined the levels of cytokines using ELISA. The phytochemical properties were determined by CG-MS and DPPH assay. Results: We determined of LC50 of 0.408 mg/mL of MEBA for in vitro anti-leishmanial activity. MEBA induced apoptosis in promastigotes (15.3% ± 0.86). Treated mice exhibited smaller lesions and contained significantly fewer parasites than did untreated mice; in addition, we found that IFN-γ and TNF-α increased in the sera of MEBA-treated mice. GC-MS analysis showed that podophyllotoxin was the most abundant compound. Evaluation of the activity by DPPH assay demonstrated an SC50 of 11.72 μg/mL. Conclusion: Based on the above data, it was concluded that MEBA is a good candidate in the search for new anti-leishmanial agents. PMID:28573235

Optimized transitory ectopic expression of promastigote surface antigen protein in Nicotiana benthamiana, a potential anti-leishmaniasis vaccine candidate.

PubMed

Lacombe, Séverine; Bangratz, Martine; Brizard, Jean-Paul; Petitdidier, Elodie; Pagniez, Julie; Sérémé, Drissa; Lemesre, Jean-Loup; Brugidou, Christophe

2018-01-01

In recent years, plants have been shown to be an efficient alternative expression system for high-value pharmaceuticals such as vaccines. However, constitutive expression of recombinant protein remains uncertain on their level of production and biological activity. To overcome these problems, transitory expression systems have been developed. Here, a series of experiments were performed to determine the most effective conditions to enhance vaccine antigen transient accumulation in Nicotiana benthamiana leaves using the promastigote surface antigen (PSA) from the parasitic protozoan Leishmania infantum. This protein has been previously identified as the major antigen of a licensed canine anti-leishmaniasis vaccine. The classical prokaryote Escherichia coli biosystem failed in accumulating PSA. Consequently, the standard plant system based on N. benthamiana has been optimized for the production of putatively active PSA. First, the RNA silencing defense mechanism set up by the plant against PSA ectopic expression was abolished by using three viral suppressors acting at different steps of the RNA silencing pathway. Then, we demonstrated that the signal peptide at the N-terminal side of the PSA is required for its accumulation. The PSA ER signaling and retention with the PSA signal peptide and the KDEL motif, respectively were optimized to significantly increase its accumulation. Finally, we demonstrate that the production of recombinant PSA in N. benthamiana leaves allows the conservation of its immunogenic property. These approaches demonstrate that based on these optimizations, plant based systems can be used to effectively produce the biological active PSA protein. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Crotoxin stimulates an M1 activation profile in murine macrophages during Leishmania amazonensis infection.

PubMed

Farias, L H S; Rodrigues, A P D; Coêlho, E C; Santos, M F; Sampaio, S C; Silva, E O

2017-09-01

American tegumentary leishmaniasis is caused by different species of Leishmania. This protozoan employs several mechanisms to subvert the microbicidal activity of macrophages and, given the limited efficacy of current therapies, the development of alternative treatments is essential. Animal venoms are known to exhibit a variety of pharmacological activities, including antiparasitic effects. Crotoxin (CTX) is the main component of Crotalus durissus terrificus venom, and it has several biological effects. Nevertheless, there is no report of CTX activity during macrophage - Leishmania interactions. Thus, the main objective of this study was to evaluate whether CTX has a role in macrophage M1 polarization during Leishmania infection murine macrophages, Leishmania amazonensis promastigotes and L. amazonensis-infected macrophages were challenged with CTX. MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] toxicity assays were performed on murine macrophages, and no damage was observed in these cells. Promastigotes, however, were affected by treatment with CTX (IC50 = 22·86 µg mL-1) as were intracellular amastigotes. Macrophages treated with CTX also demonstrated increased reactive oxygen species production. After they were infected with Leishmania, macrophages exhibited an increase in nitric oxide production that converged into an M1 activation profile, as suggested by their elevated production of the cytokines interleukin-6 and tumour necrosis factor-α and changes in their morphology. CTX was able to reverse the L. amazonensis-mediated inhibition of macrophage immune responses and is capable of polarizing macrophages to the M1 profile, which is associated with a better prognosis for cutaneous leishmaniasis treatment.

Inhibition of Fumarate Reductase in Leishmania major and L. donovani by Chalcones

PubMed Central

Chen, Ming; Zhai, Lin; Christensen, Søren Brøgger; Theander, Thor G.; Kharazmi, Arsalan

2001-01-01

Our previous studies have shown that chalcones exhibit potent antileishmanial and antimalarial activities in vitro and in vivo. Preliminary studies showed that these compounds destroyed the ultrastructure of Leishmania parasite mitochondria and inhibited the respiration and the activity of mitochondrial dehydrogenases of Leishmania parasites. The present study was designed to further investigate the mechanism of action of chalcones, focusing on the parasite respiratory chain. The data show that licochalcone A inhibited the activity of fumarate reductase (FRD) in the permeabilized Leishmania major promastigote and in the parasite mitochondria, and it also inhibited solubilized FRD and a purified FRD from L. donovani. Two other chalcones, 2,4-dimethoxy-4′-allyloxychalcone (24m4ac) and 2,4-dimethoxy-4′-butoxychalcone (24mbc), also exhibited inhibitory effects on the activity of solubilized FRD in L. major promastigotes. Although licochalcone A inhibited the activities of succinate dehydrogenase (SDH), NADH dehydrogenase (NDH), and succinate- and NADH-cytochrome c reductases in the parasite mitochondria, the 50% inhibitory concentrations (IC50) of licochalcone A for these enzymes were at least 20 times higher than that for FRD. The IC50 of licochalcone A for SDH and NDH in human peripheral blood mononuclear cells were at least 70 times higher than that for FRD. These findings indicate that FRD, one of the enzymes of the parasite respiratory chain, might be the specific target for the chalcones tested. Since FRD exists in the Leishmania parasite and does not exist in mammalian cells, it could be an excellent target for antiprotozoal drugs. PMID:11408218

Synthesis, crystal structures and properties of new homoleptic Ni(II)/Pd(II) β-oxodithioester chelates

NASA Astrophysics Data System (ADS)

Yadav, Chote L.; Manar, Krishna K.; Yadav, Manoj K.; Tiwari, Neeraj; Singh, Rakesh K.; Drew, Michael G. B.; Singh, Nanhai

2018-05-01

Six new cis-chelate complexes, [M(L)2] (L = methyl-3-hydroxy-3-(furyl)-2-propenedithioate L1, M = Ni(II) 1, Pd(II) 4; methyl-3-hydroxy-3-(thiophenyl)-2-propenedithioate L2, M = Ni(II) 2, Pd(II) 5 and methyl-3-hydroxy-3-(phenyl)-2-propenedithioate L3, M = Ni(II) 3, Pd(II) 6 have been prepared and characterized by elemental analyses, spectroscopy (IR, UV-Vis., 1H and 13C{1H} NMR). The structures of 2-6 have been revealed by X-ray crystallography. In all the crystal structures, the metal has four-coordinate slightly distorted square planar geometry with a cis-configuration of the ligands. Anti-leishmanial properties of the complexes have been studied; 2, 3 and 6 showed potential anti-promastigote and anti-amastigote activities with IC50 values of 1.70 ± 0.50, 1.62 ± 0.19, 9.20 ± 2.16 μg/mL and IC50 2.50 ± 0.10, 2.05 ± 0.40, 12.84 ± 3.46 μg/mL respectively. Cytotoxicity assays on these complexes showed toxicity on the promastigotes but less toxicity against RAW 264.7 cell lines at different concentrations. Palladium complexes 4, 5 and 6 show luminescent characteristics in CH2Cl2 solution at room temperature. Complexes 1-6 are weakly conducting (σrt = 10-4-10-6 S cm-1, Ea = 0.19-1.13 eV) but show semiconducting behavior in the solid phase.

Leishmania major survival in selective Phlebotomus papatasi sand fly vector requires a specific SCG-encoded lipophosphoglycan galactosylation pattern.

PubMed

Dobson, Deborah E; Kamhawi, Shaden; Lawyer, Phillip; Turco, Salvatore J; Beverley, Stephen M; Sacks, David L

2010-11-11

Phlebotomine sand flies that transmit the protozoan parasite Leishmania differ greatly in their ability to support different parasite species or strains in the laboratory: while some show considerable selectivity, others are more permissive. In "selective" sand flies, Leishmania binding and survival in the fly midgut typically depends upon the abundant promastigote surface adhesin lipophosphoglycan (LPG), which exhibits species- and strain-specific modifications of the dominant phosphoglycan (PG) repeat units. For the "selective" fly Phlebotomus papatasi PpapJ, side chain galactosyl-modifications (scGal) of PG repeats play key roles in parasite binding. We probed the specificity and properties of this scGal-LPG PAMP (Pathogen Associated Molecular Pattern) through studies of natural isolates exhibiting a wide range of galactosylation patterns, and of a panel of isogenic L. major engineered to express similar scGal-LPG diversity by transfection of SCG-encoded β1,3-galactosyltransferases with different activities. Surprisingly, both 'poly-scGal' and 'null-scGal' lines survived poorly relative to PpapJ-sympatric L. major FV1 and other 'mono-scGal' lines. However, survival of all lines was equivalent in P. duboscqi, which naturally transmit L. major strains bearing 'null-scGal'-LPG PAMPs. We then asked whether scGal-LPG-mediated interactions were sufficient for PpapJ midgut survival by engineering Leishmania donovani, which normally express unsubstituted LPG, to express a 'PpapJ-optimal' scGal-LPG PAMP. Unexpectedly, these "L. major FV1-cloaked" L. donovani-SCG lines remained unable to survive within PpapJ flies. These studies establish that midgut survival of L. major in PpapJ flies is exquisitely sensitive to the scGal-LPG PAMP, requiring a specific 'mono-scGal' pattern. However, failure of 'mono-scGal' L. donovani-SCG lines to survive in selective PpapJ flies suggests a requirement for an additional, as yet unidentified L. major-specific parasite factor(s). The interplay of the LPG PAMP and additional factor(s) with sand fly midgut receptors may determine whether a given sand fly host is "selective" or "permissive", with important consequences to both disease transmission and the natural co-evolution of sand flies and Leishmania.

Trypanosome transmitted by Phlebotomus: first report from the Americas.

PubMed

Anderson, J R; Ayala, S C

1968-09-06

Transmission studies carried out in the laboratory incriminated Phlebotomus vexator occidentis as a vector of a species of trypanosome that infects Bufo boreas halophilus. Toads free of parasites contracted the trypanosome after eating infected flies and after intraperitoneal inoculation of flagellates cultured from the hindgut of flies that had fed on infected toads. Discovery of this vectorhost-parasite system in the Americas, and the localization of promastigote flagellates (leptomonads) in the hindgut of the vector, should assist in clarifying interpretative problems associated with infection of wild-caught flies in studies on leishmaniasis in the Americas and elsewhere.

Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut.

PubMed

Verma, Sudha; Das, Sushmita; Mandal, Abhishek; Ansari, Md Yousuf; Kumari, Sujata; Mansuri, Rani; Kumar, Ajay; Singh, Ruby; Saini, Savita; Abhishek, Kumar; Kumar, Vijay; Sahoo, Ganesh Chandra; Das, Pradeep

2017-06-23

In vector-borne diseases such as leishmaniasis, the sand fly midgut is considered to be an important site for vector-parasite interaction. Digestive enzymes including serine peptidases such as trypsin and chymotrypsin, which are secreted in the midgut are one of the obstacles for Leishmania in establishing a successful infection. The presence of some natural inhibitors of serine peptidases (ISPs) has recently been reported in Leishmania. In the present study, we deciphered the role of these ISPs in the survival of Leishmania donovani in the hostile sand fly midgut environment. In silico and co-immunoprecipitation studies were performed to observe the interaction of L. donovani ISPs with trypsin and chymotrypsin. Zymography and in vitro enzyme assays were carried out to observe the inhibitory effect of purified recombinant ISPs of L. donovani (rLdISPs) on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of ISPs in the amastigote to promastigote transition stages were studied by semi-quantitative RT-PCR and Western blot. The role of LdISP on the survival of ISP overexpressed (OE) and ISP knocked down (KD) Leishmania parasites inside the sand fly gut was investigated by in vitro and in vivo cell viability assays. We identified two ecotin-like genes in L. donovani, LdISP1 and LdISP2. In silico and co-immunoprecipitation results clearly suggest a strong interaction of LdISP molecules with trypsin and chymotrypsin. Zymography and in vitro enzyme assay confirmed the inhibitory effect of rLdISP on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of LdISP2 was found to be strongly associated with the amastigote to promastigote phase transition. The activities of the digestive enzymes were found to be significantly reduced in the infected sand flies when compared to uninfected. To our knowledge, our study is the first report showing the possible reduction of chymotrypsin activity in L. donovani infected sand flies compared to uninfected. Interestingly, during the early transition stage, substantial killing was observed in ISP2 knocked down (ISP2KD) parasites compared to wild type (WT), whereas ISP1 knocked down (ISP1KD) parasites remained viable. Therefore, our study clearly indicates that LdISP2 is a more effective inhibitor of serine peptidases than LdISP1. Our results suggest that the lack of ISP2 is detrimental to the parasites during the early transition from amastigotes to promastigotes. Moreover, the results of the present study demonstrated for the first time that LdISP2 has an important role in the inhibition of peptidases and promoting L. donovani survival inside the Phlebotomus argentipes midgut.

Evaluation of Antileishmanial Activity of Selected Brazilian Plants and Identification of the Active Principles

PubMed Central

Filho, Valdir Cechinel; Meyre-Silva, Christiane; Niero, Rivaldo; Bolda Mariano, Luisa Nathália; Gomes do Nascimento, Fabiana; Vicente Farias, Ingrid; Gazoni, Vanessa Fátima; dos Santos Silva, Bruna; Giménez, Alberto; Gutierrez-Yapu, David; Salamanca, Efrain; Malheiros, Angela

2013-01-01

This study evaluated extracts, fractions, and isolated compounds from some selected Brazilian medicinal plants against strains of promastigotes of Leishmania amazonensis and L. brasiliensis in vitro. The cell viability was determined, comparing the results with reference standards. The dichloromethane fractions of the roots, stems, and leaves of Allamanda schottii showed IC50 values between 14.0 and 2.0 μg/mL. Plumericin was the main active compound, with IC50 of 0.3 and 0.04 μg/mL against the two species of Leishmania analyzed. The hexane extract of Eugenia umbelliflora fruits showed IC50 of 14.3 and 5.7 μg/mL against L. amazonensis and L. brasiliensis, respectively. The methanolic extracts of the seeds of Garcinia achachairu and guttiferone A presented IC50 values of 35.9 and 10.4 μg/mL, against L. amazonensis, respectively. The ethanolic extracts of the stem barks of Rapanea ferruginea and the isolated compound, myrsinoic acid B, presented activity against L. brasiliensis with IC50 of 24.1 and 6.1 μg/mL. Chloroform fraction of Solanum sisymbriifolium exhibited IC50 of 33.8 and 20.5 μg/mL, and cilistol A was the main active principle, with IC50 of 6.6 and 3.1 μg/mL against L. amazonensis and L. brasiliensis, respectively. It is concluded that the analyzed plants are promising as new and effective antiparasitic agents. PMID:23840252

Leishmania donovani Argininosuccinate Synthase Is an Active Enzyme Associated with Parasite Pathogenesis

PubMed Central

Lakhal-Naouar, Ines; Jardim, Armando; Strasser, Rona; Luo, Shen; Kozakai, Yukiko; Nakhasi, Hira L.; Duncan, Robert C.

2012-01-01

Background Gene expression analysis in Leishmania donovani (Ld) identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (LdASS), that was more abundantly expressed in amastigotes than in promastigotes. In order to characterize in detail this newly identified protein in Leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo. Methodology/Principal Findings Two parasite cell lines either over expressing wild type LdASS or a mutant form (G128S) associated with severe cases of citrullinemia in humans were developed. In addition we also produced bacterially expressed recombinant forms of the same proteins. Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid). However, the mutant form of the protein is inactive. We demonstrate that though LdASS has a glycosomal targeting signal that binds the targeting apparatus in vitro, only a small proportion of the total cellular ASS is localized in a vesicle, as indicated by protection from protease digestion of the crude organelle fraction. The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA. Surprisingly, comparison to known glycosomal proteins by IFA revealed that LdASS was located in a structure different from the known glycosomal vesicles. Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver. Conclusion/Significance Our study suggests that LdASS is an active enzyme, with unique localization and essential for parasite survival and growth in the mammalian host. Based on these observations LdASS could be further explored as a potential drug target. PMID:23094117

Propolis reduces Leishmania amazonensis-induced inflammation in the liver of BALB/c mice.

PubMed

da Silva, Suelen S; Mizokami, Sandra S; Fanti, Jacqueline R; Miranda, Milena M; Kawakami, Natalia Y; Teixeira, Fernanda Humel; Araújo, Eduardo J A; Panis, Carolina; Watanabe, Maria A E; Sforcin, José M; Pavanelli, Wander R; Verri, Waldiceu A; Felipe, Ionice; Conchon-Costa, Ivete

2016-04-01

Experimental models of mouse paw infection with L. amazonensis show an induction of a strong inflammatory response in the skin, and parasitic migration may occur to secondary organs with consequent tissue injury. There are few studies focusing on the resolution of damage in secondary organs caused by Leishmania species-related cutaneous leishmaniasis. We investigated the propolis treatment effect on liver inflammation induced by Leishmania amazonensis infection in the mouse paw. BALB/c mice were infected in the hind paw with L. amazonensis (10(7)) promastigote forms. After 15 days, animals were treated daily with propolis (5 mg/kg), Glucantime (10 mg/kg), or with propolis plus Glucantime combined. After 60 days, mice were euthanized and livers were collected for inflammatory process analysis. Liver microscopic analysis showed that propolis reduced the inflammatory process compared to untreated infected control. There was a decrease of liver myeloperoxidase and N-acetyl-β-glucosaminidase activity levels, collagen fiber deposition, pro-inflammatory cytokine production, and plasma aspartate transaminase and alanine transaminase levels. Furthermore, propolis treatment enhanced anti-inflammatory cytokine levels and reversed hepatosplenomegaly. Our data demonstrated that daily low doses of Brazilian propolis reduced the secondary chronic inflammatory process in the liver caused by L. amazonensis subcutaneous infection in a susceptible mice strain.

Antiparasitic Activity and Essential Oil Chemical Analysis of the Piper Tuberculatum Jacq Fruit

PubMed Central

dos Santos Sales, Valterlúcio; Monteiro, Álefe Brito; Delmondes, Gyllyandeson de Araújo; do Nascimento, Emmily Petícia; Sobreira Dantas Nóbrega de Figuêiredo, Francisco Rodolpho; de Souza Rodrigues, Cristina Kelly; Evangelista de Lacerda, Josefa Fernanda; Fernandes, Cícera Norma; Barbosa, Maysa de Oliveira; Brasil, Adamo Xenofonte; Tintino, Saulo Relison; Vega Gomez, Maria Celeste; Coronel, Cathia; Melo Coutinho, Henrique Douglas; Martins da Costa, José Galberto; Bezerra Felipe, Cícero Francisco; Alencar de Menezes, Irwin Rose; Kerntopf, Marta Regina

2018-01-01

With the increase of neglected diseases such as leishmaniasis and Chagas disease, there was a need for the search for new therapeutic alternatives that reduce the harm caused by medicine available for treatment. Thus, this study was performed to investigate the antiparasitic activity of the essential oil from the fruits of Piper tuberculatum Jacq, against lines of Leishmania braziliensis (MHOM/CO/88/UA301), Leishmania infantum (MHOM/ES/92/BCN83) and Trypanosoma cruzi (LC-B5 clone). Before running protocols, an analysis of the chemical composition of essential oil was conducted, which presented monoterpenes and sesquiterpenes. As major constituents, β-pinene and α-pinene were identified. Regarding to antiparasitic activity, the essential oil had an EC50 values of 133.97 µg/mL and 143.59 µg/mL against variations promastigotes of L. infantum and L. braziliensis, respectively. As for trypanocidal activity, the oil showed EC50 value of 140.31 µg/mL against epimastigote form of T. cruzi. Moreover, it showed moderate cytotoxicity in fibroblasts with LC50 value of 204.71 µg/mL. The observed effect may be related to the presence of terpenes contained in the essential oil, since it has its antiparasitic activity proven in the literature.

The lignan glycosides lyoniside and saracoside poison the unusual type IB topoisomerase of Leishmania donovani and kill the parasite both in vitro and in vivo.

PubMed

Saha, Sourav; Mukherjee, Tulika; Chowdhury, Sayan; Mishra, Amartya; Chowdhury, Somenath Roy; Jaisankar, Parasuraman; Mukhopadhyay, Sibabrata; Majumder, Hemanta K

2013-12-15

Lignans are diphenyl propanoids with vast range of biological activities. The present study provides an important insight into the anti-leishmanial activities of two lignan glycosides, viz. lyoniside and saracoside. These compounds inhibit catalytic activities of topoisomerase IB (LdTopIB) of Leishmania donovani in non-competitive manner and stabilize the LdTopIB mediated cleavage complex formation both in vitro and in Leishmania promastigotes and subsequently inhibit the religation of cleaved strand. These two compounds not only poison LdTopIB but also can interact with the free enzyme LdTopIB. We have also shown that lyoniside and saracoside are cytotoxic to promastigotes and intracellular amastigotes. The protein-DNA complex formation leads to double strand breaks in DNA which ultimately triggers apoptosis-like cell death in the parasite. Along with their cytotoxicity towards sodium antimony gluconate (SAG) sensitive AG83 strain, their ability to kill SAG resistant GE1 strain makes these two compounds potential anti-leishmanial candidates. Not only they effectively kill L. donovani amastigotes inside macrophages in vitro, lyoniside and saracoside demonstrated strong anti-leishmanial efficacies in BALB/c mice model of leishmaniasis. Treatment with these lignan glycosides produce nitric oxide and reactive oxygen species which result in almost complete clearance of the liver and splenic parasite burden. These compounds do not inhibit human topoisomerase IB upto 200μM concentrations and had poor cytotoxic effect on uninfected cultured murine peritoneal macrophages upto 100μM concentrations. Taken together it can be concluded that these compounds can be developed into excellent therapeutic agent against deadly disease leishmaniasis. Copyright © 2013 Elsevier Inc. All rights reserved.

GRIND2-based 3D-QSAR and prediction of activity spectra for symmetrical bis-pyridinium salts with promastigote antileishmanial activity.

PubMed

Diniz, Evelyn Mirella Lopes Pina; Tomich de Paula da Silva, Carlos Henrique; Gómez-Perez, Verónica; Federico, Leonardo Bruno; Campos Rosa, Joaquín María

2017-08-01

Leishmaniasis is a major group of neglected tropical diseases caused by the protozoan parasite Leishmania. About 12 million people are affected in 98 countries and 350 million people worldwide are at risk of infection. Current leishmaniasis treatments rely on a relatively small arsenal of drugs, including amphotericin B, pentamidine and others, which in general have some type of inconvenience. Recently, we have synthesized antileishmanial bis-pyridinium derivatives and symmetrical bis-pyridinium cyclophanes. These compounds are considered structural analogues of pentamidine, where the amidino moiety, protonated at physiological pH, is replaced by a positively charged nitrogen atom as a pyridinium ring. In this work, a statistically significant GRIND2-based 3D-QSAR model was built and biological activity predictions were in silico carried out allowing rationalization of the different activities recently obtained against Leishmania donovani (in L. donovani promastigotes) for a data set of 19 bis-pyridinium compounds. We will emphasize the most important structural requirements to improve the biological activity and probable interactions with the biological receptor as a guide for lead and prototype optimization. In addition, since no information about the actual biological target for this series of active compounds is provided, we have used Prediction of Activity Spectra for Biologically Active Substances to propose our compounds as potential nicotinic α6β3β4α5 receptor antagonists. This proposal is reinforced by the high structural similarity observed between our compounds and several anthelmintic drugs in current clinical use, which have the same drug action mechanism here predicted. Such new findings would be confirmed with further and additional experimental assays.

Isolation, characterization and antimicrobial evaluation of a novel compound N-octacosan 7β ol, from Fumaria parviflora Lam.

PubMed

Jameel, Mohammad; Islamuddin, Mohammad; Ali, Abuzer; Afrin, Farhat; Ali, Mohammed

2014-03-12

Fumaria parviflora Lam. (Fumaraceae) is widely used in traditional as well as folkloric system of medicine from ancient. It is commonly known as 'Pitpapra' or 'Shahtrah' in Indian traditional system of medicine and used for treating numerous ailments like diarrhea, fever, influenza, blood purifier and other complications. The object of the present study was to evaluate the Antileishmanial, antibacterial, antifungal and cytotoxic potential of isolated compound. Methanolic extract of whole plant of Fumaria parviflora was dried under reduced pressure to obtain a dark brown residue which was adsorbed on silica gel column grade (60-120 mesh) to obtain a slurry and chromatographed over silica gel loaded column in petroleum ether-chloroform (3:1, 1:1 and 1:3 v/v). The in vitro antileishmanial evaluation of isolated compound against Leishmania donovani promastigotes was investigated by growth kinetics assay, reversibility assay, analysis of cellular morphology, adverse toxicity and determination of 50% growth inhibitory concentration (GI50). Disc diffusion and broth micro dilution methods were used to study the antibacterial (Gram + Staphylococcus epidermidis and Bacillus subtilis; Gram - Escherichia coli and Salmonella typhimurium) and antifungal (Candida albicans and Aspergillus niger) potential in vitro. Structure elucidation by spectral data analysis revealed a novel compound, n-octacosan-7β-ol (OC), yield (0.471%), having significant antimicrobial activity against Leishmania donovani promastigotes, Staphylococcus epidermidis, Escherichia coli, Candida albicans and Aspergillus niger in vitro with GI50 = 5.35, MIC 250, MIC 250 and MFC 500 and MIC 250 μg ml(-1) respectively. The isolated compound did not show adverse effect against mammalian macrophages. The available evidence of compound suggested that it may be used as antimicrobial agent in future and may provide new platform for drug discovery programmes for leishmaniasis.

Aminophthalocyanine-Mediated Photodynamic Inactivation of Leishmania tropica

PubMed Central

Al-Qahtani, Ahmed; Alkahtani, Saad; Kolli, Bala; Tripathi, Pankaj; Dutta, Sujoy; Al-Kahtane, Abdullah A.; Jiang, Xiong-Jie; Ng, Dennis K. P.

2016-01-01

Photodynamic inactivation of Leishmania spp. requires the cellular uptake of photosensitizers, e.g., endocytosis of silicon(IV)-phthalocyanines (PC) axially substituted with bulky ligands. We report here that when substituted with amino-containing ligands, the PCs (PC1 and PC2) were endocytosed and displayed improved potency against Leishmania tropica promastigotes and axenic amastigotes in vitro. The uptake of these PCs by both Leishmania stages followed saturation kinetics, as expected. Sensitive assays were developed for assessing the photodynamic inactivation of Leishmania spp. by rendering them fluorescent in two ways: transfecting promastigotes to express green fluorescent protein (GFP) and loading them with carboxyfluorescein succinimidyl ester (CFSE). PC-sensitized Leishmania tropica strains were seen microscopically to lose their motility, structural integrity, and GFP/CFSE fluorescence after exposure to red light (wavelength, ∼650 nm) at a fluence of 1 to 2 J cm−2. Quantitative fluorescence assays based on the loss of GFP/CFSE from live Leishmania tropica showed that PC1 and PC2 dose dependently sensitized both stages for photoinactivation, consistent with the results of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. Leishmania tropica strains are >100 times more sensitive than their host cells or macrophages to PC1- and PC2-mediated photoinactivation, judging from the estimated 50% effective concentrations (EC50s) of these cells. Axial substitution of the PC with amino groups instead of other ligands appears to increase its leishmanial photolytic activity by up to 40-fold. PC1 and PC2 are thus potentially useful for photodynamic therapy of leishmaniasis and for oxidative photoinactivation of Leishmania spp. for use as vaccines or vaccine carriers. PMID:26824938

The sesquiterpene (-)-α-bisabolol is active against the causative agents of Old World cutaneous leishmaniasis through the induction of mitochondrial-dependent apoptosis.

PubMed

Corpas-López, V; Merino-Espinosa, G; Díaz-Sáez, V; Morillas-Márquez, F; Navarro-Moll, M C; Martín-Sánchez, J

2016-10-01

Cutaneous leishmaniasis treatment remains challenging due to the absence of a satisfactory treatment. The screening of natural compounds is a valuable strategy in the search of new drugs against leishmaniasis. The sesquiterpene (-)-α-bisabolol is effective in vivo against visceral leishmaniasis due to Leishmania infantum, but its mechanism of action remains elusive. The aim of this study is to validate this promising compound against the causative species of Old World cutaneous leishmaniasis and to get an insight into its antileishmanial mode of action. The compound was evaluated on L. tropica promastigotes and intracellular amastigotes using bone marrow-derived macrophages and its cytotoxicity was evaluated on L929 fibroblasts. The reactive oxygen species generation was evaluated using a sensitive probe. Mitochondrial depolarization was assessed evaluating the fluorescence due to rhodamine 123 in a flow cytometer. Apoptosis was investigated by measuring the fluorescence due to annexin V and propidium iodide in a flow cytometer. The ultrastructure of treated promastigotes and intracellular amastigotes was analysed through transmission electron microscopy. (-)-α-Bisabolol was active against L. tropica intracellular amastigotes displaying an inhibitory concentration 50 % of 25.2 µM and showing low cytotoxicity. This compound induced time and dose-dependent oxidative stress, mitochondrial depolarization and phosphatidilserine externalization (a marker of apoptosis). These effects were noticed at a low concentration and short exposure time. In the ultrastructural analyses, the treated parasites showed mitochondrial disruption, presence of electron-dense structures and chromatin condensation. These results suggest that this natural compound induces oxidative stress and mitochondrial-dependent apoptosis on Leishmania without disturbing the plasma membrane.

Apoptosis-like death in Leishmania donovani promastigotes induced by eugenol-rich oil of Syzygium aromaticum.

PubMed

Islamuddin, Mohammad; Sahal, Dinkar; Afrin, Farhat

2014-01-01

Leishmaniasis consists of a complex spectrum of infectious diseases with worldwide distribution of which visceral leishmaniasis or kala-azar caused by Leishmania donovani is the most devastating. In the absence of vaccines, chemotherapy remains the mainstay for the control of leishmaniasis. The drugs of choice are expensive and associated with multiple adverse side effects. Because of these limitations, the development of new antileishmanial compounds is imperative and plants offer prospects in this regard. The present work was conducted to study the antileishmanial potential of oil from Syzygium aromaticum flower buds (clove). The S. aromaticum oil was characterized by gas chromatography and GC-MS and eugenol as well as eugenyl acetate were found to be the most abundant compounds, composing 59.75 % and 29.24 %, respectively of the oil. Our findings have shown that eugenol-rich essential oil from S. aromaticum (EROSA) possesses significant activity against L. donovani, with 50 % inhibitory concentration of 21 ± 0.16 µg ml(-1) and 15.24 ± 0.14 µg ml(-1), respectively, against promastigotes and intracellular amastigotes. Alterations in cellular morphology and growth reversibility assay substantiated the leishmanicidal activity of EROSA. The leishmanicidal effect was mediated via apoptosis as confirmed by externalization of phosphatidylserine, DNA nicking by TdT-mediated dUTP nick-end labelling (TUNEL) assay, dyskinetoplastidy, cell cycle arrest at sub-G0-G1 phase, loss of mitochondrial membrane potential and reactive oxygen species generation. EROSA presented no adverse cytotoxic effects against murine macrophages even at 200 µg ml(-1). Our studies authenticate the promising antileishmanial activity of EROSA, which is mediated by programmed cell death, and, accordingly, EROSA may be a source of novel agents for the treatment of leishmaniasis.

Histamine H1-receptor antagonists against Leishmania (L.) infantum: an in vitro and in vivo evaluation using phosphatidylserine-liposomes.

PubMed

Pinto, Erika G; da Costa-Silva, Thais A; Tempone, Andre Gustavo

2014-09-01

Considering the limited and toxic therapeutic arsenal available for visceral leishmaniasis (VL), the drug repositioning approach could represent a promising tool to the introduction of alternative therapies. Histamine H1-receptor antagonists are drugs belonging to different therapeutic classes, including antiallergics and anxyolitics. In this work, we described for the first time the activity of H1-antagonists against L. (L.) infantum and their potential effectiveness in an experimental hamster model. The evaluation against promastigotes demonstrated that chlorpheniramine, cinnarizine, hydroxyzine, ketotifen, loratadine, quetiapine and risperidone exerted a leishmanicidal effect against promastigotes, with IC50 values in the range of 13-84μM. The antihistaminic drug cinnarizine demonstrated effectiveness against the intracellular amastigotes, with an IC50 value of 21μM. The mammalian cytotoxicity was investigated in NCTC cells, resulting in IC50 values in the range of 57-229μM. Cinnarizine was in vivo studied as a free formulation and entrapped into phosphatidylserine-liposomes. The free drug was administered for eight consecutive days at 50mg/kg by intraperitoneal route (i.p.) and at 100mg/kg by oral route to L. infantum-infected hamsters, but showed lack of effectiveness in both regimens, as detected by real time PCR. The liposomal formulation was administered by i.p. route at 3mg/kg for eight days and reduced the parasite burden to 54% in liver when compared to untreated group; no improvement was observed in the spleen of infected hamsters. Cinnarizine is the first antihistaminic drug with antileishmanial activity and could be used as scaffold for drug design studies for VL. Copyright © 2014 Elsevier B.V. All rights reserved.

Natural infection of Lutzomyia neivai and Lutzomyia sallesi (Diptera: Psychodidae) by Leishmania infantum chagasi in Brazil.

PubMed

Saraiva, Lara; Carvalho, Gustavo M L; Gontijo, Célia M F; Quaresma, Patrícia F; Lima, Ana C V M R; Falcão, Alda L; Andrade Filho, José D

2009-09-01

Natural infections with Leishmania were found in females of the phlebotomine sand flies Lutzomyia neivai (Pinto) (= Nyssomyia neivai) and Lutzomyia sallesi (Galvão & Coutinho) (= Evandromyia sallesi) (Diptera: Psychodidae) from Lassance, in the Brazilian state of Minas Gerais. Promastigotes were found in the pyloric region of the former species and in the abdominal midgut of the latter species. Insects found to be infected by microscopic examination were macerated in saline solution and inoculated into hamsters. Subsequent analysis by polymerase chain reaction-restriction fragment length polymorphism revealed both isolates to belong to the species Leishmania infantum chagasi Cunha & Chagas.

Leishmania tropica isolates from non-healed and healed patients in Iran: A molecular typing and phylogenetic analysis.

PubMed

Bamorovat, Mehdi; Sharifi, Iraj; Mohammadi, Mohammad Ali; Eybpoosh, Sana; Nasibi, Saeid; Aflatoonian, Mohammad Reza; Khosravi, Ahmad

2018-03-01

The precise identification of the parasite species causing leishmaniasis is essential for selecting proper treatment modality. The present study aims to compare the nucleotide variations of the ITS1, 7SL RNA, and Hsp70 sequences between non-healed and healed anthroponotic cutaneous leishmaniasis (ACL) patients in major foci in Iran. A case-control study was carried out from September 2015 to October 2016 in the cities of Kerman and Bam, in the southeast of Iran. Randomly selected skin-scraping lesions of 40 patients (20 non-healed and 20 healed) were examined and the organisms were grown in a culture medium. Promastigotes were collected by centrifugation and kept for further molecular examinations. The extracted DNA was amplified and sequenced. After global sequence alignment with BioEdit software, maximum likelihood phylogenetic analysis was performed in PhyML for typing of Leishmania isolates. Nucleotide composition of each genetic region was also compared between non-healed and healed patients. Our results showed that all isolates belonged to the Leishmania tropica complex, with their genetic composition in the ITS1 region being different among non-healed and healed patients. 7SL RNA and Hsp70 regions were genetically identical between both groups. Variability in nucleotide patterns observed between both groups in the ITS1 region may serve to encourage future research on the function of these polymorphisms and may improve our understanding of the role of parasite genome properties on patients' response to Leishmania treatment. Our results also do not support future use of 7SL RNA and Hsp70 regions of the parasite for comparative genomic analyses. Copyright © 2018 Elsevier Ltd. All rights reserved.

Effect of clinically approved HDAC inhibitors on Plasmodium, Leishmania and Schistosoma parasite growth.

PubMed

Chua, Ming Jang; Arnold, Megan S J; Xu, Weijun; Lancelot, Julien; Lamotte, Suzanne; Späth, Gerald F; Prina, Eric; Pierce, Raymond J; Fairlie, David P; Skinner-Adams, Tina S; Andrews, Katherine T

2017-04-01

Malaria, schistosomiasis and leishmaniases are among the most prevalent tropical parasitic diseases and each requires new innovative treatments. Targeting essential parasite pathways, such as those that regulate gene expression and cell cycle progression, is a key strategy for discovering new drug leads. In this study, four clinically approved anti-cancer drugs (Vorinostat, Belinostat, Panobinostat and Romidepsin) that target histone/lysine deacetylase enzymes were examined for in vitro activity against Plasmodium knowlesi, Schistosoma mansoni, Leishmania amazonensis and L. donovani parasites and two for in vivo activity in a mouse malaria model. All four compounds were potent inhibitors of P. knowlesi malaria parasites (IC 50 9-370 nM), with belinostat, panobinostat and vorinostat having 8-45 fold selectivity for the parasite over human neonatal foreskin fibroblast (NFF) or human embryonic kidney (HEK 293) cells, while romidepsin was not selective. Each of the HDAC inhibitor drugs caused hyperacetylation of P. knowlesi histone H4. None of the drugs was active against Leishmania amastigote or promastigote parasites (IC 50  > 20 μM) or S. mansoni schistosomula (IC 50  > 10 μM), however romidepsin inhibited S. mansoni adult worm parings and egg production (IC 50 ∼10 μM). Modest in vivo activity was observed in P. berghei infected mice dosed orally with vorinostat or panobinostat (25 mg/kg twice daily for four days), with a significant reduction in parasitemia observed on days 4-7 and 4-10 after infection (P < 0.05), respectively. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Synthesis and leishmanicidal activity of eugenol derivatives bearing 1,2,3-triazole functionalities.

PubMed

Teixeira, Róbson Ricardo; Gazolla, Poliana Aparecida Rodrigues; da Silva, Adalberto Manoel; Borsodi, Maria Paula Gonçalves; Bergmann, Bartira Rossi; Ferreira, Rafaela Salgado; Vaz, Boniek Gontijo; Vasconcelos, Géssica Adriana; Lima, Wallace Pacienza

2018-02-25

In this paper, it is described the synthesis and the evaluation of the leishmanicidal activity of twenty-six eugenol derivatives bearing 1,2,3-triazole functionalities. The evaluation of the compounds on promastigotes of Leishmania amazonensis (WHOM/BR/75/Josefa) showed that eugenol derivatives present leishmanicidal activities with varying degrees of effectiveness. The most active compound, namely 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-methylbenzyl)-1H-1,2,3-triazole (7k) (IC 50  = 7.4 ± 0.8 μmol L -1 ), also targeted Leishmania parasites inside peritoneal macrophages (IC 50  = 1.6 μmol L -1 ) without interfering with cell viability. The cytotoxicity of 7k against macrophage cells presented IC 50 of 211.9 μmol L -1 and the selective index was equal to 132.5. Under similar conditions, compound 7k was more effective than glucantime and pentamidine, two drugs currently in the clinic. In addition, theoretical calculations showed that this compound also presents most physicochemical and pharmacokinetic properties within the ranges expected for orally available drugs. It is believed that eugenol bearing 1,2,3-triazole functionalities may represent a scaffold to be explored toward the development of new agents to treat leishmaniasis. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Cutaneous leishmaniasis in the dorsal skin of hamsters: a useful model for the screening of antileishmanial drugs.

PubMed

Robledo, Sara M; Carrillo, Lina M; Daza, Alejandro; Restrepo, Adriana M; Muñoz, Diana L; Tobón, Jairo; Murillo, Javier D; López, Anderson; Ríos, Carolina; Mesa, Carol V; Upegui, Yulieth A; Valencia-Tobón, Alejandro; Mondragón-Shem, Karina; Rodríguez, Berardo; Vélez, Iván D

2012-04-21

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l0(7) promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Natural Terpenoids from Ambrosia Species Are Active In Vitro and In Vivo against Human Pathogenic Trypanosomatids

PubMed Central

Frank, Fernanda M.; Laurella, Laura C.; Muschietti, Liliana V.; Catalán, Cesar A.; Martino, Virginia S.; Malchiodi, Emilio L.

2013-01-01

Among the natural compounds, terpenoids play an important role in the drug discovery process for tropical diseases. The aim of the present work was to isolate antiprotozoal compounds from Ambrosia elatior and A. scabra. The sesquiterpene lactone (STL) cumanin was isolated from A. elatior whereas two other STLs, psilostachyin and cordilin, and one sterol glycoside, daucosterol, were isolated from A. scabra. Cumanin and cordilin were active against Trypanosoma cruzi epimastigotes showing 50% inhibition concentrations (IC50) values of 12 µM and 26 µM, respectively. Moreover, these compounds are active against bloodstrean trypomastigotes, regardless of the T. cruzi strain tested. Psilostachyin and cumanin were also active against amastigote forms with IC50 values of 21 µM and 8 µM, respectively. By contrast, daucosterol showed moderate activity on epimastigotes and trypomastigotes and was inactive against amastigote forms. We also found that cumanin and psilostachyin exhibited an additive effect in their trypanocidal activity when these two drugs were tested together. Cumanin has leishmanicidal activity with growth inhibition values greater than 80% at a concentration of 5 µg/ml (19 µM), against both L. braziliensis and L. amazonensis promastigotes. In an in vivo model of T. cruzi infection, cumanin was more active than benznidazole, producing an 8-fold reduction in parasitemia levels during the acute phase of the infection compared with the control group, and more importantly, a reduction in mortality with 66% of the animals surviving, in comparison with 100% mortality in the control group. Cumanin also showed nontoxic effects at the doses assayed in vivo, as determined using markers of hepatic damage. PMID:24130916

Characterization of cell cultures derived from Lutzomyia spinicrassa (Diptera: Psychodidae) and their susceptibility to infection with Leishmania (Viannia) braziliensis.

PubMed

Zapata Lesmes, Angela Cristina; Cárdenas Castro, Estrella; Bello, Felio

2005-12-01

The sand fly Lutzomyia spinicrassa (Morales, Osorno-Mesa, Osorno & de Hoyos, 1969) is a vector of Leishmania (Viannia) braziliensis, an etiological agent of cutaneous leishmaniasis in Colombia. The present article describes, for the first time, the morphological, karyotypical, and isozymatic characteristics of cell cultures derived from L. Spinicrassa embryonic tissues as well as the interaction of L. Braziliensis with these cell cultures. L. Spinicrassa embryonated eggs and neonate larvae were taken for tissue explants. These were seeded in Grace, L-15, Grace/L-15, MM/VP12, and MK/VP12 culture media. The pH range in these media was 6.7 to 6.9 and the cultures were incubated at 28 degrees C. The MHOM/CO/86/CL250 strain of L. Braziliensis was used for experimental infection of cell cultures of L. Spinicrassa. Cell growth was achieved in L-15 medium and a confluent monolayer was obtained 180 days after the embryonated eggs were explanted. The cell morphology of the primary cell cultures was initially heterogeneous, but in the confluent monolayer of these cell cultures and in the subcultures the predominant cell types were later fibroblast-like and epithelial-like. Cultured cells were predominantly diploid (2n=8); however, significant percentages of aneuploids were also recorded. The cell culture isozyme patterns of L. Spinicrassa coincided with pupae samples from the same species. Promastigote forms of L. Braziliensis could invade cells and transform into amastigote-like forms inside them. The characteristics of cell cultures derived from L. Spinicrassa embryonic tissues were determined. These cultures emerge as a new model to study the life-cycle of L. Braziliensis.

Crovirin, a snake venom cysteine-rich secretory protein (CRISP) with promising activity against Trypanosomes and Leishmania.

PubMed

Adade, Camila M; Carvalho, Ana Lúcia O; Tomaz, Marcelo A; Costa, Tatiana F R; Godinho, Joseane L; Melo, Paulo A; Lima, Ana Paula C A; Rodrigues, Juliany C F; Zingali, Russolina B; Souto-Padrón, Thaïs

2014-10-01

The neglected human diseases caused by trypanosomatids are currently treated with toxic therapy with limited efficacy. In search for novel anti-trypanosomatid agents, we showed previously that the Crotalus viridis viridis (Cvv) snake venom was active against infective forms of Trypanosoma cruzi. Here, we describe the purification of crovirin, a cysteine-rich secretory protein (CRISP) from Cvv venom with promising activity against trypanosomes and Leishmania. Crude venom extract was loaded onto a reverse phase analytical (C8) column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak containing the isolated protein (confirmed by SDS-PAGE and mass spectrometry) was collected and its protein content was measured. T. cruzi trypomastigotes and amastigotes, L. amazonensis promastigotes and amastigotes and T. brucei rhodesiense procyclic and bloodstream trypomastigotes were challenged with crovirin, whose toxicity was tested against LLC-MK2 cells, peritoneal macrophages and isolated murine extensor digitorum longus muscle. We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin. Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10-2.38 µg/ml). A considerably higher concentration (20 µg/ml) of crovirin was required to elicit only limited toxicity on mammalian cells. This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases.

Crovirin, a Snake Venom Cysteine-Rich Secretory Protein (CRISP) with Promising Activity against Trypanosomes and Leishmania

PubMed Central

Adade, Camila M.; Carvalho, Ana Lúcia O.; Tomaz, Marcelo A.; Costa, Tatiana F. R.; Godinho, Joseane L.; Melo, Paulo A.; Lima, Ana Paula C. A.; Rodrigues, Juliany C. F.; Zingali, Russolina B.; Souto-Padrón, Thaïs

2014-01-01

Background The neglected human diseases caused by trypanosomatids are currently treated with toxic therapy with limited efficacy. In search for novel anti-trypanosomatid agents, we showed previously that the Crotalus viridis viridis (Cvv) snake venom was active against infective forms of Trypanosoma cruzi. Here, we describe the purification of crovirin, a cysteine-rich secretory protein (CRISP) from Cvv venom with promising activity against trypanosomes and Leishmania. Methodology/Principal Findings Crude venom extract was loaded onto a reverse phase analytical (C8) column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak containing the isolated protein (confirmed by SDS-PAGE and mass spectrometry) was collected and its protein content was measured. T. cruzi trypomastigotes and amastigotes, L. amazonensis promastigotes and amastigotes and T. brucei rhodesiense procyclic and bloodstream trypomastigotes were challenged with crovirin, whose toxicity was tested against LLC-MK2 cells, peritoneal macrophages and isolated murine extensor digitorum longus muscle. We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin. Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10–2.38 µg/ml). A considerably higher concentration (20 µg/ml) of crovirin was required to elicit only limited toxicity on mammalian cells. Conclusions This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases. PMID:25330220

The leishmanicidal effect of (3S)-16,17-didehydrofalcarinol, an oxylipin isolated from Tridax procumbens, is independent of NO production.

PubMed

Martín-Quintal, Zhelmy; del Rosario García-Miss, María; Mut-Martín, Mirza; Matus-Moo, Abril; Torres-Tapia, Luis W; Peraza-Sánchez, Sergio R

2010-07-01

The in vitro leishmanicidal effect of (3S)-16,17-didehydrofalcarinol (1) isolated from Tridax procumbens whole plant against Leishmania mexicana, the causative agent of cutaneous leishmaniasis (chiclero's ulcer) in the New World, was investigated. This oxylipin showed significant in vitro activity against promastigotes and intracellular amastigotes of L. mexicana. Its inhibitory effect on amastigotes was not due to activation of NO in recombinant gamma-interferon-stimulated macrophages, since the production of NO was decreased in presence of the oxylipin. This is the first report on the leishmanicidal activity against the intracellular stage (amastigote) of the oxylipin (3S)-16,17-didehydrofalcarinol.

CK2 Secreted by Leishmania braziliensis Mediates Macrophage Association Invasion: A Comparative Study between Virulent and Avirulent Promastigotes.

PubMed

Zylbersztejn, Ana Madeira Brito; de Morais, Carlos Gustavo Vieira; Lima, Ana Karina Castro; Souza, Joyce Eliza de Oliveira; Lopes, Angela Hampshire; Da-Silva, Sílvia Amaral Gonçalves; Silva-Neto, Mário Alberto Cardoso; Dutra, Patrícia Maria Lourenço

2015-01-01

CK2 is a protein kinase distributed in different compartments of Leishmania braziliensis: an externally oriented ecto-CK2, an intracellular CK2, and a secreted CK2. This latter form is constitutively secreted from the parasite (CsCK2), but such secretion may be highly enhanced by the association of specific molecules, including enzyme substrates, which lead to a higher enzymatic activity, called inductively secreted CK2 (IsCK2). Here, we examined the influence of secreted CK2 (sCK2) activity on the infectivity of a virulent L. braziliensis strain. The virulent strain presented 121-fold higher total CK2 activity than those found in an avirulent strain. The use of specific CK2 inhibitors (TBB, DRB, or heparin) inhibited virulent parasite growth, whereas no effect was observed in the avirulent parasites. When these inhibitors were added to the interaction assays between the virulent L. braziliensis strain and macrophages, association index was drastically inhibited. Polyamines enhanced sCK2 activity and increased the association index between parasites and macrophages. Finally, sCK2 and the supernatant of the virulent strain increased the association index between the avirulent strain and macrophages, which was inhibited by TBB. Thus, the kinase enzyme CK2 seems to be important to invasion mechanisms of L. braziliensis.

Digital holographic microscopy as a technique to monitor macrophages infected by leishmania

NASA Astrophysics Data System (ADS)

Mendoza-Rodríguez, E.; Organista-Castelblanco, C.; Camacho, M.; Monroy-Ramírez, F.

2017-06-01

The Digital Holographic Microscopy in Transmission technique (DHM) is considered a useful tool in the noninvasive quantifying of transparent biological objects like living cells. In this work, we propose this technique to study and to monitor control macrophages infected by Leishmania (mouse lineJ774.A1). When the promastigotes enter in contact with healthy macrophages, they got phagocytosed and latterly confined in the formed parasitophorous vacuole. These processes change the morphology and density of the host macrophage. Both parameters can be measured in a label-free analysis of cells with the aid of the DHM technique. Our technique begins with the optical record of the holograms using a modified Mach-Zehnder interferometer and the reconstruction of the complex optical field transmitted by macrophages. In the latter point, we employ the angular spectrum algorithm. With the complex optical field reconstruction, we compute the field amplitude and the phase difference maps, which leads to describe one morphological characterization for the samples. Using phase difference maps is possible to measure internal variations for the integral refractive index, estimating the infection level of macrophages. Through the changes in the integral refractive index, it is also possible to describe and quantify in two different states the evolution of the infection. With these results some parameters of cells have been quantified, making the DHM technique a viable tool for diagnosis of biological samples under the presence of some pathogen.

Leishmanicidal activity of lipophilic extracts of some Hypericum species.

PubMed

Dagnino, Ana Paula; Barros, Francisco Maikon Corrêa de; Ccana-Ccapatinta, Gari Vidal; Prophiro, Josiane Somariva; Poser, Gilsane Lino von; Romão, Pedro R T

2015-01-15

Leishmaniasis has emerged as the third most prevalent parasite-borne disease worldwide after malaria and filariasis, with about 350 million people at risk of infection. Antileishmanial drugs currently available have various limitations, mainly because of the parasite resistance and side effects. The search of new antileishmanial drugs is ventured throughout the world. The purpose of this study was to assess the leishmanicidal activity of lipophilic extracts of eight Hypericum species against promastigote forms of Leishmania (Leishmania) amazonensis. The dried and powered materials of aerial parts of H. andinum Gleason, H. brevistylum Choisy, H. caprifoliatum Cham. & Schltdl., H. carinatum Griseb., H. linoides A. St.-Hil., H. myrianthum Cham. & Schltdl., H. polyanthemum Klotzsch ex Reichardt and H. silenoides Juss. were extracted by static maceration with n-hexane. Extracts were evaporated to dryness under reduced pressure and stored at -20°C until biological evaluation and HPLC analysis. The metabolites investigated were dimeric phloroglucinol derivatives, benzophenones and benzopyrans. The yields were expressed as mean of three injections in mg of compound per g of extract (mg/g extract). The effect of Hypericum species on the viability of infective forms of L. (L.) amazonensis was determined using a hemocytometer. Amphotericin B was used as a standard drug. The 50% inhibitory concentration (IC50) values for each extract were determined by linear regression analysis. The cytotoxic effects of extracts were assessed on peritoneal macrophages of BALB/c mice by MTT assay. The concentration that causes 50% of macrophage cytotoxicity (CC50) was determined by linear regression analysis. The selectivity index (SI) of the extracts was determined considering the following equation: CC50 against mammalian cells/IC50 against L. amazonensis. We demonstrated that H. carinatum, H. linoides and H. polyanthemum were able to kill the parasites in a dose dependent manner. These extracts presented low cytotoxicity against murine macrophages. At 48h of incubation H. polyanthemum presented significant leishmanicidal activity with a 50% inhibitory concentration (IC50) of 36.1µg/ml. The leishmanicidal activity of H. myrianthum was significantly lower than that presented by H. polyanthemum, H. carinatum and H. linoides extracts. H. brevistylum and H. caprifoliatum showed significant leishmanicidal activity only at high concentrations (500 and 1000µg/ml), while H. andinum and H. silenoides were ineffective. The promising results demonstrate the importance of the species of the genus Hypericum as source of compounds potentially useful for the treatment of leishmaniasis. Copyright © 2014 Elsevier GmbH. All rights reserved.

Supplementation of host response by targeting nitric oxide to the macrophage cytosol is efficacious in the hamster model of visceral leishmaniasis and adds to efficacy of amphotericin B.

PubMed

Pandya, Sanketkumar; Verma, Rahul Kumar; Khare, Prashant; Tiwari, Brajendra; Srinivasarao, Dadi A; Dube, Anuradha; Goyal, Neena; Misra, Amit

2016-08-01

We investigated efficacy of nitric oxide (NO) against Leishmania donovani. NO is a mediator of host response to infection, with direct parasiticidal activity in addition to its role in signalling to evoke innate macrophage responses. However, it is short-lived and volatile, and is therefore difficult to introduce into infected cells and maintain inracellular concentrations for meaningful periods of time. We incorporated diethylenetriamine NO adduct (DETA/NO), a prodrug, into poly(lactide-co-glycolide) particles of ∼200 nm, with or without amphotericin B (AMB). These particles sustained NO levels in mouse macrophage culture supernatants, generating an area under curve (AUC0.08-24h) of 591.2 ± 95.1 mM × h. Free DETA/NO resulted in NO peaking at 3 h and declining rapidly to yield an AUC of 462.5 ± 193.4. Particles containing AMB and DETA/NO were able to kill ∼98% of promastigotes and ∼76% of amastigotes in 12 h when tested in vitro. Promastigotes and amastigotes were killed less efficiently by particles containing a single drug- either DETA/NO (∼42%, 35%) or AMB (∼90%, 50%) alone, or by equivalent concentrations of drugs in solution. In a pre-clinical efficacy study of power >0.95 in the hamster model, DETA/NO particles were non-inferior to Fungizone® but not Ambisome®, resulting in significant (∼73%) reduction in spleen parasites in 7 days. Particles containing both DETA/NO and AMB were superior (∼93% reduction) to Ambisome®. We conclude that NO delivered to the cytosol of macrophages infected with Leishmania possesses intrinsic activity and adds significantly to the efficacy of AMB. Copyright © 2016. Published by Elsevier Ltd.

Different host complement systems and their interactions with saliva from Lutzomyia longipalpis (Diptera, Psychodidae) and Leishmania infantum promastigotes.

PubMed

Mendes-Sousa, Antonio Ferreira; Nascimento, Alexandre Alves Sousa; Queiroz, Daniel Costa; Vale, Vladimir Fazito; Fujiwara, Ricardo Toshio; Araújo, Ricardo Nascimento; Pereira, Marcos Horácio; Gontijo, Nelder Figueiredo

2013-01-01

Lutzomyia longipalpis is the vector of Leishmania infantum in the New World, and its saliva inhibits classical and alternative human complement system pathways. This inhibition is important in protecting the insect´s midgut from damage by the complement. L. longipalpis is a promiscuous blood feeder and must be protected against its host's complement. The objective of this study was to investigate the action of salivary complement inhibitors on the sera of different host species, such as dogs, guinea pigs, rats and chickens, at a pH of 7.4 (normal blood pH) and 8.15 (the midgut pH immediately after a blood meal). We also investigated the role of the chicken complement system in Leishmania clearance in the presence and absence of vector saliva. The saliva was capable of inhibiting classical pathways in dogs, guinea pigs and rats at both pHs. The alternative pathway was not inhibited except in dogs at a pH of 8.15. The chicken classical pathway was inhibited only by high concentrations of saliva and it was better inhibited by the midgut contents of sand flies. Neither the saliva nor the midgut contents had any effect on the avian alternative pathway. Fowl sera killed L. infantum promastigotes, even at a low concentration (2%), and the addition of L. longipalpis saliva did not protect the parasites. The high body temperature of chickens (40°C) had no effect on Leishmania viability during our assays. Salivary inhibitors act in a species-specific manner. It is important to determine their effects in the natural hosts of Leishmania infantum because they act on canid and rodent complements but not on chickens (which do not harbour the parasite). Moreover, we concluded that the avian complement system is the probable mechanism through which chickens eliminate Leishmania and that their high body temperature does not influence this parasite.

Interaction between cysteine synthase and serine O-acetyltransferase proteins and their stage specific expression in Leishmania donovani.

PubMed

Singh, Kuljit; Singh, Krishn Pratap; Equbal, Asif; Suman, Shashi S; Zaidi, Amir; Garg, Gaurav; Pandey, Krishna; Das, Pradeep; Ali, Vahab

2016-12-01

Leishmania possess a unique trypanothione redox metabolism with undebated roles in protection from oxidative damage and drug resistance. The biosynthesis of trypanothione depends on l-cysteine bioavailability which is regulated by cysteine biosynthesis pathway. The de novo cysteine biosynthesis pathway is comprised of serine O-acetyltransferase (SAT) and cysteine synthase (CS) enzymes which sequentially mediate two consecutive steps of cysteine biosynthesis, and is absent in mammalian host. However, despite the apparent dependency of redox metabolism on cysteine biosynthesis pathway, the role of SAT and CS in redox homeostasis has been unexplored in Leishmania parasites. Herein, we have characterized CS and SAT to investigate their interaction and relative abundance of these proteins in promastigote vs. amastigote growth stages of L. donovani. CS and SAT genes of L. donovani (LdCS and LdSAT) were cloned, expressed, and fusion proteins purified to homogeneity with affinity column chromatography. Purified LdCS contains PLP as cofactor and showed optimum enzymatic activity at pH 7.5. Enzyme kinetics showed that LdCS catalyses the synthesis of cysteine using O-acetylserine and sulfide with a K m of 15.86 mM and 0.17 mM, respectively. Digitonin fractionation and indirect immunofluorescence microscopy showed that LdCS and LdSAT are localized in the cytoplasm of promastigotes. Size exclusion chromatography, co-purification, pull down and immuno-precipitation assays demonstrated a stable complex formation between LdCS and LdSAT proteins. Furthermore, LdCS and LdSAT proteins expression/activity was upregulated in amastigote growth stage of the parasite. Thus, the stage specific differential expression of LdCS and LdSAT suggests that it may have a role in the redox homeostasis of Leishmania. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Histological and Immunological Description of the Leishmanin Skin Test in Ibizan Hounds.

PubMed

Ordeix, L; Silva, J E Dos S; Llull, J; Quirola, P; Montserrat-Sangrà, S; Martínez-Orellana, P; Solano-Gallego, L

2018-01-01

The leishmanin skin test (LST), a delayed-type hypersensitivity (DTH) reaction to Leishmania infantum, can specifically identify dogs that have made a cell-mediated immune response to L. infantum infection. The Ibizan hound appears to be more resistant to L. infantum infection than other breeds of dog. The aim of this study was to describe the histological and immunohistochemical changes induced by the LST in Ibizan hounds living in an area highly endemic for leishmaniosis. The majority of dogs were apparently healthy, lacked serum antibody to L. infantum and blood parasitaemia, but had marked specific interferon gamma production after in-vitro blood stimulation with L. infantum. Leishmanin (3 × 10 8 killed promastigotes of L. infantum/ml) was injected intradermally and biopsy samples were obtained from a positive reaction at 72 h from nine Ibizan hounds. A moderate to intense, perivascular to interstitial dermatitis and panniculitis characterized the inflammatory response at the injection site. In addition, three samples had diffuse inflammation in the deep dermis and panniculus. Oedema and necrosis were present in the deep dermis and panniculus. Congestion and haemorrhage were observed in five biopsies. T lymphocytes (CD3 + ) and large mononuclear cells (lysozyme - ) were the most prevalent cells. CD3 + cells were significantly more numerous than CD20 + B cells and lysozyme + cells. B cells were sparsely distributed, especially in the deep dermis and panniculus. Rare neutrophils and macrophages (lysozyme + ) were observed with few eosinophils. Toll-like receptor (TLR)-2 protein was expressed in large mononuclear cells mainly located in the superficial dermis. Leishmania immunohistochemistry was negative and quantitative polymerase chain reaction was positive in all cases. The intradermal injection of killed L. infantum promastigotes in Ibizan hounds causes similar histological and immunohistochemical findings to those described for human subjects and are indicative of a DTH response. Moreover, TLR2 protein is expressed in inflammatory cells similar to findings in clinically affected skin biopsy samples. Copyright © 2017 Elsevier Ltd. All rights reserved.

Leishmanicidal Activity of Piper nigrum Bioactive Fractions is Interceded via Apoptosis In Vitro and Substantiated by Th1 Immunostimulatory Potential In Vivo.

PubMed

Chouhan, Garima; Islamuddin, Mohammad; Want, Muzamil Y; Ozbak, Hani A; Hemeg, Hassan A; Sahal, Dinkar; Afrin, Farhat

2015-01-01

Visceral leishmaniasis (VL) is a life-threatening protozoal infection chiefly impinging the rural and poor population in the tropical and sub-tropical countries. The deadly affliction is rapidly expanding after its association with AIDS, swiftly defying its status of a neglected disease. Despite successful formulation of vaccine against canine leishmaniasis, no licensed vaccine is yet available for human VL, chemotherapy is in appalling state, and the development of new candidate drugs has been painfully slow. In face of lack of proper incentives, immunostimulatory plant preparations owing antileishmanial efficacy bear potential to rejuvenate awful antileishmanial chemotherapy. We have earlier reported profound leishmanicidal activity of Piper nigrum hexane (PNH) seeds and P. nigrum ethanolic (PNE) fractions derived from P. nigrum seeds against Leishmania donovani promastigotes and amastigotes. In the present study, we illustrate that the remarkable anti-promastigote activity exhibited by PNH and PNE is mediated via apoptosis as evidenced by phosphatidylserine externalization, DNA fragmentation, arrest in sub G0/G1 phase, loss of mitochondrial membrane potential and generation of reactive oxygen species. Further, P. nigrum bioactive fractions rendered significant protection to L. donovani infected BALB/c mice in comparison to piperine, a known compound present in Piper species. The substantial therapeutic potential of PNH and PNE was accompanied by elicitation of cell-mediated immune response. The bioactive fractions elevated the secretion of Th1 (INF-γ, TNF-α, and IL-2) cytokines and declined IL-4 and IL-10. PNH and PNE enhanced the production of IgG2a, upregulated the expression of co-stimulatory molecules CD80 and CD86, augmented splenic CD4(+) and CD8(+) T cell population, induced strong lymphoproliferative and DTH responses and partially stimulated NO production. PNH and PNE were devoid of any hepatic or renal toxicity. These encouraging findings merit further exploration of P. nigrum bioactive fractions as a source of potent and non-toxic antileishmanials.

Leishmanicidal Activity of Piper nigrum Bioactive Fractions is Interceded via Apoptosis In Vitro and Substantiated by Th1 Immunostimulatory Potential In Vivo

PubMed Central

Chouhan, Garima; Islamuddin, Mohammad; Want, Muzamil Y.; Ozbak, Hani A.; Hemeg, Hassan A.; Sahal, Dinkar; Afrin, Farhat

2015-01-01

Visceral leishmaniasis (VL) is a life-threatening protozoal infection chiefly impinging the rural and poor population in the tropical and sub-tropical countries. The deadly affliction is rapidly expanding after its association with AIDS, swiftly defying its status of a neglected disease. Despite successful formulation of vaccine against canine leishmaniasis, no licensed vaccine is yet available for human VL, chemotherapy is in appalling state, and the development of new candidate drugs has been painfully slow. In face of lack of proper incentives, immunostimulatory plant preparations owing antileishmanial efficacy bear potential to rejuvenate awful antileishmanial chemotherapy. We have earlier reported profound leishmanicidal activity of Piper nigrum hexane (PNH) seeds and P. nigrum ethanolic (PNE) fractions derived from P. nigrum seeds against Leishmania donovani promastigotes and amastigotes. In the present study, we illustrate that the remarkable anti-promastigote activity exhibited by PNH and PNE is mediated via apoptosis as evidenced by phosphatidylserine externalization, DNA fragmentation, arrest in sub G0/G1 phase, loss of mitochondrial membrane potential and generation of reactive oxygen species. Further, P. nigrum bioactive fractions rendered significant protection to L. donovani infected BALB/c mice in comparison to piperine, a known compound present in Piper species. The substantial therapeutic potential of PNH and PNE was accompanied by elicitation of cell-mediated immune response. The bioactive fractions elevated the secretion of Th1 (INF-γ, TNF-α, and IL-2) cytokines and declined IL-4 and IL-10. PNH and PNE enhanced the production of IgG2a, upregulated the expression of co-stimulatory molecules CD80 and CD86, augmented splenic CD4+ and CD8+ T cell population, induced strong lymphoproliferative and DTH responses and partially stimulated NO production. PNH and PNE were devoid of any hepatic or renal toxicity. These encouraging findings merit further exploration of P. nigrum bioactive fractions as a source of potent and non-toxic antileishmanials. PMID:26696979

Treatment Failure and Miltefosine Susceptibility in Dermal Leishmaniasis Caused by Leishmania Subgenus Viannia Species

PubMed Central

Obonaga, Ricardo; Fernández, Olga Lucía; Valderrama, Liliana; Rubiano, Luisa Consuelo; Castro, Maria del Mar; Barrera, Maria Claudia; Gomez, Maria Adelaida

2014-01-01

Treatment failure and parasite drug susceptibility in dermal leishmaniasis caused by Leishmania (Viannia) species are poorly understood. Prospective evaluation of drug susceptibility of strains isolated from individual patients before drug exposure and at clinical failure allows intrinsic and acquired differences in susceptibility to be discerned and analyzed. To determine whether intrinsic susceptibility or loss of susceptibility to miltefosine contributed to treatment failure, we evaluated the miltefosine susceptibility of intracellular amastigotes and promastigotes of six Leishmania (Viannia) braziliensis and six Leishmania (Viannia) panamensis strains isolated sequentially, at diagnosis and treatment failure, from two children and four adults ≥55 years old with concurrent conditions. Four patients presented only cutaneous lesions, one had mucosal disease, and one had disseminated mucocutaneous disease. Expression of the Leishmania drug transporter genes abca2, abca3, abcc2, abcc3, abcg4, abcg6, and LbMT was evaluated by quantitative reverse transcription-PCR (qRT-PCR). Intracellular amastigotes (median 50% effective concentration [EC50], 10.7 μmol/liter) were more susceptible to miltefosine than promastigotes (median EC50, 55.3 μmol/liter) (P < 0.0001). Loss of susceptibility at failure, demonstrated by a miltefosine EC50 of >32 μmol/liter (the upper limit of intracellular amastigote assay), occurred in L. panamensis infection in a child and in L. braziliensis infection in an adult and was accompanied by decreased expression of the miltefosine transporter LbMT (LbMT/β-tubulin, 0.42- to 0.26-fold [P = 0.039] and 0.70- to 0.57-fold [P = 0.009], respectively). LbMT gene polymorphisms were not associated with susceptibility phenotype. Leishmania ABCA3 transporter expression was inversely correlated with miltefosine susceptibility (r = −0.605; P = 0.037). Loss of susceptibility is one of multiple factors involved in failure of miltefosine treatment in dermal leishmaniasis. PMID:24145529

Studies on the CPA cysteine peptidase in the Leishmania infantum genome strain JPCM5.

PubMed

Denise, Hubert; Poot, Jacqueline; Jiménez, Maribel; Ambit, Audrey; Herrmann, Daland C; Vermeulen, Arno N; Coombs, Graham H; Mottram, Jeremy C

2006-11-13

Visceral leishmaniasis caused by members of the Leishmania donovani complex is often fatal in the absence of treatment. Research has been hampered by the lack of good laboratory models and tools for genetic manipulation. In this study, we have characterised a L. infantum line (JPCM5) that was isolated from a naturally infected dog and then cloned. We found that JPCM5 has attributes that make it an excellent laboratory model; different stages of the parasite life cycle can be studied in vitro, it is accessible to genetic manipulation and it has retained its virulence. Furthermore, the L. infantum JPCM5 genome has now been fully sequenced. We have further focused our studies on LiCPA, the L. infantum homologue to L. mexicana cysteine peptidase CPA. LiCPA was found to share a high percentage of amino acid identity with CPA proteins of other Leishmania species. Two independent LiCPA-deficient promastigote clones (DeltaLicpa) were generated and their phenotype characterised. In contrast to L. mexicana CPA-deficient mutants, both clones of DeltaLicpa were found to have significantly reduced virulence in vitro and in vivo. Re-expression of just one LiCPA allele (giving DeltaLicpa::CPA) was sufficient to complement the reduced infectivity of both DeltaLicpa mutants for human macrophages, which confirms the importance of LiCPA for L. infantum virulence. In contrast, in vivo experiments did not show any virulence recovery of the re-expressor clone DeltaLicpaC1::CPA compared with the CPA-deficient mutant DeltaLicpaC1. The data suggest that CPA is not essential for replication of L. infantum promastigotes, but is important for the host-parasite interaction. Further studies will be necessary to elucidate the precise roles that LiCPA plays and why the re-expression of LiCPA in the DeltaLicpa mutants complemented the gene deletion phenotype only in in vitro and not in in vivo infection of hamsters.

Genetic Manipulation of Leishmania donovani to Explore the Involvement of Argininosuccinate Synthase in Oxidative Stress Management

PubMed Central

Sardar, Abul Hasan; Jardim, Armando; Ghosh, Ayan Kumar; Mandal, Abhishek; Das, Sushmita; Saini, Savita; Abhishek, Kumar; Singh, Ruby; Verma, Sudha; Kumar, Ajay; Das, Pradeep

2016-01-01

Reactive oxygen and nitrogen species (ROS and RNS) produced by the phagocytic cells are the most common arsenals used to kill the intracellular pathogens. However, Leishmania, an intracellular pathogen, has evolved mechanisms to survive by counterbalancing the toxic oxygen metabolites produced during infection. Polyamines, the major contributor in this anti-oxidant machinery, are largely dependent on the availability of L-arginine in the intracellular milieu. Argininosuccinate synthase (ASS) plays an important role as the rate-limiting step required for converting L-citrulline to argininosuccinate to provide arginine for an assortment of metabolic processes. Leishmania produce an active ASS enzyme, yet it has an incomplete urea cycle as it lacks an argininosuccinate lyase (ASL). There is no evidence for endogenous synthesis of L-arginine in Leishmania, which suggests that these parasites salvage L-arginine from extracellular milieu and makes the biological function of ASS and the production of argininosuccinate in Leishmania unclear. Our previous quantitative proteomic analysis of Leishmania promastigotes treated with sub-lethal doses of ROS, RNS, or a combination of both, led to the identification of several differentially expressed proteins which included ASS. To assess the involvement of ASS in stress management, a mutant cell line with greatly reduced ASS activity was created by a double-targeted gene replacement strategy in L. donovani promastigote. Interestingly, LdASS is encoded by three copies of allele, but Western blot analysis showed the third allele did not appear to express ASS. The free thiol levels in the mutant LdASS-/-/+ cell line were decreased. Furthermore, the cell viability in L-arginine depleted medium was greatly attenuated on exposure to different stress environments and was adversely impacted in its ability to infect mice. These findings suggest that ASS is important for Leishmania donovani to counterbalance the stressed environments encountered during infection and can be targeted for chemotherapeutic purpose to treat visceral leishmaniasis. PMID:26939071

Design of copper DNA intercalators with leishmanicidal activity.

PubMed

Navarro, Maribel; Cisneros-Fajardo, Efrén José; Sierralta, Aníbal; Fernández-Mestre, Mercedes; Silva, Pedro; Arrieche, Dwight; Marchán, Edgar

2003-04-01

The complexes [Cu(dppz)(NO(3))]NO(3) (1), [Cu(dppz)(2)(NO(3))]NO(3) (2), [Cu(dpq)(NO(3))]NO(3) (3), and [Cu(dpq)(2)(NO(3))]NO(3) (4) were synthesized and characterized by elemental analysis, FAB-mass spectrometry, EPR, UV, and IR spectroscopies, and molar conductivity. DNA interaction studies showed that intercalation is an important way of interacting with DNA for these complexes. The biological activity of these copper complexes was evaluated on Leishmania braziliensis promastigotes, and the results showed leishmanicidal activity. Preliminary ultrastructural studies with the most active complex (2) at 1 h revealed parasite swelling and binucleated cells. This finding suggests that the leishmanicidal activity of the copper complexes could be associated with their interaction with the parasitic DNA.

Interaction of ascaridole, carvacrol, and caryophyllene oxide from essential oil of Chenopodium ambrosioides L. with mitochondria in Leishmania and other eukaryotes.

PubMed

Monzote, Lianet; Geroldinger, Gerald; Tonner, Matthias; Scull, Ramón; De Sarkar, Sritama; Bergmann, Sophie; Bacher, Markus; Staniek, Katrin; Chatterjee, Mitali; Rosenau, Thomas; Gille, Lars

2018-04-19

The antileishmanial activity of the essential oil (EO) from Chenopodium ambrosioides L. has been demonstrated in vitro and in animal models, attributed to the major components of the EO. This study focused on the effects of the three major EO compounds carvacrol, caryophyllene oxide (Caryo), and the antileishmanial endoperoxide ascaridole (Asc) on mitochondrial functions in Leishmania tarentolae promastigotes (LtP). EO and Caryo were able to partially inhibit the leishmanial electron transport chain, whereas other components failed to demonstrate a direct immediate effect. Caryo demonstrated inhibition of complex III activity in LtP and in isolated complex III from other species. The formation of superoxide radicals was studied in Leishmania by electron spin resonance spectroscopy in the presence of iron chelators wherein selected compounds failed to trigger a significant immediate additional superoxide production in LtP. However, upon prolonged incubation of Leishmania with Asc and especially in the absence of iron chelators (allowing the activation of Asc), an increased superoxide radical production and significant impairment of mitochondrial coupling in Leishmania was observed. Prolonged incubation with all EO components resulted in thiol depletion. Taken together, the major components of EO mediate their leishmanicidal activity via different mitochondrial targets and time profiles. Further studies are required to elucidate possible synergistic effects of carvacrol and Asc and the influence of minor compounds. © 2018 The Authors Phytotherapy Research Published by John Wiley & Sons Ltd.

Antibacterial and leishmanicidal activities of temporin-SHd, a 17-residue long membrane-damaging peptide.

PubMed

Abbassi, Feten; Raja, Zahid; Oury, Bruno; Gazanion, Elodie; Piesse, Christophe; Sereno, Denis; Nicolas, Pierre; Foulon, Thierry; Ladram, Ali

2013-02-01

Temporins are a family of short antimicrobial peptides (8-17 residues) that mostly show potent activity against Gram-positive bacteria. Herein, we demonstrate that temporin-SHd, a 17-residue peptide with a net charge of +2 (FLPAALAGIGGILGKLF(amide)), expressed a broad spectrum of antimicrobial activity. This peptide displayed potent antibacterial activities against Gram-negative and Gram-positive bacteria, including multi-drug resistant Staphylococcus aureus strains, as well as antiparasitic activity against promastigote and the intracellular stage (amastigote) of Leishmania infantum, at concentration not toxic for the macrophages. Temporin-SHd that is structured in a non-amphipathic α-helix in anionic membrane-mimetic environments, strongly and selectively perturbs anionic bilayer membranes by interacting with the polar head groups and acyl region of the phospholipids, with formation of regions of two coexisting phases: one phase rich in peptide and the other lipid-rich. The disruption of lipid packing within the bilayer may lead to the formation of transient pores and membrane permeation/disruption once a threshold peptide accumulation is reached. To our knowledge, Temporin-SHd represents the first known 17-residue long temporin expressing such broad spectrum of antimicrobial activity including members of the trypanosomatidae family. Additionally, since only a few shorter members (13 residues) of the temporin family are known to display antileishmanial activity (temporins-TA, -TB and -SHa), SHd is an interesting tool to analyze the antiparasitic mechanism of action of temporins. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

The protective immune response produced in dogs after primary vaccination with the LiESP/QA-21 vaccine (CaniLeish®) remains effective against an experimental challenge one year later

PubMed Central

2014-01-01

Control of canine leishmaniasis is an important objective for the benefit of dogs living in or visiting endemic areas and for public health because of the zoonotic nature of this disease. Resistance or susceptibility to developing canine leishmaniasis after exposure to Leishmania infantum is primarily determined by the ability of the immune system to develop an appropriate Th1-dominated specific response to the parasite. For this reason there is a need for effective canine vaccines that can decrease the number of dogs developing progressive infections. In this study, we followed the impact of the LiESP/QA-21 canine vaccine (composed of excreted-secreted proteins of L. infantum and the QA-21 saponin adjuvant), recently launched commercially in Europe, on selected humoral and cellular immune parameters following an infectious intravenous challenge with L. infantum promastigotes administered one year after the primary vaccine course. We also followed parasitological parameters to determine the parasitological status of the challenged dogs. In contrast to controls, vaccinated dogs retained significantly stronger cell-mediated immune responses against the parasite despite a virulent challenge and had significantly lower mean parasite burdens at the end of the study, associated with a lower probability of developing active infections. These results confirm that the immune responses generated by vaccination with LiESP/QA-21 are still effective against an intravenous challenge one year after the primary vaccine course. PMID:24964736

Pathogenicity of Leishmania donovani is associated with the high expression of a group low molecular weight proteins

PubMed Central

Mitra, Partha

2015-01-01

Background: With few exceptions, members of the Leishmania donovani complex such as L. donovani, L. infantum and L. chagashi are the etiological agents of visceral leishmaniasis or kala-azar. Promastigotes of Leishmania spp. lose their Pathogenicity; the ability to establish infection in a susceptible host, after prolonged culture. The molecular basis of this evolution of pathogenic to nonpathogenic culture has not been very well understood. It has been proposed that the loss of pathogenicity is associated with the gradual disappearance of selective parasite proteins. An alternative hypothesis is that during prolonged culture, the pathogenic clonal population of the parasite is deleted from the mixed population due to their selection pressure. This clonal deletion is proposed to be responsible for the emergence of the nonpathogenic population. Study Methodology and Results: We have a done a series of two-dimensional polyacrylamide gel electrophoresis followed by western blot experiments to study the antigenic profile of few L. donovani isolates of Indian origin. We observed a gradual and significant downregulation of expression of a group of low molecular weight proteins (LMW, molecular weight 20–30 kDa) which are associated with loss of pathogenicity. These proteins are recognized only by antiserum raised against the whole cell extract of one of the pathogenic Indian L. donovani isolates, Ag83, and remained undetected by antiserum raised against the nonpathogenic AG83 isolates. These LMW proteins were also present in the nonpathogenic extract in very low levels and remained undetected by the virulent serum, indicating a phenomenon of simultaneous downregulation of the expression and altered immunogenicity. LMW proteins were universally expressed in all early passage Indian isolate we tested and also detected in two clones obtained from pathogenic parasite culture. The antigenic patterns of none of the eight clones obtained from nonpathogenic culture were not exactly similar with the pathogenic clones. Conclusion: Therefore, our data strongly support the hypothesis that the loss of pathogenicity of L. donovani is associated with a change in antigenic profile, but not due the selective deletion of pathogenic clones. PMID:26629453

Pathogenicity of Leishmania donovani is associated with the high expression of a group low molecular weight proteins.

PubMed

Mitra, Partha

2015-01-01

With few exceptions, members of the Leishmania donovani complex such as L. donovani, L. infantum and L. chagashi are the etiological agents of visceral leishmaniasis or kala-azar. Promastigotes of Leishmania spp. lose their Pathogenicity; the ability to establish infection in a susceptible host, after prolonged culture. The molecular basis of this evolution of pathogenic to nonpathogenic culture has not been very well understood. It has been proposed that the loss of pathogenicity is associated with the gradual disappearance of selective parasite proteins. An alternative hypothesis is that during prolonged culture, the pathogenic clonal population of the parasite is deleted from the mixed population due to their selection pressure. This clonal deletion is proposed to be responsible for the emergence of the nonpathogenic population. We have a done a series of two-dimensional polyacrylamide gel electrophoresis followed by western blot experiments to study the antigenic profile of few L. donovani isolates of Indian origin. We observed a gradual and significant downregulation of expression of a group of low molecular weight proteins (LMW, molecular weight 20-30 kDa) which are associated with loss of pathogenicity. These proteins are recognized only by antiserum raised against the whole cell extract of one of the pathogenic Indian L. donovani isolates, Ag83, and remained undetected by antiserum raised against the nonpathogenic AG83 isolates. These LMW proteins were also present in the nonpathogenic extract in very low levels and remained undetected by the virulent serum, indicating a phenomenon of simultaneous downregulation of the expression and altered immunogenicity. LMW proteins were universally expressed in all early passage Indian isolate we tested and also detected in two clones obtained from pathogenic parasite culture. The antigenic patterns of none of the eight clones obtained from nonpathogenic culture were not exactly similar with the pathogenic clones. Therefore, our data strongly support the hypothesis that the loss of pathogenicity of L. donovani is associated with a change in antigenic profile, but not due the selective deletion of pathogenic clones.

AMP-acetyl CoA synthetase from Leishmania donovani: identification and functional analysis of 'PX4GK' motif.

PubMed

Soumya, Neelagiri; Kumar, I Sravan; Shivaprasad, S; Gorakh, Landage Nitin; Dinesh, Neeradi; Swamy, Kayala Kambagiri; Singh, Sushma

2015-04-01

An adenosine monophosphate forming acetyl CoA synthetase (AceCS) which is the key enzyme involved in the conversion of acetate to acetyl CoA has been identified from Leishmania donovani for the first time. Sequence analysis of L. donovani AceCS (LdAceCS) revealed the presence of a 'PX4GK' motif which is highly conserved throughout organisms with higher sequence identity (96%) to lower sequence identity (38%). A ∼ 77 kDa heterologous protein with C-terminal 6X His-tag was expressed in Escherichia coli. Expression of LdAceCS in promastigotes was confirmed by western blot and RT-PCR analysis. Immunolocalization studies revealed that it is a cytosolic protein. We also report the kinetic characterization of recombinant LdAceCS with acetate, adenosine 5'-triphosphate, coenzyme A and propionate as substrates. Site directed mutagenesis of residues in conserved PX4GK motif of LdAceCS was performed to gain insight into its potential role in substrate binding, catalysis and its role in maintaining structural integrity of the protein. P646A, G651A and K652R exhibited more than 90% loss in activity signifying its indispensible role in the enzyme activity. Substitution of other residues in this motif resulted in altered substrate specificity and catalysis. However, none of them had any role in modulation of the secondary structure of the protein except G651A mutant. Copyright © 2015 Elsevier B.V. All rights reserved.

Expression and purification of a new recombinant camel hepcidin able to promote the degradation of the iron exporter ferroportin1.

PubMed

Boumaiza, Mohamed; Jaouen, Maryse; Deschemin, Jean-Christophe; Ezzine, Aymen; Ben Khalaf, Noureddine; Vaulont, Sophie; Marzouki, Mohamed Nèjib; Sari, Marie Agnès

2015-11-01

Hepcidin, a 25-amino-acid and highly disulfide bonded antimicrobial peptide, is the central regulator of iron homeostasis. This hormone is expressed in response to iron and inflammation and interacts with ferroportin1 (FPN1), the only known iron exporter in vertebrates, inducing its internalization and degradation. Thus, the export of iron from cells to plasma will be significantly diminished. Thereby, hepcidin has become the target of intense research studies due to its profound biomedical significance. This study describes the functional expression of recombinant camel hepcidin in Escherichia coli. Biologically active recombinant camel hepcidin was obtained thanks to the production of a hepcidin-thioredoxin fusion protein (TRX-HepcD) and a purified camel hepcidin, with an extra methionine at the N-terminus, was obtained after enterokinase cleavage of the fusion protein. Presence of the four disulfide bridges was verified using MALDI-ToF spectrometry. The recombinant camel hepcidin was compared to related synthetic bioactive peptides, including human hepcidin, and was found equally able to promote ferroportin degradation of mouse macrophages. Furthermore, camel hepcidins exhibits a high capacity to inhibit the growth of Leishmania major promastigotes. These results proved that production of functional camel hepcidin can be achieved in E. coli, this is a major interest for the production of cysteine rich peptides or proteins that can be purified under their functional form without the need of a refolding process. Copyright © 2015 Elsevier Inc. All rights reserved.

Purification and biochemical characterization of three myotoxins from Bothrops mattogrossensis snake venom with toxicity against Leishmania and tumor cells.

PubMed

de Moura, Andréa A; Kayano, Anderson M; Oliveira, George A; Setúbal, Sulamita S; Ribeiro, João G; Barros, Neuza B; Nicolete, Roberto; Moura, Laura A; Fuly, Andre L; Nomizo, Auro; da Silva, Saulo L; Fernandes, Carla F C; Zuliani, Juliana P; Stábeli, Rodrigo G; Soares, Andreimar M; Calderon, Leonardo A

2014-01-01

Bothrops mattogrossensis snake is widely distributed throughout eastern South America and is responsible for snakebites in this region. This paper reports the purification and biochemical characterization of three new phospholipases A2 (PLA2s), one of which is presumably an enzymatically active Asp49 and two are very likely enzymatically inactive Lys49 PLA2 homologues. The purification was obtained after two chromatographic steps on ion exchange and reverse phase column. The 2D SDS-PAGE analysis revealed that the proteins have pI values around 10, are each made of a single chain, and have molecular masses near 13 kDa, which was confirmed by MALDI-TOF mass spectrometry. The N-terminal similarity analysis of the sequences showed that the proteins are highly homologous with other Lys49 and Asp49 PLA2s from Bothrops species. The PLA2s isolated were named BmatTX-I (Lys49 PLA2-like), BmatTX-II (Lys49 PLA2-like), and BmatTX-III (Asp49 PLA2). The PLA2s induced cytokine release from mouse neutrophils and showed cytotoxicity towards JURKAT (leukemia T) and SK-BR-3 (breast adenocarcinoma) cell lines and promastigote forms of Leishmania amazonensis. The structural and functional elucidation of snake venoms components may contribute to a better understanding of the mechanism of action of these proteins during envenomation and their potential pharmacological and therapeutic applications.

Comparison between three adjuvants for a vaccine against canine leishmaniasis: In vitro evaluation of macrophage killing ability.

PubMed

Trotta, T; Fasanella, A; Scaltrito, D; Gradoni, L; Mitolo, V; Brandonisio, O; Acquafredda, A; Panaro, M A

2010-03-01

The aim of this study was to evaluate, in terms of dog macrophage killing ability in vitro, a vaccine based on Leishmania infantum promastigote soluble antigen (LSA) formulated with three different adjuvants (BCG, AdjuPrime, MPL/TDM/CWS). A significant increase of the macrophage killing ability was observed in dogs vaccinated with LSA+MPL/TDM/CWS after 1 month from vaccination. A similar increase of macrophage parasitocidal ability was present only after 5 months in dogs vaccinated with LSA+BCG or LSA+AdjuPrime. In all dogs the augmented killing percentage was still present after 12 months from vaccination. Therefore, in particular LSA+MPL/TDM/CWS vaccine seems promising for further studies in dogs. 2009 Elsevier Ltd. All rights reserved.

In Vitro Activities of Hexaazatrinaphthylenes against Leishmania spp.

PubMed Central

García-Velázquez, Daniel; Martín-Navarro, Carmen M.; Sifaoui, Ines; Reyes-Batlle, María; Lorenzo-Morales, Jacob; Gutiérrez-Ravelo, Ángel; Piñero, José E.

2015-01-01

The in vitro activity of a novel group of compounds, hexaazatrinaphthylene derivatives, against two species of Leishmania is described in this study. These compounds showed a significant dose-dependent inhibition effect on the proliferation of the parasites, with 50% inhibitory concentrations (IC50s) ranging from 1.23 to 25.05 μM against the promastigote stage and 0.5 to 0.7 μM against intracellular amastigotes. Also, a cytotoxicity assay was carried out to in order to evaluate the possible toxic effects of these compounds. Moreover, different assays were performed to determine the type of cell death induced after incubation with these compounds. The obtained results highlight the potential use of hexaazatrinaphthylene derivatives against Leishmania species, and further studies should be undertaken to establish them as novel leishmanicidal therapeutic agents. PMID:25753635

Cytotoxicity of three new triazolo-pyrimidine derivatives against the plant trypanosomatid: Phytomonas sp. isolated from Euphorbia characias.

PubMed

Magán, Rosa; Marín, Clotilde; Salas, Juan M; Barrera-Pérez, Mario; Rosales, Maria J; Sánchez-Moreno, Manuel

2004-10-01

There is no effective chemotherapy against diseases caused by Phytomonas sp., a plant trypanosomatid responsible for economic losses in major crops. We tested three triazolo-pyrimidine complexes [two with Pt(II), and another with Ru(III)] against promastigotes of Phytomonas sp. isolated from Euphorbia characias. The incorporation of radiolabelled precursors, ultrastructural alterations and changes in the pattern of metabolite excretion were examined. Different degrees of toxicity were found for each complex: the platinum compound showed an inhibition effect on nucleic acid synthesis, provoking alterations on the levels of mitochondria, nucleus and glycosomes. These results, together with others reported previously in our laboratory about the activity of pyrimidine derivatives, reflect the potential of these compounds as agents in the treatment of Phytomonas sp.

Tafenoquine, an Antiplasmodial 8-Aminoquinoline, Targets Leishmania Respiratory Complex III and Induces Apoptosis ▿

PubMed Central

Carvalho, Luis; Luque-Ortega, Juan Román; Manzano, José Ignacio; Castanys, Santiago; Rivas, Luis; Gamarro, Francisco

2010-01-01

Tafenoquine (TFQ), an 8-aminoquinoline analogue of primaquine, which is currently under clinical trial (phase IIb/III) for the treatment and prevention of malaria, may represent an alternative treatment for leishmaniasis. In this work, we have studied the mechanism of action of TFQ against Leishmania parasites. TFQ impaired the overall bioenergetic metabolism of Leishmania promastigotes, causing a rapid drop in intracellular ATP levels without affecting plasma membrane permeability. TFQ induced mitochondrial dysfunction through the inhibition of cytochrome c reductase (respiratory complex III) with a decrease in the oxygen consumption rate and depolarization of mitochondrial membrane potential. This was accompanied by ROS production, elevation of intracellular Ca2+ levels and concomitant nuclear DNA fragmentation. We conclude that TFQ targets Leishmania mitochondria, leading to an apoptosis-like death process. PMID:20837758

Tafenoquine, an antiplasmodial 8-aminoquinoline, targets leishmania respiratory complex III and induces apoptosis.

PubMed

Carvalho, Luis; Luque-Ortega, Juan Román; Manzano, José Ignacio; Castanys, Santiago; Rivas, Luis; Gamarro, Francisco

2010-12-01

Tafenoquine (TFQ), an 8-aminoquinoline analogue of primaquine, which is currently under clinical trial (phase IIb/III) for the treatment and prevention of malaria, may represent an alternative treatment for leishmaniasis. In this work, we have studied the mechanism of action of TFQ against Leishmania parasites. TFQ impaired the overall bioenergetic metabolism of Leishmania promastigotes, causing a rapid drop in intracellular ATP levels without affecting plasma membrane permeability. TFQ induced mitochondrial dysfunction through the inhibition of cytochrome c reductase (respiratory complex III) with a decrease in the oxygen consumption rate and depolarization of mitochondrial membrane potential. This was accompanied by ROS production, elevation of intracellular Ca(2+) levels and concomitant nuclear DNA fragmentation. We conclude that TFQ targets Leishmania mitochondria, leading to an apoptosis-like death process.

Insight into the mechanism of action of temporin-SHa, a new broad-spectrum antiparasitic and antibacterial agent

PubMed Central

Abbassi, Feten; Humblot, Vincent; Bouceba, Tahar; Correia, Isabelle; Casale, Sandra; Foulon, Thierry; Sereno, Denis; Oury, Bruno; Ladram, Ali

2017-01-01

Antimicrobial peptides (AMPs) are promising drugs to kill resistant pathogens. In contrast to bacteria, protozoan parasites, such as Leishmania, were little studied. Therefore, the antiparasitic mechanism of AMPs is still unclear. In this study, we sought to get further insight into this mechanism by focusing our attention on temporin-SHa (SHa), a small broad-spectrum AMP previously shown to be active against Leishmania infantum. To improve activity, we designed analogs of SHa and compared the antibacterial and antiparasitic mechanisms. [K3]SHa emerged as a highly potent compound active against a wide range of bacteria, yeasts/fungi, and trypanosomatids (Leishmania and Trypanosoma), with leishmanicidal intramacrophagic activity and efficiency toward antibiotic-resistant strains of S. aureus and antimony-resistant L. infantum. Multipassage resistance selection demonstrated that temporins-SH, particularly [K3]SHa, are not prone to induce resistance in Escherichia coli. Analysis of the mode of action revealed that bacterial and parasite killing occur through a similar membranolytic mechanism involving rapid membrane permeabilization and depolarization. This was confirmed by high-resolution imaging (atomic force microscopy and field emission gun-scanning electron microscopy). Multiple combined techniques (nuclear magnetic resonance, surface plasmon resonance, differential scanning calorimetry) allowed us to detail peptide-membrane interactions. [K3]SHa was shown to interact selectively with anionic model membranes with a 4-fold higher affinity (KD = 3 x 10−8 M) than SHa. The amphipathic α-helical peptide inserts in-plane in the hydrophobic lipid bilayer and disrupts the acyl chain packing via a detergent-like effect. Interestingly, cellular events, such as mitochondrial membrane depolarization or DNA fragmentation, were observed in L. infantum promastigotes after exposure to SHa and [K3]SHa at concentrations above IC50. Our results indicate that these temporins exert leishmanicidal activity via a primary membranolytic mechanism but can also trigger apoptotis-like death. The many assets demonstrated for [K3]SHa make this small analog an attractive template to develop new antibacterial/antiparasitic drugs. PMID:28319176

Leishmanicidal activity of the Agaricus blazei Murill in different Leishmania species.

PubMed

Valadares, Diogo G; Duarte, Mariana C; Oliveira, Jamil S; Chávez-Fumagalli, Miguel A; Martins, Vivian T; Costa, Lourena E; Leite, João Paulo V; Santoro, Marcelo M; Régis, Wiliam C B; Tavares, Carlos A P; Coelho, Eduardo A F

2011-12-01

Leishmaniasis is a major public health problem, and the alarming spread of parasite resistance underlines the importance of discovering new therapeutic products. The present study aims to investigate the in vitro leishmanicidal activity of an Agaricus blazei Murill mushroom extract as compared to different Leishmania species and stages. The water extract proved to be effective against promastigote and amastigote-like stages of Leishmania amazonensis, L. chagasi, and L. major, with IC(50) (50% inhibitory concentration) values of 67.5, 65.8, and 56.8 μg/mL for promastigotes, and 115.4, 112.3, and 108.4 μg/mL for amastigotes-like respectively. The infectivity of the three Leishmania species before and after treatment with the water extract was analyzed, and it could be observed that 82%, 57%, and 73% of the macrophages were infected with L. amazonensis, L. major, and L. chagasi, respectively. However, when parasites were pre-incubated with the water extract, and later used to infect macrophages, they were able to infect only 12.7%, 24.5%, and 19.7% of the phagocytic cells for L. amazonensis, L. chagasi, and L. major, respectively. In other experiments, macrophages were infected with L. amazonensis, L. chagasi, or L. major, and later treated with the aforementioned extract, presented reductions of 84.4%, 79.6%, and 85.3% in the parasite burden after treatment. A confocal microscopy revealed the loss of the viability of the parasites within the infected macrophages after treatment with the water extract. The applied extract presented a low cytotoxicity in murine macrophages and a null hemolytic activity in type O(+) human red blood cells. No nitric oxide (NO) production, nor inducible nitric oxide syntase expression, could be observed in macrophages after stimulation with the water extract, suggesting that biological activity may be due to direct mechanisms other than macrophage activation by means of NO production. In conclusion, the results demonstrate that the A. blazei Murill water extract can potentially be used as a therapeutic alternative on its own, or in association with other drugs, to treat Visceral and Cutaneous Leishmaniasis. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Arginase activity in pathogenic and non-pathogenic species of Leishmania parasites.

PubMed

Badirzadeh, Alireza; Taheri, Tahereh; Taslimi, Yasaman; Abdossamadi, Zahra; Heidari-Kharaji, Maryam; Gholami, Elham; Sedaghat, Baharehsadat; Niyyati, Maryam; Rafati, Sima

2017-07-01

Proliferation of Leishmania (L.) parasites depends on polyamine availability, which can be generated by the L-arginine catabolism and the enzymatic activity of arginase (ARG) of the parasites and of the mammalian hosts. In the present study, we characterized and compared the arginase (arg) genes from pathogenic L. major and L. tropica and from non-pathogenic L. tarentolae. We quantified the level of the ARG activity in promastigotes and macrophages infected with pathogenic L. major and L. tropica and non-pathogenic L. tarentolae amastigotes. The ARG's amino acid sequences of the pathogenic and non-pathogenic Leishmania demonstrated virtually 98.6% and 88% identities with the reference L. major Friedlin ARG. Higher ARG activity was observed in all pathogenic promastigotes as compared to non-pathogenic L. tarentolae. In vitro infection of human macrophage cell line (THP1) with pathogenic and non-pathogenic Leishmania spp. resulted in increased ARG activities in the infected macrophages. The ARG activities present in vivo were assessed in susceptible BALB/c and resistant C57BL/6 mice infected with L. major, L. tropica and L. tarentolae. We demonstrated that during the development of the infection, ARG is induced in both strains of mice infected with pathogenic Leishmania. However, in L. major infected BALB/c mice, the induction of ARG and parasite load increased simultaneously according to the time course of infection, whereas in C57BL/6 mice, the enzyme is upregulated solely during the period of footpad swelling. In L. tropica infected mice, the footpads' swellings were slow to develop and demonstrated minimal cutaneous pathology and ARG activity. In contrast, ARG activity was undetectable in mice inoculated with the non-pathogenic L. tarentolae. Our data suggest that infection by Leishmania parasites can increase ARG activity of the host and provides essential polyamines for parasite salvage and its replication. Moreover, the ARG of Leishmania is vital for parasite proliferation and required for infection in mice. ARG activity can be used as one of the main marker of the disease severity.

Antigenicity, Immunogenicity and Protective Efficacy of Three Proteins Expressed in the Promastigote and Amastigote Stages of Leishmania infantum against Visceral Leishmaniasis

PubMed Central

Martins, Vivian Tamietti; Chávez-Fumagalli, Miguel Angel; Lage, Daniela Pagliara; Duarte, Mariana Costa; Garde, Esther; Costa, Lourena Emanuele; da Silva, Viviane Gomes; Oliveira, Jamil Silvano; de Magalhães-Soares, Danielle Ferreira; Teixeira, Santuza Maria Ribeiro; Fernandes, Ana Paula; Soto, Manuel; Tavares, Carlos Alberto Pereira; Coelho, Eduardo Antonio Ferraz

2015-01-01

In the present study, two Leishmania infantum hypothetical proteins present in the amastigote stage, LiHyp1 and LiHyp6, were combined with a promastigote protein, IgE-dependent histamine-releasing factor (HRF); to compose a polyproteins vaccine to be evaluated against L. infantum infection. Also, the antigenicity of the three proteins was analyzed, and their use for the serodiagnosis of canine visceral leishmaniasis (CVL) was evaluated. The LiHyp1, LiHyp6, and HRF DNA coding sequences were cloned in prokaryotic expression vectors and the recombinant proteins were purified. When employed in ELISA assays, all proteins were recognized by sera from visceral leishmaniasis (VL) dogs, and presented no cross-reactivity with either sera from dogs vaccinated with a Brazilian commercial vaccine, or sera of Trypanosoma cruzi-infected or Ehrlichia canis-infected animals. In addition, the antigens were not recognized by antibodies from non-infected animals living in endemic or non-endemic areas for leishmaniasis. The immunogenicity and protective efficacy of the three proteins administered in the presence of saponin, individually or in combination (composing a polyproteins vaccine), were evaluated in a VL murine model: BALB/c mice infected with L. infantum. Spleen cells from mice inoculated with the individual proteins or with the polyproteins vaccine plus saponin showed a protein-specific production of IFN-γ, IL-12, and GM-CSF after an in vitro stimulation, which was maintained after infection. These animals presented significant reductions in the parasite burden in different evaluated organs, when compared to mice inoculated with saline or saponin. The decrease in parasite burden was associated with an IL-12-dependent production of IFN-γ against parasite total extracts (produced mainly by CD4+ T cells), correlated to the induction of parasite proteins-driven NO production. Mice inoculated with the recombinant protein-based vaccines showed also high levels of parasite-specific IgG2a antibodies. The polyproteins vaccine administration induced a more pronounced Th1 response before and after challenge infection than individual vaccines, which was correlated to a higher control of parasite dissemination to internal organs. PMID:26367128

Arginase activity in pathogenic and non-pathogenic species of Leishmania parasites

PubMed Central

Badirzadeh, Alireza; Taheri, Tahereh; Taslimi, Yasaman; Abdossamadi, Zahra; Heidari-Kharaji, Maryam; Gholami, Elham; Sedaghat, Baharehsadat; Niyyati, Maryam

2017-01-01

Proliferation of Leishmania (L.) parasites depends on polyamine availability, which can be generated by the L-arginine catabolism and the enzymatic activity of arginase (ARG) of the parasites and of the mammalian hosts. In the present study, we characterized and compared the arginase (arg) genes from pathogenic L. major and L. tropica and from non-pathogenic L. tarentolae. We quantified the level of the ARG activity in promastigotes and macrophages infected with pathogenic L. major and L. tropica and non-pathogenic L. tarentolae amastigotes. The ARG's amino acid sequences of the pathogenic and non-pathogenic Leishmania demonstrated virtually 98.6% and 88% identities with the reference L. major Friedlin ARG. Higher ARG activity was observed in all pathogenic promastigotes as compared to non-pathogenic L. tarentolae. In vitro infection of human macrophage cell line (THP1) with pathogenic and non-pathogenic Leishmania spp. resulted in increased ARG activities in the infected macrophages. The ARG activities present in vivo were assessed in susceptible BALB/c and resistant C57BL/6 mice infected with L. major, L. tropica and L. tarentolae. We demonstrated that during the development of the infection, ARG is induced in both strains of mice infected with pathogenic Leishmania. However, in L. major infected BALB/c mice, the induction of ARG and parasite load increased simultaneously according to the time course of infection, whereas in C57BL/6 mice, the enzyme is upregulated solely during the period of footpad swelling. In L. tropica infected mice, the footpads' swellings were slow to develop and demonstrated minimal cutaneous pathology and ARG activity. In contrast, ARG activity was undetectable in mice inoculated with the non-pathogenic L. tarentolae. Our data suggest that infection by Leishmania parasites can increase ARG activity of the host and provides essential polyamines for parasite salvage and its replication. Moreover, the ARG of Leishmania is vital for parasite proliferation and required for infection in mice. ARG activity can be used as one of the main marker of the disease severity. PMID:28708893

In Vitro Evaluation of a Soluble Leishmania Promastigote Surface Antigen as a Potential Vaccine Candidate against Human Leishmaniasis

PubMed Central

Bahi-Jaber, Narges; Petitdidier, Elodie; Markikou-Ouni, Wafa; Aoun, Karim; Moreno, Javier; Carrillo, Eugenia; Salotra, Poonam; Kaushal, Himanshu; Negi, Narender Singh; Arevalo, Jorge; Falconi-Agapito, Francesca; Privat, Angela; Cruz, Maria; Pagniez, Julie; Papierok, Gérard-Marie; Rhouma, Faten Bel Haj; Torres, Pilar; Lemesre, Jean-Loup; Chenik, Mehdi; Meddeb-Garnaoui, Amel

2014-01-01

PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high or low levels of IFN-γ in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-γ, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-α in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-γ after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-γ-producing CD4+ T cells and the released IFN-γ. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection. PMID:24786587

HPLC-DAD phenolic profile, cytotoxic and anti-kinetoplastidae activity of Melissa officinalis.

PubMed

Cunha, Francisco; Tintino, Saulo R; Figueredo, Fernando; Barros, Luiz; Duarte, Antonia E; Vega Gomez, Maria Celeste; Coronel, Cathia Cecilia; Rolón, Mírian; Leite, Nadghia; Sobral-Souza, Celestina E; Brito, S V; Waczuc, Emily Pansera; Boligon, Aline Augusti; Athayde, Margareth; Kamdem, Jean Paul; Coutinho, Henrique Douglas Melo; Franco, Jéferson

2016-09-01

Context Melissa officinalis subsp. inodora Bornm. (Lamiaceae) has been used since ancient times in folk medicine against various diseases, but it has not been investigated against protozoa. Objective To evaluate the activities of M. officinalis against Leishmania braziliensis, Leishmania infantum and Trypanosoma cruzi as well as its cytotoxicity in fibroblast cell line. Materials and methods The fresh leaves were chopped into 1 cm(2) pieces, washed and macerated with 99.9% of ethanol for 72 h at room temperature. Antiparasitic activity of M. officinalis was accessed by direct counting of cells after serial dilution, while the cytotoxicity of M. officinalis was evaluated in fibroblast cell line (NCTC929) by measuring the reduction of resazurin. The test duration was 24 h. High-performance liquid chromatography (HPLC) was used to characterise the extract. Results The extract at concentrations of 250 and 125 μg/mL inhibited 80.39 and 54.27% of promastigote (LC50  value = 105.78 μg/mL) form of L. infantum, 80.59 and 68.61% of L. brasiliensis (LC50 value  = 110.69 μg/mL) and against epimastigote (LC50 value  = 245.23 μg/mL) forms of T. cruzi with an inhibition of 54.45 and 22.26%, respectively, was observed. The maximum toxicity was noted at 500 μg/mL with 95.41% (LC50  value = 141.01 μg/mL). The HPLC analysis identified caffeic acid and rutin as the major compounds. Discussion The inhibition of the parasites is considered clinically relevant (< 500 μg/mL). Rutin and caffeic acids may be responsible for the antiprotozoal effect of the extract. Conclusion The ethanol extract of M. officinalis can be considered a potential alternative source of natural products with antileishmania and antitrypanosoma activities.

Comparative analysis of Leishmania exoproteomes: implication for host-pathogen interactions.

PubMed

Peysselon, Franck; Launay, Guillaume; Lisacek, Frédérique; Duclos, Bertrand; Ricard-Blum, Sylvie

2013-12-01

Leishmaniasis is a vector-borne disease caused by the protozoa Leishmania. We have analyzed and compared the sequences of three experimental exoproteomes of Leishmania promastigotes from different species to determine their specific features and to identify new candidate proteins involved in interactions of Leishmania with the host. The exoproteomes differ from the proteomes by a decrease in the average molecular weight per protein, in disordered amino acid residues and in basic proteins. The exoproteome of the visceral species is significantly enriched in sites predicted to be phosphorylated as well as in features frequently associated with molecular interactions (intrinsic disorder, number of disordered binding regions per protein, interaction and/or trafficking motifs) compared to the other species. The visceral species might thus have a larger interaction repertoire with the host than the other species. Less than 10% of the exoproteomes contain heparin-binding and RGD sequences, and ~30% the host targeting signal RXLXE/D/Q. These latter proteins might thus be exported inside the host cell during the intracellular stage of the infection. Furthermore we have identified nine protein families conserved in the three exoproteomes with specific combinations of Pfam domains and selected eleven proteins containing at least three interaction and/or trafficking motifs including two splicing factors, phosphomannomutase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, the paraflagellar rod protein-1D and a putative helicase. Their role in host-Leishmania interactions warrants further investigation but the putative ATP-dependent DEAD/H RNA helicase, which contains numerous interaction motifs, a host targeting signal and two disordered regions, is a very promising candidate. © 2013.

NYVAC vector modified by C7L viral gene insertion improves T cell immune responses and effectiveness against leishmaniasis.

PubMed

Sánchez-Sampedro, L; Mejías-Pérez, E; S Sorzano, Carlos Óscar; Nájera, J L; Esteban, M

2016-07-15

The NYVAC poxvirus vector is used as vaccine candidate for HIV and other diseases, although there is only limited experimental information on its immunogenicity and effectiveness for use against human pathogens. Here we defined the selective advantage of NYVAC vectors in a mouse model by comparing the immune responses and protection induced by vectors that express the LACK (Leishmania-activated C-kinase antigen), alone or with insertion of the viral host range gene C7L that allows the virus to replicate in human cells. Using DNA prime/virus boost protocols, we show that replication-competent NYVAC-LACK that expresses C7L (NYVAC-LACK-C7L) induced higher-magnitude polyfunctional CD8(+) and CD4(+) primary adaptive and effector memory T cell responses (IFNγ, TNFα, IL-2, CD107a) to LACK antigen than non-replicating NYVAC-LACK. Compared to NYVAC-LACK, the NYVAC-LACK-C7L-induced CD8(+) T cell population also showed higher proliferation when stimulated with LACK antigen. After a challenge by subcutaneous Leishmania major metacyclic promastigotes, NYVAC-LACK-C7L-vaccinated mouse groups showed greater protection than the NYVAC-LACK-vaccinated group. Our results indicate that the type and potency of immune responses induced by LACK-expressing NYVAC vectors is improved by insertion of the C7L gene, and that a replication-competent vector as a vaccine renders greater protection against a human pathogen than a non-replicating vector. Copyright © 2016 Elsevier B.V. All rights reserved.

Discovery of novel inhibitors for Leishmania nucleoside diphosphatase kinase (NDK) based on its structural and functional characterization

NASA Astrophysics Data System (ADS)

Mishra, Arjun K.; Singh, Nidhi; Agnihotri, Pragati; Mishra, Shikha; Singh, Saurabh P.; Kolli, Bala K.; Chang, Kwang Poo; Sahasrabuddhe, Amogh A.; Siddiqi, M. I.; Pratap, J. Venkatesh

2017-06-01

Nucleoside diphosphate kinases (NDKs) are ubiquitous enzymes that catalyze the transfer of the γ-phosphate moiety from an NTP donor to an NDP acceptor, crucial for maintaining the cellular level of nucleoside triphosphates (NTPs). The inability of trypanosomatids to synthesize purines de novo and their dependence on the salvage pathway makes NDK an attractive target to develop drugs for the diseases they cause. Here we report the discovery of novel inhibitors for Leishmania NDK based on the structural and functional characterization of purified recombinant NDK from Leishmania amazonensis. Recombinant LaNDK possesses auto-phosphorylation, phosphotransferase and kinase activities with Histidine 117 playing an essential role. LaNDK crystals were grown by hanging drop vapour diffusion method in a solution containing 18% PEG-MME 500, 100 mM Bis-Tris propane pH 6.0 and 50 mM MgCl2. It belongs to the hexagonal space group P6322 with unit cell parameters a = b = 115.18, c = 62.18 Å and α = β = 90°, γ = 120°. The structure solved by molecular replacement methods was refined to crystallographic R-factor and Rfree values of 22.54 and 26.52%, respectively. Molecular docking and dynamics simulation -based virtual screening identified putative binding compounds. Protein inhibition studies of selected hits identified five inhibitors effective at micromolar concentrations. One of the compounds showed 45% inhibition of Leishmania promastigotes proliferation. Analysis of inhibitor-NDK complexes reveals the mode of their binding, facilitating design of new compounds for optimization of activities as drugs against leishmaniasis.

Uptake of the antileishmania drug tafenoquine follows a sterol-dependent diffusion process in Leishmania.

PubMed

Manzano, José Ignacio; Carvalho, Luis; García-Hernández, Raquel; Poveda, José Antonio; Ferragut, José Antonio; Castanys, Santiago; Gamarro, Francisco

2011-11-01

The present study was designed to elucidate the mechanism of tafenoquine uptake in Leishmania and its sterol dependence. Because tafenoquine is a fluorescent compound, spectrofluorimetric analysis allowed us to monitor its uptake by Leishmania promastigotes and intracellular amastigotes, and to evaluate the effect of temperature, energy and H+ gradient on drug entry. The influence of sterols on tafenoquine uptake in Leishmania parasites was determined in experiments using sterol-depleting agents such as methyl-β-cyclodextrin or cholesterol oxidase. Tafenoquine exhibited fast entry kinetics into Leishmania in an energy-independent, but pH- and temperature-dependent, non-saturable process. Furthermore, sterol depletion decreased tafenoquine uptake. These findings suggest that Leishmania takes up tafenoquine by a diffusion process and that decreases in membrane sterol content may induce a decrease in drug uptake.

Synthesis, spectroscopic characterization and in vitro antimicrobial, anticancer and antileishmanial activities as well interaction with Salmon sperm DNA of newly synthesized carboxylic acid derivative, 4-(4-methoxy-2-nitrophenylamino)-4-oxobutanoic acid

NASA Astrophysics Data System (ADS)

Sirajuddin, Muhammad; Ali, Saqib; McKee, Vickie; Ullah, Hameed

2015-03-01

This paper stresses on the synthesis, characterization of novel carboxylic acid derivative and its application in pharmaceutics. Carboxylic acid derivatives have a growing importance in medicine, particularly in oncology. A novel carboxylic acid, 4-(4-methoxy-2-nitrophenylamino)-4-oxobutanoic acid, was synthesized and characterized by elemental analysis, FT-IR, NMR (1H, and 13C), mass spectrometry and single crystal X-ray structural analysis. The structure of the title compound, C11H12N2O6, shows the molecules dimerised by short intramolecular Osbnd H⋯O hydrogen bonds. The compound was screened for in vitro antimicrobial, anticancer, and antileishmanial activities as well as interaction with SS-DNA. The compound was also checked for in vitro anticancer activity against BHK-21, H-157 and HCEC cell lines, and showed significant anticancer activity. The compound was almost non-toxic towards human corneal epithelial cells (HCEC) and did not show more than 7.4% antiproliferative activity when used at the 2.0 μg/mL end concentration. It was also tested for antileishmanial activity against the promastigote form of leishmania major and obtained attractive result. DNA interaction study exposes that the binding mode of the compound with SS-DNA is an intercalative as it results in hypochromism along with minor red shift. A new and efficient strategy to identify pharmacophores sites in carboxylic acid derivative for antibacterial/antifungal activity using Petra, Osiris and Molinspiration (POM) analyses was also carried out.

Leishmanicidal activity of the crude extract, fractions and major piperidine alkaloids from the flowers of Senna spectabilis.

PubMed

de Albuquerque Melo, Gabriela Muniz; Silva, Marcela Campelo Rodrigues; Guimarães, Thaís Pereira; Pinheiro, Kátia Mantovani; da Matta, Carolina Barbosa Brito; de Queiroz, Aline Cavalcanti; Pivatto, Marcos; Bolzani, Vanderlan da Silva; Alexandre-Moreira, Magna Suzana; Viegas, Claudio

2014-02-15

Senna spectabilis (sin. Cassia excelsa, C. spectabilis) is an endemic tree of South America and Africa, very common in Brazil, where it is known as "canafistula-de-besouro" and "cassia-do-nordeste". In folk medicine, this plant is indicated for the treatment of constipation, insomnia, anxiety, epilepsy, malaria, dysentery and headache. Phytopharmacological studies have also confirmed anticonvulsive, sedative, anti-malarial, antimicrobial and cytotoxic properties of many parts of S. spectabilis. In this communication, we present a comparative study of the leishmanicidal activity of the crude ethanolic extract, its fractions and also the two major alkaloidal metabolites (-)-cassine/(-)-spectaline, trying to establish a relationship between the presence of piperidine alkaloidal constituents and leishmanicidal activity. The growth inhibitory effect of promastigote forms of Leishmania major was determined for the crude extract, fractions of the flowers of S. spectabilis and a mixture of (-)-cassine/(-)-spectaline in comparison to pentamidine used as standard drug. The cytotoxic effects were assessed on macrophage strain J774 by lactate dehydrogenase assay. Fractions dichloromethane (FL-DCM) and n-butanol (FL-Bu) and a mixture of (-)-cassine/(-)-spectaline (∼7:3) exhibited significant activity against the parasite Leishmania major (IC50 values of 0.6±0.1 μg/ml, 1.6±0.9 μg/ml and 24.9±1.4 μg/ml, respectively), without toxic effects on murine macrophages. Due to the promising results elicited, further studies in vivo need to be performed to confirm the therapeutic potential of Senna spectabilis. Copyright © 2013 Elsevier GmbH. All rights reserved.

A Target-Based High Throughput Screen Yields Trypanosoma brucei Hexokinase Small Molecule Inhibitors with Antiparasitic Activity

PubMed Central

Sharlow, Elizabeth R.; Lyda, Todd A.; Dodson, Heidi C.; Mustata, Gabriela; Morris, Meredith T.; Leimgruber, Stephanie S.; Lee, Kuo-Hsiung; Kashiwada, Yoshiki; Close, David; Lazo, John S.; Morris, James C.

2010-01-01

Background The parasitic protozoan Trypanosoma brucei utilizes glycolysis exclusively for ATP production during infection of the mammalian host. The first step in this metabolic pathway is mediated by hexokinase (TbHK), an enzyme essential to the parasite that transfers the γ-phospho of ATP to a hexose. Here we describe the identification and confirmation of novel small molecule inhibitors of bacterially expressed TbHK1, one of two TbHKs expressed by T. brucei, using a high throughput screening assay. Methodology/Principal Findings Exploiting optimized high throughput screening assay procedures, we interrogated 220,233 unique compounds and identified 239 active compounds from which ten small molecules were further characterized. Computation chemical cluster analyses indicated that six compounds were structurally related while the remaining four compounds were classified as unrelated or singletons. All ten compounds were ∼20-17,000-fold more potent than lonidamine, a previously identified TbHK1 inhibitor. Seven compounds inhibited T. brucei blood stage form parasite growth (0.03≤EC50<3 µM) with parasite specificity of the compounds being demonstrated using insect stage T. brucei parasites, Leishmania promastigotes, and mammalian cell lines. Analysis of two structurally related compounds, ebselen and SID 17387000, revealed that both were mixed inhibitors of TbHK1 with respect to ATP. Additionally, both compounds inhibited parasite lysate-derived HK activity. None of the compounds displayed structural similarity to known hexokinase inhibitors or human African trypanosomiasis therapeutics. Conclusions/Significance The novel chemotypes identified here could represent leads for future therapeutic development against the African trypanosome. PMID:20405000

Leishmania major Survival in Selective Phlebotomus papatasi Sand Fly Vector Requires a Specific SCG-Encoded Lipophosphoglycan Galactosylation Pattern

PubMed Central

Dobson, Deborah E.; Kamhawi, Shaden; Lawyer, Phillip; Turco, Salvatore J.; Beverley, Stephen M.; Sacks, David L.

2010-01-01

Phlebotomine sand flies that transmit the protozoan parasite Leishmania differ greatly in their ability to support different parasite species or strains in the laboratory: while some show considerable selectivity, others are more permissive. In “selective” sand flies, Leishmania binding and survival in the fly midgut typically depends upon the abundant promastigote surface adhesin lipophosphoglycan (LPG), which exhibits species- and strain-specific modifications of the dominant phosphoglycan (PG) repeat units. For the “selective” fly Phlebotomus papatasi PpapJ, side chain galactosyl-modifications (scGal) of PG repeats play key roles in parasite binding. We probed the specificity and properties of this scGal-LPG PAMP (Pathogen Associated Molecular Pattern) through studies of natural isolates exhibiting a wide range of galactosylation patterns, and of a panel of isogenic L. major engineered to express similar scGal-LPG diversity by transfection of SCG-encoded β1,3-galactosyltransferases with different activities. Surprisingly, both ‘poly-scGal’ and ‘null-scGal’ lines survived poorly relative to PpapJ-sympatric L. major FV1 and other ‘mono-scGal’ lines. However, survival of all lines was equivalent in P. duboscqi, which naturally transmit L. major strains bearing ‘null-scGal’-LPG PAMPs. We then asked whether scGal-LPG-mediated interactions were sufficient for PpapJ midgut survival by engineering Leishmania donovani, which normally express unsubstituted LPG, to express a ‘PpapJ-optimal’ scGal-LPG PAMP. Unexpectedly, these “L. major FV1-cloaked” L. donovani-SCG lines remained unable to survive within PpapJ flies. These studies establish that midgut survival of L. major in PpapJ flies is exquisitely sensitive to the scGal-LPG PAMP, requiring a specific ‘mono-scGal’ pattern. However, failure of ‘mono-scGal’ L. donovani-SCG lines to survive in selective PpapJ flies suggests a requirement for an additional, as yet unidentified L. major-specific parasite factor(s). The interplay of the LPG PAMP and additional factor(s) with sand fly midgut receptors may determine whether a given sand fly host is “selective” or “permissive”, with important consequences to both disease transmission and the natural co-evolution of sand flies and Leishmania. PMID:21085609

Optical detection of parasitic protozoa in sol-gel matrices

NASA Astrophysics Data System (ADS)

Livage, Jacques; Barreau, J. Y.; Da Costa, J. M.; Desportes, I.

1994-10-01

Whole cell parasitic protozoa have been entrapped within sol-gel porous silica matrices. Stationary phase promastigote cells of Leishmania donovani infantum are mixed with a silica sol before gelation occurs. They remain trapped within the growing oxide network and their cellular organization appears to be well preserved. Moreover protozoa retain their antigenic properties in the porous gel. They are still able to detect parasite specific antibodies in serum samples from infected patients via an enzyme linked immunosorbent assay (ELISA). Antigen- antibody associations occurring in the gel are optically detected via the reactions of a peroxidase conjugate with ortho-phenylenediamine leading to the formation of a yellow coloration. A clear-cut difference in optical density is measured between positive and negative sera. Such an entrapment of antigenic species into porous sol-gel matrices avoids the main problems due to non specific binding and could be advantageously used in diagnostic kits.

Back to monoxeny: Phytomonas nordicus descended from dixenous plant parasites.

PubMed

Frolov, Alexander O; Malysheva, Marina N; Yurchenko, Vyacheslav; Kostygov, Alexei Yu

2016-02-01

The trypanosomatid Phytomonas nordicus parasitizing the predatory bug Troilus luridus was described at the twilight of the morphotype-based systematics. Despite its monoxenous life cycle, this species was attributed to the dixenous genus Phytomonas due to the presence of long twisted promastigotes and development of flagellates in salivary glands. However, these characteristics were considered insufficient for proving the phytomonad nature of the species and therefore its description remained virtually unnoticed. Here, we performed molecular phylogenetic analyses using 18S ribosomal RNA (rRNA) gene and region containing internal trascribed spacers (ITS) 1 and 2 and convincingly demonstrated the affinity of P. nordicus to the genus Phytomonas. In addition, we investigated its development in the salivary glands. We argue that in many aspects the life cycle of monoxenous P. nordicus resembles that of its dixenous relatives represented by tomato-parasitizing Phytomonas serpens. Copyright © 2015 Elsevier GmbH. All rights reserved.

Biochemical and ultrastructural alterations caused by newly synthesized 1,2,4-triazole[1,5a]pyrimidine derivatives against Phytomonas staheli (Trypanosomatidae).

PubMed

Luque, F; Fernandez-Ramos, C; Entrala, E; Rosales, M J; Salas, M C; Navarro, J; Sánchez-Moreno, M

2000-12-01

Six compounds, all newly synthesized triazole-pyrimidine derivatives that proved inhibitory of in in vitro growth of epimastigotes in Trypanosoma cruzi and of promastigotes of Leishmania donovani and Phytomonas staheli, were studied to investigate their toxic effects. As a biological model, the plant trypanosome P. staheli, which causes sudden wilt in the oil palm and Hartrot in the coconut palm, was used. The six compounds markedly inhibited macromolecule synthesis (nucleic acids and proteins) by the parasite. The cells treated with these compounds present severe damage in their ultrastructure-intense 'vacuolization, and appearance of lysosomes as well as other residual bodies. The mitochondrial section appeared larger in size. with a swollen matrix. In addition, these compounds changed the excretion of end metabolites, primarily affecting ethanol and acetate excretion, possibly by directly influencing certain enzymes (alcohol dehydrogenase and acetate synthetase) or their synthesis. 2000 Elsevier Science Ltd.

Inhibitory Activity of Eleven Artemisia Species from Iran against Leishmania Major Parasites

PubMed Central

Emami, Seyed Ahmad; Zamanai Taghizadeh Rabe, Shahrzad; Ahi, Ali; Mahmoudi, Mahmoud

2012-01-01

Objective(s) Annual incidence of cutaneous leishmaniasis is increasingly growing and development of the alternative drugs against it is a major concern. Artemisia genus is a traditional medicinal plant in Iran. The aim of this study was to examine the leishmanicidal activity of various Iranian Artemisia species extracts. Materials and Methods Different extracts were gathered from eleven Iranian Artemisia species. Their leishmanicidal activities against the growth of Leishmania major (L. major) promastigotes were examined as the half maximal inhibitory concentration (IC50) using MTT assay. Results Obtained results showed that ethanol extracts especially those taken from A. ciniformis, A. santolina and A. kulbadica have the strongest effects. Conclusion Looking for the effective leishmanicidal agents from natural resources in Iran, we found that the ethanol extract of collected Artemisia species had significant effect on in vitro leishmanicidal activity and may be suitable candidates in the treatment of leishmaniasis. PMID:23493354

Synthesis and evaluation of novel triazolyl quinoline derivatives as potential antileishmanial agents.

PubMed

Upadhyay, Akanksha; Kushwaha, Pragati; Gupta, Sampa; Dodda, Ranga Prasad; Ramalingam, Karthik; Kant, Ruchir; Goyal, Neena; Sashidhara, Koneni V

2018-05-12

The high potential of quinoline containing natural products and their derivatives in medicinal chemistry led us to discover novel series of 25 compounds for the development of new antileishmanial agents. A series of triazolyl 2-methyl-4-phenylquinoline-3-carboxylate derivatives has been synthesized via click chemistry inspired molecular hybridization approach and evaluated against Leishmania donovani. Most of the screened derivatives exhibited significant in vitro anti-leishmanial activity against promastigote (IC 50 ranging from 2.43 to 45.75 μM) and intracellular amastigotes (IC 50 ranging from 7.06 to 34.9 μM) than the control, miltefosine (IC 50  = 8.4 μM), with less cytotoxicity in comparison to the standard drugs. Overall results revealed that prototype signify a new structural lead for antileishmanial chemotherapy. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

2-propen-1-amine derivatives and their synthetic intermediates: activity against pathogenic trypanosomatids.

PubMed

de Souza, A O; Hemerly, F P; Gomes-Cardoso, L; Santa-Rita, R M; Leon, L L; de Castro, S L; Durán, N

2004-12-01

The potential activity of three new derivatives of 3-(4'-Y-[1,1'-biphenyl]-4-yl)-3-(4-X-phenyl)-N,N-dimethyl-2-propen-1-amine (2-PAMs) was assayed against Trypanosoma cruzi and Leishmania amazonensis. They showed higher activity against trypomastigotes and epimastigotes of T. cruzi than the standard drugs, crystal violet and nifurtimox. Besides these derivatives, a series of eleven 2-PAMs derivatives and the corresponding intermediates, biphenyl methanones (BPMs) were assayed against promastigotes of L. amazonensis, showing that the 2-PAMs were remarkably more active than the BPMs. The PAMs 2c, 2e and 2j were about 2-fold more active that pentamidine isothionate and between 27.2- and 46.4-fold less toxic to V79 mammalian cells. The present results encourage further studies, especially against intracellular parasites and in experimental animals.

In Vitro Evaluation of Antileishmanial Activity of Computationally Screened Compounds against Ascorbate Peroxidase To Combat Amphotericin B Drug Resistance

PubMed Central

Mansuri, Rani; Kumar, Ashish; Rana, Sindhuprava; Panthi, Bhavana; Ansari, M. Yousuf; Das, Sushmita; Dikhit, Manas Ranjan

2017-01-01

ABSTRACT In visceral leishmaniasis (VL), the host macrophages generate oxidative stress to destroy the pathogen, while Leishmania combats the harmful effect of radicals by redox homeostasis through its unique trypanothione cascade. Leishmania donovani ascorbate peroxidase (LdAPx) is a redox enzyme that regulates the trypanothione cascade and detoxifies the effect of H2O2. The absence of an LdAPx homologue in humans makes it an excellent drug target. In this study, the homology model of LdAPx was built, including heme, and diverse compounds were prefiltered (PAINS, ADMET, and Lipinski's rule of five) and thereafter screened against the LdAPx model. Compounds having good affinity in terms of the Glide XP (extra precision) score were clustered to select diverse compounds for experimental validation. A total of 26 cluster representatives were procured and tested on promastigote culture, yielding 12 compounds with good antileishmanial activity. Out of them, six compounds were safer on the BALB/c peritoneal macrophages and were also effective against disease-causing intracellular amastigotes. Three out of six compounds inhibited recombinant LdAPx in a noncompetitive manner and also demonstrated partial reversion of the resistance property in an amphotericin B (AmB)-resistant strain, which may be due to an increased level of reactive oxygen species (ROS) and decrease of glutathione (GSH) content. However, inhibition of LdAPx in resistant parasites enhanced annexin V staining and activation of metacaspase-like protease activity, which may help in DNA fragmentation and apoptosis-like cell death. Thus, the present study will help in the search for specific hits and templates of potential therapeutic interest and therefore may facilitate the development of new drugs for combination therapy against VL. PMID:28461317

Triphenylmethane Derivatives Have High In Vitro and In Vivo Activity against the Main Causative Agents of Cutaneous Leishmaniasis

PubMed Central

de Souza Pietra, Renata Celi Carvalho; Rodrigues, Lucas Fonseca; Teixeira, Eliane; Fried, Levi; Lefkove, Benjamin; Rabello, Ana; Arbiser, Jack; Ferreira, Lucas Antônio Miranda; Fernandes, Ana Paula

2013-01-01

The current standard of care for cutaneous leishmaniasis (CL) is organic antimonial compounds, but the administration of these compounds is complicated by a low therapeutic - toxic index, as well as parenteral administration. Thus, there is an urgent need for the development of new and inexpensive therapies for the treatment of CL. In this study, we evaluate the activity of the triphenylmethane (TPM) class of compounds against three species of Leishmania which are pathogenic in humans. The TPM have a history of safe use in humans, dating back to the use of the original member of this class, gentian violet (GV), from the early 20th century. Initially, the in vitro efficacy against Leishmania (Viannia) braziliensis, L. (Leishmania) amazonensis and L. (L.) major of 9 newly synthesized TPM, in addition to GV, was tested. Inhibitory concentrations (IC) IC50 of 0.025 to 0.84 µM had been found in promastigotes in vitro assays. The four most effective compounds were then tested in amastigote intracellular assays, resulting in IC50 of 0.10 to 1.59 µM. A high degree of selectivity of antiparasitic activity over toxicity to mammalian cells was observed. Afterwards, GV and TPM 6 were tested in a topical formulation in mice infected with L. (L.) amazonensis leading to elimination of parasite burdens at the site of lesion/infection. These results demonstrated that TPM present significant anti-leishmanial activities and provide a rationale for human clinical trials of GV and other TPM. TPM are inexpensive and safe, thus using them for treatment of CL may have a major impact on public health. PMID:23341885

Assessment of sesquiterpene lactones isolated from Mikania plants species for their potential efficacy against Trypanosoma cruzi and Leishmania sp.

PubMed Central

Bivona, Augusto E.; Sánchez Alberti, Andrés; Giberti, Gustavo; Malchiodi, Emilio L.; Martino, Virginia S.; Catalan, Cesar A.; Alonso, María Rosario; Cazorla, Silvia I.

2017-01-01

Four sesquiterpene lactones, mikanolide, deoxymikanolide, dihydromikanolide and scandenolide, were isolated by a bioassay-guided fractionation of Mikania variifolia and Mikania micrantha dichloromethane extracts. Mikanolide and deoxymikanolide were the major compounds in both extracts (2.2% and 0.4% for Mikania variifolia and 21.0% and 6.4% for Mikania micrantha respectively, calculated on extract dry weight). Mikanolide, deoxymikanolide and dihydromikanolide were active against Trypanosoma cruzi epimastigotes (50% inhibitory concentrations of 0.7, 0.08 and 2.5 μg/mL, for each compound respectively). These sesquiterpene lactones were also active against the bloodstream trypomastigotes (50% inhibitory concentrations for each compound were 2.1, 1.5 and 0.3 μg/mL, respectively) and against amastigotes (50% inhibitory concentrations for each compound were 4.5, 6.3 and 8.5 μg/mL, respectively). By contrast, scandenolide was not active on Trypanosoma cruzi. Besides, mikanolide and deoxymikanolide were also active on Leishmania braziliensis promastigotes (50% inhibitory concentrations of 5.1 and 11.5 μg/mL, respectively). The four sesquiterpene lactones were tested for their cytotoxicity on THP 1 cells. Deoxymikanolide presented the highest selectivity index for trypomastigotes (SI = 54) and amastigotes (SI = 12.5). In an in vivo model of Trypanosoma cruzi infection, deoxymikanolide was able to decrease the parasitemia and the weight loss associated to the acute phase of the parasite infection. More importantly, while 100% of control mice died by day 22 after receiving a lethal T. cruzi infection, 70% of deoxymikanolide-treated mice survived. We also observed that this compound increased TNF-α and IL-12 production by macrophages, which could contribute to control T. cruzi infection. PMID:28945741

Antiparasitic activity and effect of casearins isolated from Casearia sylvestris on Leishmania and Trypanosoma cruzi plasma membrane.

PubMed

Bou, Diego Dinis; Tempone, André G; Pinto, Érika G; Lago, João Henrique G; Sartorelli, Patricia

2014-04-15

Leishmaniasis and Chagas disease are infectious diseases caused by parasite Leishmania sp. and Trypanosoma cruzi, respectively, and are included among the most neglected diseases in several underdeveloped and developing countries, with an urgent demand for new drugs. Considering the antiparasitic potential of MeOH extract from leaves of Casearia sylvestris Sw. (Salicaceae), a bioguided fractionation was conducted and afforded four active clerodane diterpenes (casearins A, B, G, and J). The obtained results indicated a superior efficacy of tested casearins against trypomastigotes of T. cruzi, with IC50 values ranging from 0.53 to 2.77 μg/ml. Leishmania infantum promastigotes were also susceptible to casearins, with IC50 values in a range between 4.45 and 9.48 μg/ml. These substances were also evaluated for mammalian cytotoxicity against NCTC cells resulting in 50% cytotoxic concentrations (CC50) ranging from 1.46 to 13.76 μg/ml. Additionally, the action of casearins on parasite membranes was investigated using the fluorescent probe SYTOX Green. The obtained results demonstrated a strong interaction of casearins A and B to the plasma membrane of T. cruzi parasites, corroborating their higher efficacy against these parasites. In contrast, the tested casearins induced no alteration in the permeability of plasma membrane of Leishmania parasites, suggesting that biochemical differences between Leishmania and T. cruzi plasma membrane might have contributed to the target effect of casearins on trypomastigotes. Thus, considering the importance of studying novel and selective drug candidates against protozoans, casearins A, B, G, and J could be used as tools to future drug design studies. Copyright © 2014 Elsevier GmbH. All rights reserved.

Monocyte-Derived Signals Activate Human Natural Killer Cells in Response to Leishmania Parasites

PubMed Central

Messlinger, Helena; Sebald, Heidi; Heger, Lukas; Dudziak, Diana; Bogdan, Christian; Schleicher, Ulrike

2018-01-01

Activated natural killer (NK) cells release interferon (IFN)-γ, which is crucial for the control of intracellular pathogens such as Leishmania. In contrast to experimental murine leishmaniasis, the human NK cell response to Leishmania is still poorly characterized. Here, we investigated the interaction of human blood NK cells with promastigotes of different Leishmania species (Leishmania major, Leishmania mexicana, Leishmania infantum, and Leishmania donovani). When peripheral blood mononuclear cells or purified NK cells and monocytes (all derived from healthy blood donors from Germany without a history of leishmaniasis) were exposed to promastigotes, NK cells showed increased surface expression of the activation marker CD69. The extent of this effect varied depending on the Leishmania species; differences between dermotropic and viscerotropic L. infantum strains were not observed. Upregulation of CD69 required direct contact between monocytes and Leishmania and was partly inhibitable by anti-interleukin (IL)-18. Unexpectedly, IL-18 was undetectable in most of the supernatants (SNs) of monocyte/parasite cocultures. Confocal fluorescence microscopy of non-permeabilized cells revealed that Leishmania-infected monocytes trans-presented IL-18 to NK cells. Native, but not heat-treated SNs of monocyte/Leishmania cocultures also induced CD69 on NK cells, indicating the involvement of a soluble heat-labile factor other than IL-18. A role for the NK cell-activating cytokines IL-1β, IL-2, IL-12, IL-15, IL-21, and IFN-α/β was excluded. The increase of CD69 was not paralleled by NK cell IFN-γ production or enhanced cytotoxicity. However, prior exposure of NK cells to Leishmania parasites synergistically increased their IFN-γ release in response to IL-12, which was dependent on endogenous IL-18. CD1c+ dendritic cells were identified as possible source of Leishmania-induced IL-12. Finally, we observed that direct contact between Leishmania and NK cells reduced the expression of CD56 mRNA and protein on NK cells. We conclude that Leishmania activate NK cells via trans-presentation of IL-18 by monocytes and by a monocyte-derived soluble factor. IL-12 is needed to elicit the IFN-γ-response of NK cells, which is likely to be an important component of the innate control of the parasite. PMID:29472914

Molecular detection and identification of Leishmania spp. in naturally infected Phlebotomus tobbi and Sergentomyia dentata in a focus of human and canine leishmaniasis in western Turkey.

PubMed

Özbel, Yusuf; Karakuş, Mehmet; Arserim, Suha K; Kalkan, Şaban Orçun; Töz, Seray

2016-03-01

Human visceral leishmaniasis (VL) is reported from 38 provinces of Turkey and dogs are accepted as main reservoir hosts. Kuşadası town, belonging to Aydın province and located in western part of Turkey, is endemic for human and canine visceral leishmaniasis caused by Leishmania infantum MON1 and MON98. In this study, phlebotomine survey was conducted to determine the vector sand fly species and to identify sand fly blood meal sources. In August and September 2012, 1027 sand fly specimens were caught using CDC light traps. Eight Phlebotomus and two Sergentomyia species with the dominancy of Phlebotomus tobbi (61.34%) were detected. A total of 622 female sand flies (571 Phlebotomus; 51 Sergentomyia) were checked for Leishmania infection by direct dissection of the midgut. The half of the midgut content was inoculated into NNN culture for isolation of the parasite. Leishmania species-specific ITS1 real time PCR, conventional PCR assays of ITS1 and hsp70 genes and subsequent sequencing were performed from extracted DNAs. A region of cytochrome b (cyt-b) gene of vertebrates based PCR was used to determine the source of blood meal of sand flies. In microscopical examinations, two female specimens (0.32%) were found naturally infected with high number and different stages of promastigotes. No growth was observed in NNN culture but Leishmania DNA was obtained from both specimens. First positive specimen was identified as P. tobbi and L. infantum DNA was detected. Second specimen was Sergentomyia dentata, but Leishmania DNA could not be identified on species level. A total of 16 blood-fed female P. tobbi specimens were used for blood meal analysis and eight, three and one specimens were positive for human, dog and mouse, respectively. This is the first detection of Leishmania promastigotes using microscopical examination in P. tobbi and S. dentata in human and canine visceral leishmaniasis endemic area in western part of Turkey. Our results indicate that, (i) P. tobbi is the principal vector species and (ii) human and dogs are main blood sources. The detection of Leishmania sp. in Sergentomyia species may be an evidence for natural cycle of Sauro-leishmania agents in the area. Copyright © 2016 Elsevier B.V. All rights reserved.

Canine leishmaniosis caused by Leishmania major and Leishmania tropica: comparative findings and serology.

PubMed

Baneth, Gad; Yasur-Landau, Daniel; Gilad, Matan; Nachum-Biala, Yaarit

2017-03-13

Infection and clinical disease associated with Leishmania major and Leishmania tropica, two common agents of human cutaneous leishmaniosis, have rarely been reported in dogs. This study describes dogs infected with these Leishmania spp. prevalent in the Middle East and North Africa, and compares the serological response of dogs infected with Leishmania infantum, L. major or L. tropica to whole promastigote antigen enzyme-linked immunosorbent assay (ELISA) of each species and to rK39 dipstick. Leishmania major infection in a 5-month-old male dog was associated with alopecic and ulcerative periocular and limb skin lesions which responded to allopurinol treatment. Infection was detected by skin and blood polymerase chain reaction (PCR) and confirmed by DNA sequencing but the dog was seronegative. Leishmania tropica infection was detected in a 3-month-old female dog co-infected with Babesia vogeli and Anaplasma platys and with no skin lesions. PCR and DNA sequencing of the blood and parasite culture were positive for L. tropica. Sera from 11 dogs infected with L. infantum, L. major or L. tropica were reactive with all three Leishmania spp. antigens except for sera from a dog with L. major infection. No significant differences were found between reactivity of dog sera to the antigen of the infecting species, or to the other Leishmania spp. antigens. Sera from dogs infected with L. infantum and L. tropica were positive with the rK39 antigen kit, while dogs with L. major infection were seronegative. Skin lesions in L. major infected dogs from this study and previous reports (n = 2) were ulcerative and located on the muzzle, feet and foot pads and not associated with generalized lymphadenomegaly and splenomegaly. In previous L. tropica infections, skin lesions were proliferative mucocutaneous in young dogs (n = 2), or associated with widespread dermatitis, lymphadenomegaly and splenomegaly in older dogs with similarity to L. infantum infection (n = 2). This study suggests that ELISA serology with whole promastigote antigen is not distinctive between L. infantum, L. major and L. tropica canine infections and that some L. major infections are not seropositive. PCR with DNA sequencing should be used to discriminate between canine infections with these three species.

C-Geranylated flavonoids from Paulownia tomentosa fruits with antimicrobial potential and synergistic activity with antibiotics.

PubMed

Navrátilová, Alice; Nešuta, Ondřej; Vančatová, Irena; Čížek, Alois; Varela-M, Ruben E; López-Abán, Julio; Villa-Pulgarin, Janny A; Mollinedo, Faustino; Muro, Antonio; Žemličková, Helena; Kadlecová, Daniela; Šmejkal, Karel

2016-08-01

Context C-6-Geranylated flavonoids possess promising biological activities. These substances could be a source of lead compounds for the development of therapeutics. Objective The study was designed to evaluate their antibacterial and antileishmanial activity. Materials and methods C-6-Geranylated flavanones were tested in micromolar concentrations against promastigote forms of Leishmania brazilensis, L. donovani, L. infantum, and L. panamensis against methicillin-resistant Staphylococcus aureus (MRSA); and synergistic potential with antibiotics was analyzed. IC50 values (after 72 h) were calculated and compared with that of miltefosine. Flow cytometry and DNA fragmentation analysis were used the mechanism of the effect. Geranylated flavanones or epigallocatechin gallate were combined with oxacillin, tetracycline, and ciprofloxacin, and the effects of these two-component combinations were evaluated. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were established (after 24 h), the synergy was measured by the checkerboard titration technique, and the sums of the fractional inhibitory concentrations (∑FICs) were computed. Results 3'-O-Methyl-5'-O-methyldiplacone and 3'-O-methyldiplacone showed good antileishmanial activities (IC50 8-42 μM). 3'-O-Methyl-5'-hydroxydiplacone activates the apoptotic death at leishmanias, the effect of 3'-O-methyl-5'-O-methyldiplacone has another mechanism. The test of the antibacterial activity showed good effects of 3'-O-methyldiplacol and mimulone against MRSA (MIC 2-16 μg/mL), and in six cases, the results showed synergistic effects when combined with oxacillin. Synergistic effects were also found for the combination of epigallocatechin gallate with tetracycline or oxacillin. Conclusion This work demonstrates anti-MRSA and antileishmanial potential of geranylated flavanones and uncovers their promising synergistic activities with antibiotics. In addition, the mechanism of antileishmanial effect is proposed.

Novel features of a PIWI-like protein homolog in the parasitic protozoan Leishmania.

PubMed

Padmanabhan, Prasad K; Dumas, Carole; Samant, Mukesh; Rochette, Annie; Simard, Martin J; Papadopoulou, Barbara

2012-01-01

In contrast to nearly all eukaryotes, the Old World Leishmania species L. infantum and L. major lack the bona fide RNAi machinery genes. Interestingly, both Leishmania genomes code for an atypical Argonaute-like protein that possesses a PIWI domain but lacks the PAZ domain found in Argonautes from RNAi proficient organisms. Using sub-cellular fractionation and confocal fluorescence microscopy, we show that unlike other eukaryotes, the PIWI-like protein is mainly localized in the single mitochondrion in Leishmania. To predict PIWI function, we generated a knockout mutant for the PIWI gene in both L. infantum (Lin) and L. major species by double-targeted gene replacement. Depletion of PIWI has no effect on the viability of insect promastigote forms but leads to an important growth defect of the mammalian amastigote lifestage in vitro and significantly delays disease pathology in mice, consistent with a higher expression of the PIWI transcript in amastigotes. Moreover, amastigotes lacking PIWI display a higher sensitivity to apoptosis inducing agents than wild type parasites, suggesting that PIWI may be a sensor for apoptotic stimuli. Furthermore, a whole-genome DNA microarray analysis revealed that loss of LinPIWI in Leishmania amastigotes affects mostly the expression of specific subsets of developmentally regulated genes. Several transcripts encoding surface and membrane-bound proteins were found downregulated in the LinPIWI((-/-)) mutant whereas all histone transcripts were upregulated in the null mutant, supporting the possibility that PIWI plays a direct or indirect role in the stability of these transcripts. Although our data suggest that PIWI is not involved in the biogenesis or the stability of small noncoding RNAs, additional studies are required to gain further insights into the role of this protein on RNA regulation and amastigote development in Leishmania.

Physicochemical and in vitro biological evaluations of furazolidone-based β-cyclodextrin complexes in Leishmania amazonensis.

PubMed

Carvalho, Suzana Gonçalves; Siqueira, Larissa Ataíde; Zanini, Marcos Santos; Dos Santos Matos, Ana Paula; Quaresma, Carla Holandino; da Silva, Luisa Mota; de Andrade, Sérgio Faloni; Severi, Juliana Aparecida; Villanova, Janaina Cecília Oliveira

2018-06-15

Recently, there have been numerous cases of leishmaniasis reported in different Brazilian states. The use of furazolidone (FZD) to treat leishmaniasis has been previously described; however, the drug is associated with adverse effects such as anorexia, weight loss, incoordination, and fatigue in dogs. Thus, in the present study, we prepared and evaluated inclusion complexes between FZD and β-cyclodextrin (β-CD) to guarantee increased drug solubility and reduce the toxicity associated with high doses. The FZD:β-CD complexes were prepared by two different techniques (kneading and lyophilization) prior to incorporation in an oral pharmaceutical dosage form. Formation of the complexes was confirmed using appropriate physicochemical methods. Antileishmanial activity against L. amazonensis was tested in vitro via a microplate assay using resazurin dye and cytotoxicity was determined using the fibroblast L929 lineage. Solubility studies showed the formation of complexes with complexation efficiencies lower than 100%. Physicochemical analysis revealed that FZD was inserted into the β-CD cavity after complexation by both methods. Biological in vitro evaluations demonstrated that free FZD and the FZD:β-CD complexes presented significant leishmanicidal activity against L. amazonensis with IC 50 values of 6.16 μg/mL and 1.83 μg/mL for the complexes prepared by kneading and lyophilization, respectively. The data showed that these complexes reduced the survival of promastigotes and presented no toxicity for tested cells. Our results indicate that the new compounds could be a cost-effective alternative for use in the pharmacotherapy of leishmaniasis in dogs infected with L. amazonensis. Copyright © 2018 Elsevier Ltd. All rights reserved.

Relationship between neural response and adaptation selectivity to form and color: an ERP study.

PubMed

Rentzeperis, Ilias; Nikolaev, Andrey R; Kiper, Daniel C; van Leeuwen, Cees

2012-01-01

Adaptation is widely used as a tool for studying selectivity to visual features. In these studies it is usually assumed that the loci of feature selective neural responses and adaptation coincide. We used an adaptation paradigm to investigate the relationship between response and adaptation selectivity in event-related potentials (ERPs). ERPs were evoked by the presentation of colored Glass patterns in a form discrimination task. Response selectivities to form and, to some extent, color of the patterns were reflected in the C1 and N1 ERP components. Adaptation selectivity to color was reflected in N1 and was followed by a late (300-500 ms after stimulus onset) effect of form adaptation. Thus for form, response and adaptation selectivity were manifested in non-overlapping intervals. These results indicate that adaptation and response selectivity can be associated with different processes. Therefore, inferring selectivity from an adaptation paradigm requires analysis of both adaptation and neural response data.

Phenology and Cover of Plant Growth Forms Predict Herbivore Habitat Selection in a High Latitude Ecosystem

PubMed Central

Fauchald, Per; Langeland, Knut; Ims, Rolf A.; Yoccoz, Nigel G.; Bråthen, Kari Anne

2014-01-01

The spatial and temporal distribution of forage quality is among the most central factors affecting herbivore habitat selection. Yet, for high latitude areas, forage quantity has been found to be more important than quality. Studies on large ungulate foraging patterns are faced with methodological challenges in both assessing animal movements at the scale of forage distribution, and in assessing forage quality with relevant metrics. Here we use first-passage time analyses to assess how reindeer movements relate to forage quality and quantity measured as the phenology and cover of growth forms along reindeer tracks. The study was conducted in a high latitude ecosystem dominated by low-palatable growth forms. We found that the scale of reindeer movement was season dependent, with more extensive area use as the summer season advanced. Small-scale movement in the early season was related to selection for younger stages of phenology and for higher abundances of generally phenologically advanced palatable growth forms (grasses and deciduous shrubs). Also there was a clear selection for later phenological stages of the most dominant, yet generally phenologically slow and low-palatable growth form (evergreen shrubs). As the summer season advanced only quantity was important, with selection for higher quantities of one palatable growth form and avoidance of a low palatable growth form. We conclude that both forage quality and quantity are significant predictors to habitat selection by a large herbivore at high latitude. The early season selectivity reflected that among dominating low palatability growth forms there were palatable phenological stages and palatable growth forms available, causing herbivores to be selective in their habitat use. The diminishing selectivity and the increasing scale of movement as the season developed suggest a response by reindeer to homogenized forage availability of low quality. PMID:24972188

Phenology and cover of plant growth forms predict herbivore habitat selection in a high latitude ecosystem.

PubMed

Iversen, Marianne; Fauchald, Per; Langeland, Knut; Ims, Rolf A; Yoccoz, Nigel G; Bråthen, Kari Anne

2014-01-01

The spatial and temporal distribution of forage quality is among the most central factors affecting herbivore habitat selection. Yet, for high latitude areas, forage quantity has been found to be more important than quality. Studies on large ungulate foraging patterns are faced with methodological challenges in both assessing animal movements at the scale of forage distribution, and in assessing forage quality with relevant metrics. Here we use first-passage time analyses to assess how reindeer movements relate to forage quality and quantity measured as the phenology and cover of growth forms along reindeer tracks. The study was conducted in a high latitude ecosystem dominated by low-palatable growth forms. We found that the scale of reindeer movement was season dependent, with more extensive area use as the summer season advanced. Small-scale movement in the early season was related to selection for younger stages of phenology and for higher abundances of generally phenologically advanced palatable growth forms (grasses and deciduous shrubs). Also there was a clear selection for later phenological stages of the most dominant, yet generally phenologically slow and low-palatable growth form (evergreen shrubs). As the summer season advanced only quantity was important, with selection for higher quantities of one palatable growth form and avoidance of a low palatable growth form. We conclude that both forage quality and quantity are significant predictors to habitat selection by a large herbivore at high latitude. The early season selectivity reflected that among dominating low palatability growth forms there were palatable phenological stages and palatable growth forms available, causing herbivores to be selective in their habitat use. The diminishing selectivity and the increasing scale of movement as the season developed suggest a response by reindeer to homogenized forage availability of low quality.

78 FR 63464 - William D. Ford Federal Direct Loan Program Repayment Plan Selection Form; Extension of Public...

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Methods for producing thin film charge selective transport layers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hammond, Scott Ryan; Olson, Dana C.; van Hest, Marinus Franciscus Antonius Maria

Methods for producing thin film charge selective transport layers are provided. In one embodiment, a method for forming a thin film charge selective transport layer comprises: providing a precursor solution comprising a metal containing reactive precursor material dissolved into a complexing solvent; depositing the precursor solution onto a surface of a substrate to form a film; and forming a charge selective transport layer on the substrate by annealing the film.

Identification of Leishmania donovani Topoisomerase 1 inhibitors via intuitive scaffold hopping and bioisosteric modification of known Top 1 inhibitors

NASA Astrophysics Data System (ADS)

Mamidala, Rajinikanth; Majumdar, Papiya; Jha, Kunal Kumar; Bathula, Chandramohan; Agarwal, Rahul; Chary, M. Thirumala; Mazumdar, H. K.; Munshi, Parthapratim; Sen, Subhabrata

2016-05-01

A library of arylidenefuropyridinediones was discovered as potent inhibitors of Leishmania donovani Topoisomerase 1 (LdTop1) where the active molecules displayed considerable inhibition with single digit micromolar EC50 values. This molecular library was designed via intuitive scaffold hopping and bioisosteric modification of known topoisomerase 1 inhibitors such as camptothecin, edotecarin and etc. The design was rationalized by molecular docking analysis of the compound prototype with human topoisomerase 1 (HTop1) and Leishmania donovani topoisomerase 1(LdTop1). The most active compound 4 displayed no cytotoxicity against normal mammalian COS7 cell line (~100 fold less inhibition at the EC50). Similar to camptothecin, 4 interacted with free LdTop1 as observed in the preincubation DNA relaxation inhibition experiment. It also displayed anti-protozoal activity against Leishmania donovani promastigote. Crystal structure investigation of 4 and its molecular modelling with LdTop1 revealed putative binding sites in the enzyme that could be harnessed to generate molecules with better potency.

Genotoxicity and antileishmanial activity evaluation of Physalis angulata concentrated ethanolic extract.

PubMed

Nogueira, Renata Campos; Rocha, Vinicius Pinto Costa; Nonato, Fabiana Regina; Tomassini, Therezinha Coelho Barbosa; Ribeiro, Ivone Maria; dos Santos, Ricardo Ribeiro; Soares, Milena Botelho Pereira

2013-11-01

Antileishmanial in vitro tests, as well as Ames and micronucleus assays were performed with a concentrated ethanolic extract of Physalis angulata (EEPA) RESULTS: EEPA did not present mutagenic effect in Salmonella typhimurium strains at concentration reaching 3000 μg/plate and did not induce mutagenic effects after two oral administrations with a 24h interval at a dose level of 2000 mg/kg. EEPA presented antileishmanial activity and presented an IC₅₀ value of 5.35 ± 2.50 μg/mL and 4.50 ± 1.17 μg/mL against Leishmania amazonensis and Leishmania braziliensis promastigotes, respectively. In the cytotoxicity test against macrophages, the EEPA had a LC₅₀ of 6.14 ± 0.59 μg/mL. Importantly, the IC₅₀ against L. amazonensis intracellular amastigotes was 1.23 ± 0.11 μg/mL. EEPA extract is non-mutagenic and presented a promising pharmacological effect against Leishmania parasites. Copyright © 2013 Elsevier B.V. All rights reserved.

Nanobiotechnologic approach to a promising vaccine prototype for immunisation against leishmaniasis: a fast and effective method to incorporate GPI-anchored proteins of Leishmania amazonensis into liposomes.

PubMed

Colhone, Marcelle Carolina; Silva-Jardim, Izaltina; Stabeli, Rodrigo Guerino; Ciancaglini, Pietro

2015-01-01

Liposomes are known to be a potent adjuvant for a wide range of antigens, as well as appropriate antigen carriers for antibody generation response in vivo. In addition, liposomes are effective vehicles for peptides and proteins, thus enhancing their immunogenicity. Considering these properties of liposomes and the antigenicity of the Leishmania membrane proteins, we evaluated if liposomes carrying glycosylphosphatidylinositol (GPI)-anchored proteins of Leishmania amazonensis promastigotes could induce protective immunity in BALB/c mice. To assay protective immunity, BALB/c mice were intraperitoneally injected with liposomes, GPI-protein extract (EPSGPI) as well as with the proteoliposomes carrying GPI-proteins. Mice inoculated with EPSGPI and total protein present in constitutive proteoliposomes displayed a post-infection protection of about 70% and 90%, respectively. The liposomes are able to work as adjuvant in the EPSGPI protection. These systems seem to be a promising vaccine prototype for immunisation against leishmaniasis.

Prevalence of antibodies to Trypanosoma cruzi, Leishmania infantum, Encephalitozoon cuniculi, Sarcocystis neurona, and Neospora caninum in Capybara, Hydrochoerus hydrochaeris, from São Paulo State, Brazil.

PubMed

Valadas, Samantha; Gennari, Solange Maria; Yai, Lucia Eiko Oishi; Rosypal, Alexa C; Lindsay, David S

2010-06-01

Little is known about the importance of capybara, Hydrochoerus hydrochaeris, as reservoirs for parasites of zoonotic or veterinary importance. Sera from 63 capybaras, from 6 counties in the state of São Paulo, Brazil, were examined for antibodies to Trypanosoma cruzi, Leishmania infantum, Encephalitozoon cuniculi, Sarcocystis neurona, and Neospora caninum using an indirect immunofluorescent antibody test. Five (8%) of the 63 capybaras had antibodies to T. cruzi epimastigotes. None of the samples from capybara reacted positively with L. infantum promastigotes or with spores of E. cuniculi . Two (3%) of the serum samples were positive for antibodies to S. neurona merozoites, and 2 (3%) of the serum samples were positive for antibodies to N. caninum tachyzoites. A serum sample from 1 capybara was positive for antibodies to both T. cruzi and N. caninum. None of the remaining 62 samples reacted with more than 1 parasite.

Description of Phytomonas oxycareni n. sp. from the Salivary Glands of Oxycarenus lavaterae.

PubMed

Seward, Emily A; Votýpka, Jan; Kment, Petr; Lukeš, Julius; Kelly, Steven

2017-02-01

Phytomonas spp. (phytomonads) are a diverse and globally distributed group of unicellular eukaryotes that parasitize a wide range of plants and are transmitted by insect hosts. Here we report the discovery and characterisation of a new species of Phytomonas, named Phytomonas oxycareni n. sp., which was obtained from the salivary glands of the invasive species of true bug Oxycarenus lavaterae (Heteroptera). The new Phytomonas species exhibits a long slender promastigote morphology and can be found both within the lumen of the insect host's salivary glands as well as within the cells of the salivary gland itself. Sampling multiple individuals from the same population post-winter hibernation on two consecutive years revealed that infection was persistent over time. Finally, phylogenetic analyses of small subunit ribosomal RNA genes revealed that this species is sister to other species within the genus Phytomonas, providing new insight into the evolutionary history of the clade. Copyright © 2016 Elsevier GmbH. All rights reserved.

A gene encoding the plant-like alternative oxidase is present in Phytomonas but absent in Leishmania spp.

PubMed

Van Hellemond, J J; Simons, B; Millenaar, F F; Tielens, A G

1998-01-01

The constituents of the respiratory chain are believed to differ among the trypanosomatids; bloodstream stages of African trypanosomes and Phytomonas promastigotes oxidize ubiquinol by a ubiquinol:oxygen oxidoreductase, also known as alternative oxidase, whereas Leishmania spp. oxidize ubiquinol via a classic cytochrome-containing respiratory chain. The molecular basis for this elementary difference in ubiquinol oxidation by the mitochondrial electron-transport chain in distinct trypanosomatids was investigated. The presence of a gene encoding the plant-like alternative oxidase could be demonstrated in Phytomonas and Trypanosoma brucei, trypanosomatids that are known to contain alternative oxidase activity. Our results further demonstrated that Leishmania spp. lack a gene encoding the plant-like alternative oxidase, and therefore, all stages of Leishmania spp. will lack the alternative oxidase protein. The observed fundamental differences between the respiratory chains of distinct members of the trypanosomatid family are thus caused by the presence or absence of a gene encoding the plant-like alternative oxidase.

A Multiplatform Metabolomic Approach to the Basis of Antimonial Action and Resistance in Leishmania infantum

PubMed Central

Castilho-Martins, Emerson A.; Tavares, Marina F. M.; Barbas, Coral; López-Gonzálvez, Ángeles; Rivas, Luis

2015-01-01

There is a rising resistance against antimony drugs, the gold-standard for treatment until some years ago. That is a serious problem due to the paucity of drugs in current clinical use. In a research to reveal how these drugs affect the parasite during treatment and to unravel the underlying basis for their resistance, we have employed metabolomics to study treatment in Leishmania infantum promastigotes. This was accomplished first through the untargeted analysis of metabolic snapshots of treated and untreated parasites both resistant and responders, utilizing a multiplatform approach to give the widest as possible coverage of the metabolome, and additionally through novel monitoring of the origin of the detected alterations through a 13C traceability experiment. Our data stress a multi-target metabolic alteration with treatment, affecting in particular the cell redox system that is essential to cope with detoxification and biosynthetic processes. Additionally, relevant changes were noted in amino acid metabolism. Our results are in agreement with other authors studying other Leishmania species. PMID:26161866

Isolation and characterization of an anti-leishmanial disintegrin from Cerastes cerastes venom.

PubMed

Allane, Dihia; Oussedik-Oumehdi, Habiba; Harrat, Zoubir; Seve, Michel; Laraba-Djebari, Fatima

2018-02-01

Investigating new antimicrobial and antiparasitic components from Viperidae venoms represents an alternative therapeutic strategy. In this study, we report the characterization of a disintegrin isolated from Cerastes cerastes venom, exhibiting antiparasitic activity on Leishmania infantum promastigotes. Indeed, isolated disintegrin, referred to Disintegrin_Cc, induced 84.75% of parasiticidal activity and deep morphological alterations on the parasites. SDS-PAGE analysis indicated that this disintegrin was homogenous. This dimeric disintegrin of 14,193.97 Da contains an RGD domain and four intramolecular disulfide bridges. It presents a high percentage of identity with other related snake disintegrins. Predicted 3D structure indicated that this peptide shares partial homology with well-known active antimicrobial peptides. Disintegrin_Cc inhibited 80% of arachidonic acid-induced platelet aggregation. The obtained results suggest that the isolated molecule plays a dual role as a disintegrin and as an anti-leishmanial compound. This component could be useful as a drug in the treatment of leishmaniasis. © 2017 Wiley Periodicals, Inc.

78 FR 72139 - Forms Submitted to the Office of Management and Budget for Extension of Clearance

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9 CFR 121.4 - Overlap select agents and toxins.

Code of Federal Regulations, 2012 CFR

2012-01-01

... select agent or toxin to APHIS or CDC. (i) The seizure of any of the following overlap select agents and.... This report must be followed by submission of APHIS/CDC Form 4 within 7 calendar days after seizure of the overlap select agent or toxin. (ii) For all other overlap select agents or toxins, APHIS/CDC Form...

Modulation of Aneuploidy in Leishmania donovani during Adaptation to Different In Vitro and In Vivo Environments and Its Impact on Gene Expression.

PubMed

Dumetz, F; Imamura, H; Sanders, M; Seblova, V; Myskova, J; Pescher, P; Vanaerschot, M; Meehan, C J; Cuypers, B; De Muylder, G; Späth, G F; Bussotti, G; Vermeesch, J R; Berriman, M; Cotton, J A; Volf, P; Dujardin, J C; Domagalska, M A

2017-05-23

Aneuploidy is usually deleterious in multicellular organisms but appears to be tolerated and potentially beneficial in unicellular organisms, including pathogens. Leishmania , a major protozoan parasite, is emerging as a new model for aneuploidy, since in vitro -cultivated strains are highly aneuploid, with interstrain diversity and intrastrain mosaicism. The alternation of two life stages in different environments (extracellular promastigotes and intracellular amastigotes) offers a unique opportunity to study the impact of environment on aneuploidy and gene expression. We sequenced the whole genomes and transcriptomes of Leishmania donovani strains throughout their adaptation to in vivo conditions mimicking natural vertebrate and invertebrate host environments. The nucleotide sequences were almost unchanged within a strain, in contrast to highly variable aneuploidy. Although high in promastigotes in vitro , aneuploidy dropped significantly in hamster amastigotes, in a progressive and strain-specific manner, accompanied by the emergence of new polysomies. After a passage through a sand fly, smaller yet consistent karyotype changes were detected. Changes in chromosome copy numbers were correlated with the corresponding transcript levels, but additional aneuploidy-independent regulation of gene expression was observed. This affected stage-specific gene expression, downregulation of the entire chromosome 31, and upregulation of gene arrays on chromosomes 5 and 8. Aneuploidy changes in Leishmania are probably adaptive and exploited to modulate the dosage and expression of specific genes; they are well tolerated, but additional mechanisms may exist to regulate the transcript levels of other genes located on aneuploid chromosomes. Our model should allow studies of the impact of aneuploidy on molecular adaptations and cellular fitness. IMPORTANCE Aneuploidy is usually detrimental in multicellular organisms, but in several microorganisms, it can be tolerated and even beneficial. Leishmania -a protozoan parasite that kills more than 30,000 people each year-is emerging as a new model for aneuploidy studies, as unexpectedly high levels of aneuploidy are found in clinical isolates. Leishmania lacks classical regulation of transcription at initiation through promoters, so aneuploidy could represent a major adaptive strategy of this parasite to modulate gene dosage in response to stressful environments. For the first time, we document the dynamics of aneuploidy throughout the life cycle of the parasite, in vitro and in vivo We show its adaptive impact on transcription and its interaction with regulation. Besides offering a new model for aneuploidy studies, we show that further genomic studies should be done directly in clinical samples without parasite isolation and that adequate methods should be developed for this. Copyright © 2017 Dumetz et al.

Method of forming a chemical composition

DOEpatents

Bingham, Dennis N.; Wilding, Bruce M.; Klingler, Kerry M.; Zollinger, William T.; Wendt, Kraig M.

2007-10-09

A method of forming a chemical composition such as a chemical hydride is described and which includes the steps of selecting a composition having chemical bonds and which is capable of forming a chemical hydride; providing a source of hydrogen; and exposing the selected composition to an amount of ionizing radiation to encourage the changing of the chemical bonds of the selected composition, and chemically reacting the selected composition with the source of hydrogen to facilitate the formation of a chemical hydride.

Top down and bottom up selection drives variations in frequency and form of a visual signal.

PubMed

Yeh, Chien-Wei; Blamires, Sean J; Liao, Chen-Pan; Tso, I-Min

2015-03-30

The frequency and form of visual signals can be shaped by selection from predators, prey or both. When a signal simultaneously attracts predators and prey selection may favour a strategy that minimizes risks while attracting prey. Accordingly, varying the frequency and form of the silken decorations added to their web may be a way that Argiope spiders minimize predation while attracting prey. Nonetheless, the role of extraneous factors renders the influences of top down and bottom up selection on decoration frequency and form variation difficult to discern. Here we used dummy spiders and decorations to simulate four possible strategies that the spider Argiope aemula may choose and measured the prey and predator attraction consequences for each in the field. The strategy of decorating at a high frequency with a variable form attracted the most prey, while that of decorating at a high frequency with a fixed form attracted the most predators. These results suggest that mitigating the cost of attracting predators while maintaining prey attraction drives the use of variation in decoration form by many Argiope spp. when decorating frequently. Our study highlights the importance of considering top-down and bottom up selection pressure when devising evolutionary ecology experiments.

System for routing messages in a vertex symmetric network by using addresses formed from permutations of the transmission line indicees

DOEpatents

Faber, Vance; Moore, James W.

1992-01-01

A network of interconnected processors is formed from a vertex symmetric graph selected from graphs .GAMMA..sub.d (k) with degree d, diameter k, and (d+1)!/(d-k+1)! processors for each d.gtoreq.k and .GAMMA..sub.d (k,-1) with degree 3-1, diameter k+1, and (d+1)!/(d-k+1)! processors for each d.gtoreq.k.gtoreq.4. Each processor has an address formed by one of the permutations from a predetermined sequence of letters chosen a selected number of letters at a time, and an extended address formed by appending to the address the remaining ones of the predetermined sequence of letters. A plurality of transmission channels is provided from each of the processors, where each processor has one less channel than the selected number of letters forming the sequence. Where a network .GAMMA..sub.d (k,-1) is provided, no processor has a channel connected to form an edge in a direction .delta..sub.1. Each of the channels has an identification number selected from the sequence of letters and connected from a first processor having a first extended address to a second processor having a second address formed from a second extended address defined by moving to the front of the first extended address the letter found in the position within the first extended address defined by the channel identification number. The second address is then formed by selecting the first elements of the second extended address corresponding to the selected number used to form the address permutations.

Selective Erasure of Distinct Forms of Long-Term Synaptic Plasticity Underlying Different Forms of Memory in the Same Postsynaptic Neuron.

PubMed

Hu, Jiangyuan; Ferguson, Larissa; Adler, Kerry; Farah, Carole A; Hastings, Margaret H; Sossin, Wayne S; Schacher, Samuel

2017-07-10

Generalization of fear responses to non-threatening stimuli is a feature of anxiety disorders. It has been challenging to target maladaptive generalized memories without affecting adaptive memories. Synapse-specific long-term plasticity underlying memory involves the targeting of plasticity-related proteins (PRPs) to activated synapses. If distinct tags and PRPs are used for different forms of plasticity, one could selectively remove distinct forms of memory. Using a stimulation paradigm in which associative long-term facilitation (LTF) occurs at one input and non-associative LTF at another input to the same postsynaptic neuron in an Aplysia sensorimotor preparation, we found that each form of LTF is reversed by inhibiting distinct isoforms of protein kinase M (PKM), putative PRPs, in the postsynaptic neuron. A dominant-negative (dn) atypical PKM selectively reversed associative LTF, while a dn classical PKM selectively reversed non-associative LTF. Although both PKMs are formed from calpain-mediated cleavage of protein kinase C (PKC) isoforms, each form of LTF is sensitive to a distinct dn calpain expressed in the postsynaptic neuron. Associative LTF is blocked by dn classical calpain, whereas non-associative LTF is blocked by dn small optic lobe (SOL) calpain. Interfering with a putative synaptic tag, the adaptor protein KIBRA, which protects the atypical PKM from degradation, selectively erases associative LTF. Thus, the activity of distinct PRPs and tags in a postsynaptic neuron contribute to the maintenance of different forms of synaptic plasticity at separate inputs, allowing for selective reversal of synaptic plasticity and providing a cellular basis for developing therapeutic strategies for selectively reversing maladaptive memories. Copyright © 2017 Elsevier Ltd. All rights reserved.

Article with buffer layer and method of making the same

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCamy, James W.; Ma, Zhixun; Kabagambe, Benjamin

A method of forming a coating layer on a glass substrate in a glass manufacturing process includes: providing a first coating precursor material for a selected coating layer composition to at least one multislot coater to form a first coating region of the selected coating layer; and providing a second coating precursor material for the selected coating layer composition to the multislot coater to form a second coating region of the selected coating layer over the first region. The first coating precursor material is different than the second precursor coating material.

Preformulation considerations for controlled release dosage forms. Part III. Candidate form selection using numerical weighting and scoring.

PubMed

Chrzanowski, Frank

2008-01-01

Two numerical methods, Decision Analysis (DA) and Potential Problem Analysis (PPA) are presented as alternative selection methods to the logical method presented in Part I. In DA properties are weighted and outcomes are scored. The weighted scores for each candidate are totaled and final selection is based on the totals. Higher scores indicate better candidates. In PPA potential problems are assigned a seriousness factor and test outcomes are used to define the probability of occurrence. The seriousness-probability products are totaled and forms with minimal scores are preferred. DA and PPA have never been compared to the logical-elimination method. Additional data were available for two forms of McN-5707 to provide complete preformulation data for five candidate forms. Weight and seriousness factors (independent variables) were obtained from a survey of experienced formulators. Scores and probabilities (dependent variables) were provided independently by Preformulation. The rankings of the five candidate forms, best to worst, were similar for all three methods. These results validate the applicability of DA and PPA for candidate form selection. DA and PPA are particularly applicable in cases where there are many candidate forms and where each form has some degree of unfavorable properties.

In situ preparation of powder and the sorption behaviors of molecularly imprinted polymers through the complexation between polymer ion of methyl methacrylate/acrylic acid and Ca++ ion.

PubMed

Chough, Sung Hyo; Park, Kwang Ho; Cho, Seung Jin; Park, Hye Ryoung

2014-09-02

Molecularly imprinted polymer (MIP) powders were prepared using a simple complexation strategy between the polymer carboxylate groups and template molecule followed by metal cation cross-linking of residual polymer carboxylates. Polymer powders were formed in situ by templating carboxylic acid containing polymers with 4-ethylaniline (4-EA), followed by addition of an aqueous CaCl2 solution. The solution remained homogeneous. The powders were prepared by precipitation by slowly adding a non-solvent, H2O, to the mixture. The resulting particles were very porous with uptake capacity that approached the theoretical value. We suggest two types of complexes are formed between the template, 4-EA, and polymer. The isolated entry type forms well defined cavities for the template with high specific selectivity, while the adjacent entry type forms wider binding sites without specific sorption for isomeric molecules. To evaluate conditions for forming materials with high affinity and selectivity, three MIPs were prepared containing 0.5, 1.0, and 1.5 equivalents of template to the base polymer. The MIP containing 0.5 eq showed higher specific selectivity to 4-EA, but the MIP containing 1.5 eq had noticeably lower selectivity. The lower selectivity is attributed to poorly formed binding sites with little selective sorption to any isomer when the higher ratio of template was used. However at the lower ratio of template the isolated entry is preferably formed to produce well defined binding cavities with higher selectivity to template. Copyright © 2014 Elsevier B.V. All rights reserved.

In vitro antileishmanial and cytotoxicity activities of essential oils from Haplophyllum tuberculatum A. Juss leaves, stems and aerial parts.

PubMed

Hamdi, Assia; Bero, Joanne; Beaufay, Claire; Flamini, Guido; Marzouk, Zohra; Vander Heyden, Yvan; Quetin-Leclercq, Joelle

2018-02-14

Plants used for traditional medicine produce diverse and complex secondary metabolites exhibiting various medicinal properties. The medicinal plant Haplophyllum tuberculatum is used by native people against malaria and parasitic infections. In this study and in order to contribute for the search of new natural drugs for leishmaniasis, the essential oils of H. tuberculatum leaves, stems and aerial parts (leaves+stems) collected in two different periods, 2013 and 2015, and their components by GC/FID and GC/MS analyses were investigated. Those collected in 2013 were also re-analyzed two years later. The extracted oils were screened in vitro for anti-leishmanial activity on Leishmania mexicana mexicana (L.m.m.) promastigotes and cytotoxicity on the Chinese Hamster Ovary (CHO) cell line. Limonene (1.5 - 8%), its isomers (R- (+)-limonene and S-(-)-limonene), linalool and octanol were also tested. Results showed that the chemical composition varied according to the year of collection. Though major compounds remain almost the same, qualitative and quantitative variations in the composition of the EOs can be observed between the two years of collection, with some minor compounds identified only in one type of samples. Variation in the composition were also observed in the re-analyzed volatile oils, showing stability concerns. The essential oils and R-(+)-limonene showed moderate anti-leishmanial activity. Their IC 50 range from 6.48 to 50.28 μg/ml. Cytotoxicity assays for theses volatile extracts, R- (+)-limonene and S- (-)-limonene on CHO cells showed relatively potent cytotoxicity with a selectivity index <10. Their CC 50 range from 27.79 to 82.56 μg/ml. The findings of the present study demonstrated that H. tuberculatum might not be considered as a natural source for production of new anti-leishmanial agents without further analyzing its eventual in vivo toxicity as well as that of major pure compounds.

Top down and bottom up selection drives variations in frequency and form of a visual signal

PubMed Central

Yeh, Chien-Wei; Blamires, Sean J.; Liao, Chen-Pan; Tso, I.-Min

2015-01-01

The frequency and form of visual signals can be shaped by selection from predators, prey or both. When a signal simultaneously attracts predators and prey, selection may favour a strategy that minimizes risks while attracting prey. Accordingly, varying the frequency and form of the silken decorations added to their web may be a way that Argiope spiders minimize predation while attracting prey. Nonetheless, the role of extraneous factors renders the influences of top down and bottom up selection on decoration frequency and form variation difficult to discern. Here we used dummy spiders and decorations to simulate four possible strategies that the spider Argiope aemula may choose and measured the prey and predator attraction consequences for each in the field. The strategy of decorating at a high frequency with a variable form attracted the most prey, while that of decorating at a high frequency with a fixed form attracted the most predators. These results suggest that mitigating the cost of attracting predators while maintaining prey attraction drives the use of variation in decoration form by many Argiope spp. when decorating frequently. Our study highlights the importance of considering top-down and bottom up selection pressure when devising evolutionary ecology experiments. PMID:25828030

Adolescents' Visual Preference for Color over Form.

ERIC Educational Resources Information Center

Uba, Anselm

1985-01-01

Examines whether Nigerian adolescent girls are more likely to demonstrate superior performance in a task involving cultural differences in visual selective attention preference for color over form than boys are. Students (N=100) completed the Visual Selective Attention Color-Form Matching Experiment. Results confirmed the hypothesis. (BH)

Forms and Formalities: A Resource.

ERIC Educational Resources Information Center

Clarkson, Glenn; Warpinski, Robert

This report provides a selection of examples of National Diffusion Network participant forms utilized between Developer/Demonstrators, State Facilitators, and Local Educational Agencies. They were selected to demonstrate variety and to be a source of ideas for others. Forms are grouped in five categories: needs assessment, adoption agreement,…

H-11-linked gene has a parallel effect on Leishmania major and L. donovani infections in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blackwell, J.M.; Hale, C.; Roberts, M.B.

1985-01-01

The courses of visceral infection following intravenous injection of Leishmania donovani amastigotes, or lesion growth following subcutaneous injection of L. major promastigotes, were examined in B10.129(10M) (H-2b, H-11b) mice and compared with disease profiles observed in congenic C57BL/10ScSn(= B10) (H-2b, H-11a) and B10.D2/n (H-2d, H-11a) mice, and in BALB/mice. Possession of alternative alleles at H-11 and closely linked loci transformed the normal curing/healing phenotype of B10 mice into a characteristically different noncuring/nonhealing phenotype affecting both visceral and subcutaneous infections in B10.129(10M) mice. In reciprocal radiation bone marrow chimeras made between the congenic B10 and B10.129(10M) strains, both cure and noncuremore » phenotypes were transferable with the donor hematopoietic system. Although it was possible to demonstrate transfer of suppression with T-enriched spleen cells from day 61 L. donovani-infected B10.129(10M) donor mice into 550 rad syngeneic recipients, the pretreatment of mice with sublethal irradiation did not, as in the earlier studies of Scl-controlled L. major nonhealing or H-2-controlled L. donovani noncure phenotypes, have a clear or consistent prophylactic effect. Together with the progressive disease profile observed even for L. donovani at low parasite doses this suggests that, despite their ability to develop initial delayed-type hypersensitivity reactions to parasite antigen early in L. major infection, B10.129(10M) mice possess some inherent defect in ability to mount a cell-mediated response effective at the level of macrophage neishmanial activity in vivo even when suppressor T cells are not generated. Elucidation of this characteristically different noncuring/nonhealing phenotye may provide important insight into common events involved in the development of the cell-mediated immune response to both visceral and subcutaneous forms of leishmaniasis.« less

Characterization of a midgut mucin-like glycoconjugate of Lutzomyia longipalpis with a potential role in Leishmania attachment.

PubMed

Myšková, Jitka; Dostálová, Anna; Pěničková, Lucie; Halada, Petr; Bates, Paul A; Volf, Petr

2016-07-25

Leishmania parasites are transmitted by phlebotomine sand flies and a crucial step in their life-cycle is the binding to the sand fly midgut. Laboratory studies on sand fly competence to Leishmania parasites suggest that the sand flies fall into two groups: several species are termed "specific/restricted" vectors that support the development of one Leishmania species only, while the others belong to so-called "permissive" vectors susceptible to a wide range of Leishmania species. In a previous study we revealed a correlation between specificity vs permissivity of the vector and glycosylation of its midgut proteins. Lutzomyia longipalpis and other four permissive species tested possessed O-linked glycoproteins whereas none were detected in three specific vectors examined. We used a combination of biochemical, molecular and parasitological approaches to characterize biochemical and biological properties of O-linked glycoprotein of Lu. longipalpis. Lectin blotting and mass spectrometry revealed that this molecule with an apparent molecular weight about 45-50 kDa corresponds to a putative 19 kDa protein with unknown function detected in a midgut cDNA library of Lu. longipalpis. We produced a recombinant glycoprotein rLuloG with molecular weight around 45 kDa. Anti-rLuloG antibodies localize the native glycoprotein on epithelial midgut surface of Lu. longipalpis. Although we could not prove involvement of LuloG in Leishmania attachment by blocking the native protein with anti-rLuloG during sand fly infections, we demonstrated strong binding of rLuloG to whole surface of Leishmania promastigotes. We characterized a novel O-glycoprotein from sand fly Lutzomyia longipalpis. It has mucin-like properties and is localized on the luminal side of the midgut epithelium. Recombinant form of the protein binds to Leishmania parasites in vitro. We propose a role of this molecule in Leishmania attachment to sand fly midgut.

Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection.

PubMed

Tiwananthagorn, Saruda; Kato, Hirotomo; Yeewa, Ranchana; Muengpan, Amontip; Polseela, Raxsina; Leelayoova, Saovanee

2017-02-01

Leishmaniasis caused by Leishmania martiniquensis infection has been reported in human and domestic animals of Martinique Island, Germany, Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of disseminated cutaneous and visceral forms co-existence render the urgent need of specific diagnostic tool to identify the natural sand fly vectors for effective prevention and control strategies. Loop-mediated isothermal amplification (LAMP) of 18S rRNA gene as well as polymerase chain reaction (PCR) of minicircle kinetoplast DNA gene (PCR-mkDNA) have never been applied to detect L. martiniquensis and L. siamensis in sand fly vectors. The present study was aimed to validate malachite green-LAMP (MG-LAMP) and PCR-mkDNA techniques to detect L. martiniquensis in sand fly vectors, compared with the conventional PCR of internal transcribed spacer 1 (PCR-ITS1). We compared the validity of LAMP of 18S rRNA gene and PCR-mkDNA, to PCR-ITS1 in simulation model of L. martiniquensis infection in Sergentomyia gemmea sand flies. Attributable to the sensitivity and specificity, PCR-mkDNA was consecutively applied to detect L. martiniquensis in 380 female sand fly individuals captured in the newly identified affected region of Lamphun Province, Thailand. Results showed that PCR-mkDNA could detect at least one promastigote per sand fly, which was 10-time superior to LAMP and PCR-ITS1. In addition, PCR-mkDNA was more specific, able to differentiate L. martiniquensis from other viscerotropic Leishmania species, such as L. siamensis, L. (L.) donovani, and L. (L.) infantum. Consecutively, mass screening of L. martiniquensis in 380 female sand fly individuals by PCR-mkDNA was implemented in a new affected area of Thailand where a patient with leishmaniasis/HIV co-infection resides; however Leishmania DNA was undetected. In conclusion, PCR-mkDNA is a promising tool for molecular mass screening of L. martiniquensis infection in outbreak areas where several species of Leishmania and sand flies co-exist.

A Novel Spirooxindole Derivative Inhibits the Growth of Leishmania donovani Parasites both In Vitro and In Vivo by Targeting Type IB Topoisomerase

PubMed Central

Saha, Sourav; Acharya, Chiranjit; Pal, Uttam; Chowdhury, Somenath Roy; Sarkar, Kahini; Maiti, Nakul C.

2016-01-01

Visceral leishmaniasis is a fatal parasitic disease, and there is an emergent need for development of effective drugs against this neglected tropical disease. We report here the development of a novel spirooxindole derivative, N-benzyl-2,2′α-3,3′,5′,6′,7′,7α,α′-octahydro-2methoxycarbonyl-spiro[indole-3,3′-pyrrolizidine]-2-one (compound 4c), which inhibits Leishmania donovani topoisomerase IB (LdTopIB) and kills the wild type as well as drug-resistant parasite strains. This compound inhibits catalytic activity of LdTopIB in a competitive manner. Unlike camptothecin (CPT), the compound does not stabilize the DNA-topoisomerase IB cleavage complex; rather, it hinders drug-DNA-enzyme covalent complex formation. Fluorescence studies show that the stoichiometry of this compound binding to LdTopIB is 2:1 (mole/mole), with a dissociation constant of 6.65 μM. Molecular docking with LdTopIB using the stereoisomers of compound 4c produced two probable hits for the binding site, one in the small subunit and the other in the hinge region of the large subunit of LdTopIB. This spirooxindole is highly cytotoxic to promastigotes of L. donovani and also induces apoptosis-like cell death in the parasite. Treatment with compound 4c causes depolarization of mitochondrial membrane potential, formation of reactive oxygen species inside parasites, and ultimately fragmentation of nuclear DNA. Compound 4c also effectively clears amastigote forms of wild-type and drug-resistant parasites from infected mouse peritoneal macrophages but has less of an effect on host macrophages. Moreover, compound 4c showed strong antileishmanial efficacies in the BALB/c mouse model of leishmaniasis. This compound potentially can be used as a lead for developing excellent antileishmanial agents against emerging drug-resistant strains of the parasite. PMID:27503653

The crystal structure of the Leishmania infantum Silent Information Regulator 2 related protein 1: Implications to protein function and drug design.

PubMed

Ronin, Céline; Costa, David Mendes; Tavares, Joana; Faria, Joana; Ciesielski, Fabrice; Ciapetti, Paola; Smith, Terry K; MacDougall, Jane; Cordeiro-da-Silva, Anabela; Pemberton, Iain K

2018-01-01

The de novo crystal structure of the Leishmania infantum Silent Information Regulator 2 related protein 1 (LiSir2rp1) has been solved at 1.99Å in complex with an acetyl-lysine peptide substrate. The structure is broadly commensurate with Hst2/SIRT2 proteins of yeast and human origin, reproducing many of the structural features common to these sirtuin deacetylases, including the characteristic small zinc-binding domain, and the larger Rossmann-fold domain involved in NAD+-binding interactions. The two domains are linked via a cofactor binding loop ordered in open conformation. The peptide substrate binds to the LiSir2rp1 protein via a cleft formed between the small and large domains, with the acetyl-lysine side chain inserting further into the resultant hydrophobic tunnel. Crystals were obtained only with recombinant LiSir2rp1 possessing an extensive internal deletion of a proteolytically-sensitive region unique to the sirtuins of kinetoplastid origin. Deletion of 51 internal amino acids (P253-E303) from LiSir2rp1 did not appear to alter peptide substrate interactions in deacetylation assays, but was indispensable to obtain crystals. Removal of this potentially flexible region, that otherwise extends from the classical structural elements of the Rossmann-fold, specifically the β8-β9 connector, appears to result in lower accumulation of the protein when expressed from episomal vectors in L. infantum SIR2rp1 single knockout promastigotes. The biological function of the large serine-rich insertion in kinetoplastid/trypanosomatid sirtuins, highlighted as a disordered region with strong potential for post-translational modification, remains unknown but may confer additional cellular functions that are distinct from their human counterparts. These unique molecular features, along with the resolution of the first kinetoplastid sirtuin deacetylase structure, present novel opportunities for drug design against a protein target previously established as essential to parasite survival and proliferation.

Characterization of the biology and infectivity of Leishmania infantum viscerotropic and dermotropic strains isolated from HIV+ and HIV- patients in the murine model of visceral leishmaniasis

PubMed Central

2013-01-01

Background Leishmaniasis is a group of diseases with a variety of clinical manifestations. The form of the disease is highly dependent on the infective Leishmania species and the immunological status of the host. The infectivity of the parasite strain also plays an important role in the progression of the infection. The aim of this work is to understand the influence of the natural infectivity of Leishmania strains in the outcome of visceral leishmaniasis. Methods In this study we have characterized four strains of L. infantum in terms of molecular typing, in vitro cultivation and differentiation. Two strains were isolated from HIV+ patients with visceral leishmaniasis (Bibiano and E390M), one strain was isolated from a cutaneous lesion in an immunocompetent patient (HL) and another internal reference strain causative of visceral leishmaniasis (ST) also from an immunocompetent patient was used for comparison. For this objective, we have compared their virulence by in vitro and in vivo infectivity in a murine model of visceral leishmaniasis. Results Molecular typing unraveled a new k26 sequence attributed to MON-284 zymodeme and allowed the generation of a molecular signature for the identification of each strain. In vitro cultivation enabled the production of promastigotes with comparable growth curves and metacyclogenesis development. The HL strain was the most infective, showing the highest parasite loads in vitro that were corroborated with the in vivo assays, 6 weeks post-infection in BALB/c mice. The two strains isolated from HIV+ patients, both belonging to two different zymodemes, revealed different kinetics of infection. Conclusion Differences in in vitro and in vivo infectivity found in the murine model were then attributed to intrinsic characteristics of each strain. This work is supported by other studies that present the parasite’s inherent features as factors for the multiplicity of clinical manifestations and severity of leishmaniasis. PMID:23622683

Selecting a Short Form of the MMPI: Addendum to Faschingbauer.

ERIC Educational Resources Information Center

Poythress, Norman G., Jr.

1978-01-01

Faschingbauer offered guidelines in selecting MMPI short forms as substitutes for the MMPI. This reviews empirical studies of the clinical validity of MMPI short forms and discusses MMPI-168, not considered in Faschingbauer's earlier article. Empirical evidence favors the Faschingbauer Abbreviated MMPI and the MMPI-168 over others. (Author)

Remarks on Height-Diameter Modeling

Treesearch

Lei Yuancai; Bernard R. Parresol

2001-01-01

Height-diameter model forms in earlier published papers are examined. The selection criteria used in height-diameter model forms are not reasonable when considering tree biological growth pattern. During model selection, forms for height-diameter relationships should include consideration of both data-related and reasonable biological criteria, not just data-related...

Comparative Analysis of Cellular Immune Responses in Treated Leishmania Patients and Hamsters against Recombinant Th1 Stimulatory Proteins of Leishmania donovani

PubMed Central

Joshi, Sumit; Yadav, Narendra K.; Rawat, Keerti; Tripathi, Chandra Dev P.; Jaiswal, Anil K.; Khare, Prashant; Tandon, Rati; Baharia, Rajendra K.; Das, Sanchita; Gupta, Reema; Kushawaha, Pramod K.; Sundar, Shyam; Sahasrabuddhe, Amogh A.; Dube, Anuradha

2016-01-01

Our prior studies demonstrated that cellular response of T helper 1 (Th1) type was generated by a soluble antigenic fraction (ranging from 89.9 to 97.1 kDa) of Leishmania donovani promastigote, in treated Leishmania patients as well as hamsters and showed significant prophylactic potential against experimental visceral leishmaniasis (VL). Eighteen Th1 stimulatory proteins were identified through proteomic analysis of this subfraction, out of which 15 were developed as recombinant proteins. In the present work, we have evaluated these 15 recombinant proteins simultaneously for their comparative cellular responses in treated Leishmania patients and hamsters. Six proteins viz. elongation factor-2, enolase, aldolase, triose phosphate isomerase, protein disulfide isomerase, and p45 emerged as most immunogenic as they produced a significant lymphoproliferative response, nitric oxide generation and Th1 cytokine response in PBMCs and lymphocytes of treated Leishmania patients and hamsters respectively. The results suggested that these proteins may be exploited for developing a successful poly-protein and/or poly-epitope vaccine against VL. PMID:27047452

An ethanolic extract of leaves of Piper betle (Paan) Linn mediates its antileishmanial activity via apoptosis.

PubMed

Sarkar, Avijit; Sen, Rupashree; Saha, Piu; Ganguly, Sudipto; Mandal, Goutam; Chatterjee, Mitali

2008-05-01

An unprecedented increase in the incidence of unresponsiveness to antimonial compounds has highlighted the urgent need to develop new antileishmanial agents. The leaves of Piper betle (locally known as Paan) have long been in use in the Indian indigenous system of medicine for its antimicrobial properties but its antileishmanial potential has not been studied. Accordingly, an ethanolic extract of leaves of Piper betle (PB) was tested for its antileishmanial activity that was evidenced in both promastigotes and amastigotes, with IC50 values of 9.8 and 5.45 microg/ml, respectively; importantly, it was accompanied by a safety index of >12-fold. This leishmanicidal activity of PB was mediated via apoptosis as evidenced by morphological changes, loss of mitochondrial membrane potential, in situ labeling of DNA fragments by terminal deoxyribonucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling, and cell-cycle arrest at the sub-G0/G1 phase. Taken together, the data indicate that PB has promising antileishmanial activity that is mediated via programmed cell death and, accordingly, merits consideration and further investigation as a therapeutic option for the treatment of leishmaniasis.

Using Proteomics to Understand How Leishmania Parasites Survive inside the Host and Establish Infection

PubMed Central

Veras, Patrícia Sampaio Tavares; Bezerra de Menezes, Juliana Perrone

2016-01-01

Leishmania is a protozoan parasite that causes a wide range of different clinical manifestations in mammalian hosts. It is a major public health risk on different continents and represents one of the most important neglected diseases. Due to the high toxicity of the drugs currently used, and in the light of increasing drug resistance, there is a critical need to develop new drugs and vaccines to control Leishmania infection. Over the past few years, proteomics has become an important tool to understand the underlying biology of Leishmania parasites and host interaction. The large-scale study of proteins, both in parasites and within the host in response to infection, can accelerate the discovery of new therapeutic targets. By studying the proteomes of host cells and tissues infected with Leishmania, as well as changes in protein profiles among promastigotes and amastigotes, scientists hope to better understand the biology involved in the parasite survival and the host-parasite interaction. This review demonstrates the feasibility of proteomics as an approach to identify new proteins involved in Leishmania differentiation and intracellular survival. PMID:27548150

Using Proteomics to Understand How Leishmania Parasites Survive inside the Host and Establish Infection.

PubMed

Veras, Patrícia Sampaio Tavares; Bezerra de Menezes, Juliana Perrone

2016-08-19

Leishmania is a protozoan parasite that causes a wide range of different clinical manifestations in mammalian hosts. It is a major public health risk on different continents and represents one of the most important neglected diseases. Due to the high toxicity of the drugs currently used, and in the light of increasing drug resistance, there is a critical need to develop new drugs and vaccines to control Leishmania infection. Over the past few years, proteomics has become an important tool to understand the underlying biology of Leishmania parasites and host interaction. The large-scale study of proteins, both in parasites and within the host in response to infection, can accelerate the discovery of new therapeutic targets. By studying the proteomes of host cells and tissues infected with Leishmania, as well as changes in protein profiles among promastigotes and amastigotes, scientists hope to better understand the biology involved in the parasite survival and the host-parasite interaction. This review demonstrates the feasibility of proteomics as an approach to identify new proteins involved in Leishmania differentiation and intracellular survival.

Man-biting sand fly species and natural infection with the Leishmania promastigote in leishmaniasis-endemic areas of Ecuador.

PubMed

Gomez, Eduardo A; Kato, Hirotomo; Hashiguchi, Yoshihisa

2014-12-01

A countrywide surveillance of sand flies was performed to obtain information on their geographical distribution and natural infection by Leishmania protozoa in Ecuador. A total of 18,119 sand flies were collected by human landing collections during 32 years from 1982 to 2014, and 29 species were recognized. The most prevalent 10 species were Lutzomyia gomezi, Lu. robusta, Lu. hartmanni, Lu. shannoni, Lu. trapidoi, Lu. panamensis, Lu. maranonensis, Lu. ayacuchensis, Lu. tortura and Lu. yuilli yuilli, and their topographical and vertical distributions were identified. Among all the sand flies, only 197 (1.09%) flies of four Lutzomyia species, Lu. gomezi, Lu. trapidoi, Lu. tortura and Lu. ayacuchensis, were positive for Leishmania. Endotrypanum, a flagellate parasite not pathogenic to humans, were detected in five Lutzomyia species, Lu. robusta, Lu. hartmanni, Lu. trapidoi, Lu. panamensis and Lu. yuilli yuilli, suggesting wide vector-ranges of Endotrypanum species. These data on the genus Lutzomyia and their natural infections with Leishmania and Endotrypanum will be useful for transmission studies and surveillance of leishmaniasis. Copyright © 2014 Elsevier B.V. All rights reserved.

Establishment and characterization of a new continuous cell line from Lutzomyia longipalpis (Diptera: psychodidae) and its susceptibility to infections with arboviruses and Leishmania chagasi.

PubMed

Rey, G J; Ferro, C; Bello, F J

2000-01-01

Embryonic tissue explants of the sand fly Lutzomyia longipalpis (Lutz & Neiva 1912) the main vector of Leishmania chagasi (Cunha and Chagas), were used to obtain a continuous cell line (Lulo). The tissues were seeded in MM/VP12 medium and these were incubated at 28 masculineC. The first subculture was obtained 45 days after explanting and 96 passages have been made to date. Lulo is composed of epithelioid cells, showed a 0.04 generations/hour exponential growth rate and population doubling time at 24.7 h. The cell line isoenzymatic profiles were determined by using PGI, PGM, MPI and 6-PGDH systems, coinciding with patterns obtained from the same species and colony's pupae and adults. The species karyotype characteristics were recognized (2n = 8), in which pair 1 is subtelocentric and pairs 2, 3 and 4 are metacentric. Lulo was free from bacterial, fungal, mycoplasmic and viral infection. Susceptibility to five arbovirus was determined, the same as Lulo interaction with Leishmania promastigotes.

Expression and subcellular localization of ORC1 in Leishmania major

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Diwakar; Mukherji, Agnideep; Saha, Swati

2008-10-10

The mechanism of DNA replication is highly conserved in eukaryotes, with the process being preceded by the ordered assembly of pre-replication complexes (pre-RCs). Pre-RC formation is triggered by the association of the origin replication complex (ORC) with chromatin. Leishmania major appears to have only one ORC ortholog, ORC1. ORC1 in other eukaryotes is the largest of the ORC subunits and is believed to play a significant role in modulating replication initiation. Here we report for the first time, the cloning of ORC1 from L. major, and the analysis of its expression in L. major promastigotes. In human cells ORC1 levelsmore » have been found to be upregulated in G1 and subsequently degraded, thus playing a role in controlling replication initiation. We examine the subcellular localization of L. major ORC1 in relation to the different stages of the cell cycle. Our results show that, unlike what is widely believed to be the case with ORC1 in human cells, ORC1 in L. major is nuclear at all stages of the cell cycle.« less

Antileishmanial activity study and theoretical calculations for 4-amino-1,2,4-triazole derivatives

NASA Astrophysics Data System (ADS)

Süleymanoğlu, Nevin; Ünver, Yasemin; Ustabaş, Reşat; Direkel, Şahin; Alpaslan, Gökhan

2017-09-01

4-amino-1,2,4-triazole derivatives; 4-amino-1-((5-mercapto-1,3,4-oxadiazole-2-yl)methyl)-3-(thiophene-2-ylmethyl)-1H-1,2,4-triazole-5(4H)-one (1) and 4-amino-1-((4-amino-5 mercapto-4H-1,2,4-triazole-3-yl)methyl)-3-(thiophene-2-ylmethyl)-1H-1,2,4-triazole-5(4H)-one (2) were studied theoretically by Density Functional Theory (DFT) method with 6-311++G(d,p) basis set, structural and some spectroscopic parameters were determined. Significant differences between the experimental and calculated values of vibrational frequencies and chemical shifts were explained by the presence of intermolecular (Ssbnd H⋯O and Ssbnd H⋯N type) hydrogen bonds in structures. The Molecular Electrostatic Potential (MEP) maps obtained at B3LYP/6-311G++(d,p) support the existence of hydrogen bonds. Compounds were tested against to Leishmania infantum promastigots by microdilution broth assay with Alamar Blue Dye. Antileishmanial activity of 4-amino-1,2,4-triazole derivative (2) is remarkable.

Volatile oil profile of some lamiaceous plants growing in Saudi Arabia and their biological activities.

PubMed

Ibrahim, Sabrin R M; Abdallah, Hossam M; Mohamed, Gamal A; Farag, Mohamed A; Alshali, Khalid Z; Alsherif, Emad A; Ross, Samir A

2017-01-01

A comparative investigation of hydro-distilled essential oils from aerial parts of Mentha longifolia L. (ML), Mentha microphylla K.Koch (MM), Mentha australis R.Br. (MA), and Teucrium polium L. (TP) growing in Al Madinah Al Munawwarah, Saudi Arabia, was carried out. The total numbers of identified constituents were 22, 23, 14, and 20 in ML, MM, MA, and TP oils, representing 93.0, 99.3, 78.1, and 81.1% of the total oil composition, respectively. Pulegone (40.7%) and cineole (33.4%) were the major components in ML, whereas carvone (64.6%) was the major one in MM. Furthermore, β-linalool (22.9%) and α-terpineol (12%) were the major components in MA, whereas, (E)-3-caren-2-ol accounted for 12.1% in TP. The essential oils of TP and MA exhibited promising activities against Leishmania donovani promastigotes with IC50 values of 2.3 and 3.7 μg/mL, respectively. In contrast, MA essential oils exhibited antifungal activities towards Candida krusei and C. glabrata with IC50 values of 1 and 1.2 μg/mL, respectively.

Identification, molecular and functional characterization of calmodulin gene of Phytomonas serpens 15T that shares high similarity with its pathogenic counterparts Trypanosoma cruzi.

PubMed

de Souza, Tatiana de Arruda Campos Brasil; Graça-de Souza, Viviane Krominski; Lancheros, César Armando Contreras; Monteiro-Góes, Viviane; Krieger, Marco Aurélio; Goldenberg, Samuel; Yamauchi, Lucy Megumi; Yamada-Ogatta, Sueli Fumie

2011-03-01

In trypanosomatids, Ca²+-binding proteins can affect parasite growth, differentiation and invasion. Due to their importance for parasite maintenance, they become an attractive target for drug discovery and design. Phytomonas serpens 15T is a non-human pathogenic trypanosomatid that expresses important protein homologs of human pathogenic trypanosomatids. In this study, the coding sequence of calmodulin, a Ca²+-binding protein, of P. serpens 15T was cloned and characterized. The encoded polypeptide (CaMP) displayed high amino acid identity to homolog protein of Trypanosoma cruzi and four helix-loop-helix motifs were found. CaMP sequence analysis showed 20 amino acid substitutions compared to its mammalian counterparts. This gene is located on a chromosomal band with estimated size of 1,300 kb and two transcripts were detected by Northern blot analysis. A polyclonal antiserum raised against the recombinant protein recognized a polypeptide with an estimated size of 17 kDa in log-phase promastigote extracts. The recombinant CaMP retains its Ca²+-binding capacity.

Cell death features induced in Leishmania major by 1,3,4-thiadiazole derivatives.

PubMed

Ardestani, Sussan K; Poorrajab, Fatemeh; Razmi, Sepideh; Foroumadi, Alireza; Ajdary, Soheila; Gharegozlou, Behnaz; Behrouzi-Fardmoghadam, Mina; Shafiee, Abbas

2012-10-01

Under a variety of stress conditions, Leishmania species display some morphological and biochemical features characteristic of mammalian programmed cell death or necrosis. Nitroheteroaryl-1,3,4-thiadiazoles induce cell death in Leishmania major (L. major). Putative mechanisms of action of these compounds were investigated in vitro at cellular and molecular levels. We used colorimetric assay to measure acid phosphatase activity which is an indicator of cell viability in the promastigotes. The mode of toxicity was determined by detection of phosphatidylserine translocation to the surface, evaluation of cell membrane integrity, and in situ dUTP nick end-labeling assay. We also determined poly-ADP-ribose polymerase-like protein (PARP) level in the parasites after treatment. A significant reduction of acid phosphatase level, one of the most crucial and virulent factors of the parasite was found in parasites treated with 1,3,4-thiadiazole derivatives. In addition, 1,3,4-thiadiazole derivatives induced loss of plasma membrane integrity, DNA breakage, proteolysis of PARP and necrotic-like death in the parasites. Copyright © 2012 Elsevier Inc. All rights reserved.

Study of antileishmanial activity of 2-aminobenzoyl amino acid hydrazides and their quinazoline derivatives.

PubMed

Khattab, Sherine Nabil; Haiba, Nesreen Saied; Asal, Ahmed Mosaad; Bekhit, Adnan A; Guemei, Aida A; Amer, Adel; El-Faham, Ayman

2017-02-15

A new small library of 2-aminobenzoyl amino acid hydrazide derivatives and quinazolinones derivatives was synthesized and fully characterized by IR, NMR, and elemental analysis. The activity of the prepared compounds on the growth of Leishmania aethiopica promastigotes was evaluated. 2-Benzoyl amino acid hydrazide showed higher inhibitory effect than the quinazoline counterpart. The in vitro antipromastigote activity demonstrated that compounds 2a, 2b, 2f and 4a had IC 50 better than standard drug miltefosine and comparable activity to amphotericin B deoxycholate, which indicates their high antileishmanial activity against Leishmania. aethiopica. Among the prepared compounds; 2-amino-N-(6-hydrazinyl-6-oxohexyl)benzamide 2f (IC 50 =0.051μM) has the best activity, 154 folds more active than reference standard drug miltefosine (IC 50 =7.832μM), and half fold the activity of amphotericin B (IC 50 =0.035μM). In addition, this compound was safe and well tolerated by experimental animals orally up to 250mg/kg and parenterally up to 100mg/kg. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Gut Microbiome of the Vector Lutzomyia longipalpis Is Essential for Survival of Leishmania infantum

PubMed Central

Kelly, Patrick H.; Bahr, Sarah M.; Serafim, Tiago D.; Ajami, Nadim J.; Petrosino, Joseph F.; Meneses, Claudio; Kirby, John R.; Valenzuela, Jesus G.; Kamhawi, Shaden

2017-01-01

ABSTRACT The vector-borne disease leishmaniasis, caused by Leishmania species protozoa, is transmitted to humans by phlebotomine sand flies. Development of Leishmania to infective metacyclic promastigotes in the insect gut, a process termed metacyclogenesis, is an essential prerequisite for transmission. Based on the hypothesis that vector gut microbiota influence the development of virulent parasites, we sequenced midgut microbiomes in the sand fly Lutzomyia longipalpis with or without Leishmania infantum infection. Sucrose-fed sand flies contained a highly diverse, stable midgut microbiome. Blood feeding caused a decrease in microbial richness that eventually recovered. However, bacterial richness progressively decreased in L. infantum-infected sand flies. Acetobacteraceae spp. became dominant and numbers of Pseudomonadaceae spp. diminished coordinately as the parasite underwent metacyclogenesis and parasite numbers increased. Importantly, antibiotic-mediated perturbation of the midgut microbiome rendered sand flies unable to support parasite growth and metacyclogenesis. Together, these data suggest that the sand fly midgut microbiome is a critical factor for Leishmania growth and differentiation to its infective state prior to disease transmission. PMID:28096483

Physicochemical characterisation of natural K-clinoptilolite and heavy-metal forms from Gördes (Manisa, western Turkey)

NASA Astrophysics Data System (ADS)

Ünaldı, Tevfik; Mızrak, İbrahim; Kadir, Selahattin

2013-12-01

Physicochemical characterisation of natural K-clinoptilolite and heavy-metal (Ag+, Cd2+, Cr3+ and Co3+) forms was accomplished through ion exchange by batch, X-ray diffractometric (XRD), X-ray fluorescence (XRF), infrared-spectral (FT-IR), differential thermal analysis-thermal gravimetric (DTA-TG) and scanning-electron microscopic (SEM) methods. Increasing the normality in the cases of heavy-metal forms resulted in decrease in crystallinity and increases in unit-cell volume, rate of ion exchange, and percentage of ion selectivity. In this study, the order of ion-selectivity percentages (rather than ion selectivity) of heavy-metal forms was determined to be Ag+ > Cd2+ > Cr3+ > Co3+. This finding is consistent with the results of worldwide research on the order of ion selectivity in modified clinoptilolite.

Interconnection networks

DOEpatents

Faber, V.; Moore, J.W.

1988-06-20

A network of interconnected processors is formed from a vertex symmetric graph selected from graphs GAMMA/sub d/(k) with degree d, diameter k, and (d + 1)exclamation/ (d /minus/ k + 1)exclamation processors for each d greater than or equal to k and GAMMA/sub d/(k, /minus/1) with degree d /minus/ 1, diameter k + 1, and (d + 1)exclamation/(d /minus/ k + 1)exclamation processors for each d greater than or equal to k greater than or equal to 4. Each processor has an address formed by one of the permutations from a predetermined sequence of letters chosen a selected number of letters at a time, and an extended address formed by appending to the address the remaining ones of the predetermined sequence of letters. A plurality of transmission channels is provided from each of the processors, where each processor has one less channel than the selected number of letters forming the sequence. Where a network GAMMA/sub d/(k, /minus/1) is provided, no processor has a channel connected to form an edge in a direction delta/sub 1/. Each of the channels has an identification number selected from the sequence of letters and connected from a first processor having a first extended address to a second processor having a second address formed from a second extended address defined by moving to the front of the first extended address the letter found in the position within the first extended address defined by the channel identification number. The second address is then formed by selecting the first elements of the second extended address corresponding to the selected number used to form the address permutations. 9 figs.

Atomization methods for forming magnet powders

DOEpatents

Sellers, Charles H.; Branagan, Daniel J.; Hyde, Timothy A.

2000-01-01

The invention encompasses methods of utilizing atomization, methods for forming magnet powders, methods for forming magnets, and methods for forming bonded magnets. The invention further encompasses methods for simulating atomization conditions. In one aspect, the invention includes an atomization method for forming a magnet powder comprising: a) forming a melt comprising R.sub.2.1 Q.sub.13.9 B.sub.1, Z and X, wherein R is a rare earth element; X is an element selected from the group consisting of carbon, nitrogen, oxygen and mixtures thereof; Q is an element selected from the group consisting of Fe, Co and mixtures thereof; and Z is an element selected from the group consisting of Ti, Zr, Hf and mixtures thereof; b) atomizing the melt to form generally spherical alloy powder granules having an internal structure comprising at least one of a substantially amorphous phase or a substantially nanocrystalline phase; and c) heat treating the alloy powder to increase an energy product of the alloy powder; after the heat treatment, the alloy powder comprising an energy product of at least 10 MGOe. In another aspect, the invention includes a magnet comprising R, Q, B, Z and X, wherein R is a rare earth element; X is an element selected from the group consisting of carbon, nitrogen, oxygen and mixtures thereof; Q is an element selected from the group consisting of Fe, Co and mixtures thereof; and Z is an element selected from the group consisting of Ti, Zr, Hf and mixtures thereof; the magnet comprising an internal structure comprising R.sub.2.1 Q.sub.13.9 B.sub.1.

42 CFR 73.5 - Exemptions for HHS select agents and toxins.

Code of Federal Regulations, 2014 CFR

2014-10-01

... the select agent or toxin is reported to CDC or APHIS and to other appropriate authorities when... followed by submission of APHIS/CDC Form 4 within seven calendar days after identification. (ii) For all other HHS select agents or toxins, APHIS/CDC Form 4 must be submitted within seven calendar days after...

42 CFR 73.5 - Exemptions for HHS select agents and toxins.

Code of Federal Regulations, 2013 CFR

2013-10-01

... the select agent or toxin is reported to CDC or APHIS and to other appropriate authorities when... followed by submission of APHIS/CDC Form 4 within seven calendar days after identification. (ii) For all other HHS select agents or toxins, APHIS/CDC Form 4 must be submitted within seven calendar days after...

Automated Test-Form Generation

ERIC Educational Resources Information Center

van der Linden, Wim J.; Diao, Qi

2011-01-01

In automated test assembly (ATA), the methodology of mixed-integer programming is used to select test items from an item bank to meet the specifications for a desired test form and optimize its measurement accuracy. The same methodology can be used to automate the formatting of the set of selected items into the actual test form. Three different…

Integration of active pharmaceutical ingredient solid form selection and particle engineering into drug product design.

PubMed

Ticehurst, Martyn David; Marziano, Ivan

2015-06-01

This review seeks to offer a broad perspective that encompasses an understanding of the drug product attributes affected by active pharmaceutical ingredient (API) physical properties, their link to solid form selection and the role of particle engineering. While the crucial role of active pharmaceutical ingredient (API) solid form selection is universally acknowledged in the pharmaceutical industry, the value of increasing effort to understanding the link between solid form, API physical properties and drug product formulation and manufacture is now also being recognised. A truly holistic strategy for drug product development should focus on connecting solid form selection, particle engineering and formulation design to both exploit opportunities to access simpler manufacturing operations and prevent failures. Modelling and predictive tools that assist in establishing these links early in product development are discussed. In addition, the potential for differences between the ingoing API physical properties and those in the final product caused by drug product processing is considered. The focus of this review is on oral solid dosage forms and dry powder inhaler products for lung delivery. © 2015 Royal Pharmaceutical Society.

Sexual selection on cuticular hydrocarbons in the Australian field cricket, Teleogryllus oceanicus

PubMed Central

Thomas, Melissa L; Simmons, Leigh W

2009-01-01

Background Females in a wide range of taxa have been shown to base their choice of mates on pheromone signals. However, little research has focussed specifically on the form and intensity of selection that mate choice imposes on the pheromone signal. Using multivariate selection analysis, we characterise directly the form and intensity of sexual selection acting on cuticular hydrocarbons, chemical compounds widely used in the selection of mates in insects. Using the Australian field cricket Teleogryllus oceanicus as a model organism, we use three measures of male attractiveness to estimate fitness; mating success, the duration of courtship required to elicit copulation, and subsequent spermatophore attachment duration. Results We found that all three measures of male attractiveness generated sexual selection on male cuticular hydrocarbons, however there were differences in the form and intensity of selection among these three measures. Mating success was the only measure of attractiveness that imposed both univariate linear and quadratic selection on cuticular hydrocarbons. Although we found that all three attractiveness measures generated nonlinear selection, again only mating success was found to exert statistically significant stabilizing selection. Conclusion This study shows that sexual selection plays an important role in the evolution of male cuticular hydrocarbon signals. PMID:19594896

Preformulation considerations for controlled release dosage forms. Part I. Selecting candidates.

PubMed

Chrzanowski, Frank

2008-01-01

The physical-chemical properties of interest for controlled release (CR) dosage form development presented are based on the author's experience. Part I addresses selection of the final form based on a logical progression of physical-chemical properties evaluation of candidate forms and elimination of forms with undesirable properties from further evaluation in order to simplify final form selection. Several candidate forms which could include salt, free base or acid, polymorphic and amorphic forms of a new chemical entity (NCE) or existing drug substance (DS) are prepared and evaluated for critical properties in a scheme relevant to manufacturing processes, predictive of problems, requiring small amounts of test materials and simple analytical tools. A stability indicating assay is not needed to initiate the evaluation. This process is applicable to CR and immediate release (IR) dosage form development. The critical properties evaluated are melting, crystallinity, solubilities in water, 0.1 N HCl, and SIF, hygrodymamics, i.e., moisture sorption and loss at extremes of RH, and LOD at typical wet granulation drying conditions, and processability, i.e., corrosivity, and filming and/or sticking upon compression.

7 CFR Exhibit D to Subpart N of... - Project Selection Criteria-Outline Rating Form

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 13 2011-01-01 2009-01-01 true Project Selection Criteria-Outline Rating Form D Exhibit D to Subpart N of Part 1944 Agriculture Regulations of the Department of Agriculture (Continued... Preservation Grants Pt. 1944, Subpt. N, Exh. D Exhibit D to Subpart N of Part 1944—Project Selection Criteria...

7 CFR Exhibit D to Subpart N of... - Project Selection Criteria-Outline Rating Form

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 13 2010-01-01 2009-01-01 true Project Selection Criteria-Outline Rating Form D Exhibit D to Subpart N of Part 1944 Agriculture Regulations of the Department of Agriculture (Continued... Preservation Grants Pt. 1944, Subpt. N, Exh. D Exhibit D to Subpart N of Part 1944—Project Selection Criteria...

Local Norms and Test Characteristics for Selected Forms of the M.A.A. Placement Test.

ERIC Educational Resources Information Center

Melancon, Janet G.; Thompson, Bruce

The psychometric integrity of selected items from the Mathematics Association of America (MAA) placement tests for college students was investigated. Two alternative and parallel versions of the test were developed (Form A and Form B) for this study. Data for 539 students seeking admission into an undergraduate mathematics curriculum at a private…

Selective Attention in Web Forms: An Exploratory Case Study with Older People

ERIC Educational Resources Information Center

Sayago, Sergio; Guijarro, Jose-Maria; Blat, Josep

2012-01-01

This article reports on an exploratory study aimed to identify which ways of marking required and optional fields help older people fill in web forms correctly. Drawing on a pilot study and selective attention research in ageing, modified versions of widely used forms were created, in which standard asterisks were replaced with one of three…

18 CFR Appendix A 1 to Part 281 - Comparison of Selected Fuel Price Data, FPC Form No. 423 Versus Monthly Energy Review, 1976...

Code of Federal Regulations, 2011 CFR

2011-04-01

... Fuel Price Data, FPC Form No. 423 Versus Monthly Energy Review, 1976-January 1980 A Appendix A 1 to... Selected Fuel Price Data, FPC Form No. 423 Versus Monthly Energy Review, 1976—January 1980 Type of fuel FPC form No. 423 price data 1 1976 1977 1978 1979 January 1980 Monthly energy review price data 2 1976 1977...

18 CFR Appendix A 1 to Part 281 - Comparison of Selected Fuel Price Data, FPC Form No. 423 Versus Monthly Energy Review, 1976...

Code of Federal Regulations, 2013 CFR

2013-04-01

... Fuel Price Data, FPC Form No. 423 Versus Monthly Energy Review, 1976-January 1980 A Appendix A 1 to... Selected Fuel Price Data, FPC Form No. 423 Versus Monthly Energy Review, 1976—January 1980 Type of fuel FPC form No. 423 price data 1 1976 1977 1978 1979 January 1980 Monthly energy review price data 2 1976 1977...

18 CFR Appendix A 1 to Part 281 - Comparison of Selected Fuel Price Data, FPC Form No. 423 Versus Monthly Energy Review, 1976...

Code of Federal Regulations, 2012 CFR

2012-04-01

... Fuel Price Data, FPC Form No. 423 Versus Monthly Energy Review, 1976-January 1980 A Appendix A 1 to... Selected Fuel Price Data, FPC Form No. 423 Versus Monthly Energy Review, 1976—January 1980 Type of fuel FPC form No. 423 price data 1 1976 1977 1978 1979 January 1980 Monthly energy review price data 2 1976 1977...

18 CFR Appendix A 1 to Part 281 - Comparison of Selected Fuel Price Data, FPC Form No. 423 Versus Monthly Energy Review, 1976...

Code of Federal Regulations, 2014 CFR

2014-04-01

... Fuel Price Data, FPC Form No. 423 Versus Monthly Energy Review, 1976-January 1980 A Appendix A 1 to... Selected Fuel Price Data, FPC Form No. 423 Versus Monthly Energy Review, 1976—January 1980 Type of fuel FPC form No. 423 price data 1 1976 1977 1978 1979 January 1980 Monthly energy review price data 2 1976 1977...

Interactions between motion and form processing in the human visual system.

PubMed

Mather, George; Pavan, Andrea; Bellacosa Marotti, Rosilari; Campana, Gianluca; Casco, Clara

2013-01-01

The predominant view of motion and form processing in the human visual system assumes that these two attributes are handled by separate and independent modules. Motion processing involves filtering by direction-selective sensors, followed by integration to solve the aperture problem. Form processing involves filtering by orientation-selective and size-selective receptive fields, followed by integration to encode object shape. It has long been known that motion signals can influence form processing in the well-known Gestalt principle of common fate; texture elements which share a common motion property are grouped into a single contour or texture region. However, recent research in psychophysics and neuroscience indicates that the influence of form signals on motion processing is more extensive than previously thought. First, the salience and apparent direction of moving lines depends on how the local orientation and direction of motion combine to match the receptive field properties of motion-selective neurons. Second, orientation signals generated by "motion-streaks" influence motion processing; motion sensitivity, apparent direction and adaptation are affected by simultaneously present orientation signals. Third, form signals generated by human body shape influence biological motion processing, as revealed by studies using point-light motion stimuli. Thus, form-motion integration seems to occur at several different levels of cortical processing, from V1 to STS.

Interactions between motion and form processing in the human visual system

PubMed Central

Mather, George; Pavan, Andrea; Bellacosa Marotti, Rosilari; Campana, Gianluca; Casco, Clara

2013-01-01

The predominant view of motion and form processing in the human visual system assumes that these two attributes are handled by separate and independent modules. Motion processing involves filtering by direction-selective sensors, followed by integration to solve the aperture problem. Form processing involves filtering by orientation-selective and size-selective receptive fields, followed by integration to encode object shape. It has long been known that motion signals can influence form processing in the well-known Gestalt principle of common fate; texture elements which share a common motion property are grouped into a single contour or texture region. However, recent research in psychophysics and neuroscience indicates that the influence of form signals on motion processing is more extensive than previously thought. First, the salience and apparent direction of moving lines depends on how the local orientation and direction of motion combine to match the receptive field properties of motion-selective neurons. Second, orientation signals generated by “motion-streaks” influence motion processing; motion sensitivity, apparent direction and adaptation are affected by simultaneously present orientation signals. Third, form signals generated by human body shape influence biological motion processing, as revealed by studies using point-light motion stimuli. Thus, form-motion integration seems to occur at several different levels of cortical processing, from V1 to STS. PMID:23730286

A benefit/risk approach towards selecting appropriate pharmaceutical dosage forms - an application for paediatric dosage form selection.

PubMed

Sam, Tom; Ernest, Terry B; Walsh, Jennifer; Williams, Julie L

2012-10-05

The design and selection of new pharmaceutical dosage forms involves the careful consideration and balancing of a quality target product profile against technical challenges and development feasibility. Paediatric dosage forms present particular complexity due to the diverse patient population, patient compliance challenges and safety considerations of this vulnerable population. This paper presents a structured framework for assessing the comparative benefits and risks of different pharmaceutical design options against pre-determined criteria relating to (1) efficacy, (2) safety and (3) patient access. This benefit/risk framework has then been applied to three hypothetical, but realistic, scenarios for paediatric dosage forms in order to explore its utility in guiding dosage form design and formulation selection. The approach allows a rigorous, systematic and qualitative assessment of the merits and disadvantages of each dosage form option and helps identify mitigating strategies to modify risk. The application of a weighting and scoring system to the criteria depending on the specific case could further refine the analysis and aid decision-making. In this paper, one case study is scored for illustrative purposes. However, it is acknowledged that in real development scenarios, the generation of actual data considering the very specific situation for the patient/product/developer would come into play to drive decisions on the most appropriate dosage form strategy. Copyright © 2012 Elsevier B.V. All rights reserved.

Antiprotozoal activity of extracts and isolated triterpenoids of 'carnauba' (Copernicia prunifera) wax from Brazil.

PubMed

de Almeida, Buana C; Araújo, Bruno Q; Carvalho, Adonias A; Freitas, Sâmya Danielle L; Maciel, Dayany da S Alves; Ferreira, Ari José S; Tempone, Andre G; Martins, Ligia F; Alexandre, Tatiana R; Chaves, Mariana H; Lago, João Henrique G

2016-12-01

'Carnauba' wax is a natural product obtained from the processing of the powder exuded from Copernicia prunifera (Miller) H. E. Moore (Arecaceae). This material is widely used in the Brazilian folk medicine, including the treatment of rheumatism and syphilis. To investigate the antiprotozoal activity of hexane and EtOH extracts from the 'carnauba' wax as well as from the isolated compounds from the bioactive extracts. Two different samples of 'carnauba' (C. prunifera) waxes - types 1 and 4 - were individually extracted using hexane (EH) and EtOH (EE). Aliquots of hexane (type 1 - EH-1 and EH-4) and EtOH (type 4 - EE-1 and EE-4) extracts were tested against promastigote (2-200 μg/mL in DMSO during 48 h at 24 °C) and amastigote (3-150 μg/mL in DMSO during 120 h at 37 °C) forms of Leishmania infantum as well as against trypomastigote (3-150 μg/mL in DMSO during 24 h at 37 °C) forms of Trypanosoma cruzi. Bioactive extracts EH-1 and EE-4 were subjected to a bioactivity-guided fractionation to afford three dammarane-type triterpenoids (1-3). The in vitro antiprotozoal activities of the obtained compounds were evaluated as described above. Additionally, the cytotoxicity activity of compounds 1-3 against mammalian conjunctive cells (NCTC - 2-200 μg/mL in DMSO during 48 h at 37 °C) was determined. From the bioactive hexane and EtOH extracts from the 'carnauba' (C. prunifera) wax, were isolated three dammarane-type triterpenoids: (24R*)-methyldammar-25-ene-3β,20-diol (carnaubadiol, 1), (24R*)-methyldammara-20,25-dien-3-one (2) and (24R*)-methyldammara-20,25-dien-3α-ol (3). These compounds were identified based on the analysis of NMR and MS spectroscopic data. Compounds 1-3 were effective against the intracellular amastigotes of L. infantum, with IC 50 values ranging from 8 to 52 μM, while compounds 1 and 3 displayed activity against trypomastigote forms of T. cruzi with IC 50 values of 15 and 35 μM, respectively. The mammalian cytotoxicity assay demonstrated no damage to NCTC conjunctive cells up to 200 μM, except for compound 1, which demonstrated a CC 50 value of 34 μM. Based on the results, it was possible to conclude that the detected antiprotozoal bioactivity of 'carnauba' (C. prunifera) wax extracts could be related to the presence of the natural dammarane triterpenoid derivatives. The results suggested that these compounds could be used as promising scaffolds for drug design studies for leishmaniasis and Chagas disease.

Non-thermal plasma conversion of hydrocarbons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strohm, James J.; Skoptsov, George L.; Musselman, Evan T.

A non-thermal plasma is generated to selectively convert a precursor to a product. More specifically, plasma forming material and a precursor material are provided to a reaction zone of a vessel. The reaction zone is exposed to microwave radiation, including exposing the plasma forming material and the precursor material to the microwave radiation. The exposure of the plasma forming material to the microwave radiation selectively converts the plasma forming material to a non-thermal plasma including formation of one or more streamers. The precursor material is mixed with the plasma forming material and the precursor material is exposed to the non-thermalmore » plasma including exposing the precursor material to the one or more streamers. The exposure of the precursor material to the streamers and the microwave radiation selectively converts the precursor material to a product.« less

Glycoconjugates and methods

DOEpatents

Bertozzi, Carolyn C [Albany, CA; Yarema, Kevin J [Albany, CA; Mahal, Lara K [Berkeley, CA

2008-04-01

Methods for making the functionalized glycoconjugates include (a) contacting a cell with a first monosaccharide, and (b) incubating the cell under conditions whereby the cell (i) internalizes the first monosaccharide, (ii) biochemically processes the first monosaccharide into a second saccharide, (iii) conjugates the saccharide to a carrier to form a glycoconjugate, and (iv) extracellularly expresses the glycoconjugate to form an extracellular glycoconjugate comprising a selectively reactive functional group. Methods for forming products at a cell further comprise contacting the functional group of the extracellularly expressed glycoconjugate with an agent which selectively reacts with the functional group to form a product. Subject compositions include cyto-compatible monosaccharides comprising a nitrogen or ether linked functional group selectively reactive at a cell surface and compositions and cells comprising such saccharides.

Selective photoswitching of the binuclear spin crossover compound {[Fe(bt)(NCS)2]2(bpm)} into two distinct macroscopic phases.

PubMed

Moussa, N Ould; Molnár, G; Bonhommeau, S; Zwick, A; Mouri, S; Tanaka, K; Real, J A; Bousseksou, A

2005-03-18

The low-spin (LS-LS, S = 0) diamagnetic form of the binuclear spin crossover complex {[Fe(bt)(NCS)(2)](2)(bpm)} was selectively photoconverted into two distinct macroscopic phases at different excitation wavelengths (1342 or 647.1 nm). These long-lived metastable phases have been identified, respectively, as the symmetry-broken paramagnetic form (HS-LS, S = 2) and the antiferromagnetically coupled (HS-HS, S = 0) high-spin form of the compound. The selectivity may be explained by the strong coupling of the primary excited states to the paramagnetic state.

Method for forming metal contacts

DOEpatents

Reddington, Erik; Sutter, Thomas C; Bu, Lujia; Cannon, Alexandra; Habas, Susan E; Curtis, Calvin J; Miedaner, Alexander; Ginley, David S; Van Hest, Marinus Franciscus Antonius Maria

2013-09-17

Methods of forming metal contacts with metal inks in the manufacture of photovoltaic devices are disclosed. The metal inks are selectively deposited on semiconductor coatings by inkjet and aerosol apparatus. The composite is heated to selective temperatures where the metal inks burn through the coating to form an electrical contact with the semiconductor. Metal layers are then deposited on the electrical contacts by light induced or light assisted plating.

Validation of an Adapted French Form of the Career Adapt-Abilities Scale in Four Francophone Countries

ERIC Educational Resources Information Center

Johnston, Claire S.; Broonen, Jean-Paul; Stauffer, Sarah D.; Hamtiaux, Armanda; Pouyaud, Jacques; Zecca, Gregory; Houssemand, Claude; Rossier, Jerome

2013-01-01

This study presents the validation of a French version of the Career Adapt-Abilities Scale in four Francophone countries. The aim was to re-analyze the item selection and then compare this newly developed French-language form with the international form 2.0. Exploratory factor analysis was used as a tool for item selection, and confirmatory factor…

New Form Discovery for the Analgesics Flurbiprofen and Sulindac Facilitated by Polymer-Induced Heteronucleation

PubMed Central

GRZESIAK, ADAM L.; MATZGER, ADAM J.

2008-01-01

The selection and discovery of new crystalline forms is a longstanding issue in solid-state chemistry of critical importance because of the effect molecular packing arrangement exerts on materials properties. Polymer-induced heteronucleation has recently been developed as a powerful approach to discover and control the production of crystal modifications based on the insoluble polymer heteronucleant added to the crystallization solution. The selective nucleation and discovery of new crystal forms of the well-studied pharmaceuticals flurbiprofen (FBP) and sulindac (SUL) has been achieved utilizing this approach. For the first time, FBP form III was produced in bulk quantities and its crystal structure was also determined. Furthermore, a novel 3:2 FBP:H2O phase was discovered that nucleates selectively from only a few polymers. Crystallization of SUL in the presence of insoluble polymers facilitated the growth of form I single crystals suitable for structure determination. Additionally, a new SUL polymorph (form IV) was discovered by this method. The crystal forms of FBP and SUL are characterized by Raman and FTIR spectroscopies, X-ray diffraction, and differential scanning calorimetry. PMID:17567888

Defense Acquisitions. Assessments of Selected Weapon Programs

DTIC Science & Technology

2007-03-01

Selected Weapon Programs March 2007 GAO-07-406SP Report Documentation Page Form ApprovedOMB No. 0704-0188 Public reporting burden for...unclassified Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 What GAO Found United States Government Accountability Office Why GAO Did This...Joint Tactical Radio System Ground Mobile Radio (JTRS GMR) 93 JTRS Handheld, Manpack, Small Form Fit (JTRS HMS) 95 Kinetic Energy Interceptors (KEI) 97

Pre and Post-copulatory Selection Favor Similar Genital Phenotypes in the Male Broad Horned Beetle

PubMed Central

House, Clarissa M.; Sharma, M. D.; Okada, Kensuke; Hosken, David J.

2016-01-01

Sexual selection can operate before and after copulation and the same or different trait(s) can be targeted during these episodes of selection. The direction and form of sexual selection imposed on characters prior to mating has been relatively well described, but the same is not true after copulation. In general, when male–male competition and female choice favor the same traits then there is the expectation of reinforcing selection on male sexual traits that improve competitiveness before and after copulation. However, when male–male competition overrides pre-copulatory choice then the opposite could be true. With respect to studies of selection on genitalia there is good evidence that male genital morphology influences mating and fertilization success. However, whether genital morphology affects reproductive success in more than one context (i.e., mating versus fertilization success) is largely unknown. Here we use multivariate analysis to estimate linear and nonlinear selection on male body size and genital morphology in the flour beetle Gnatocerus cornutus, simulated in a non-competitive (i.e., monogamous) setting. This analysis estimates the form of selection on multiple traits and typically, linear (directional) selection is easiest to detect, while nonlinear selection is more complex and can be stabilizing, disruptive, or correlational. We find that mating generates stabilizing selection on male body size and genitalia, and fertilization causes a blend of directional and stabilizing selection. Differences in the form of selection across these bouts of selection result from a significant alteration of nonlinear selection on body size and a marginally significant difference in nonlinear selection on a component of genital shape. This suggests that both bouts of selection favor similar genital phenotypes, whereas the strong stabilizing selection imposed on male body size during mate acquisition is weak during fertilization. PMID:27371390

Platinum-group elements fractionation by selective complexing, the Os, Ir, Ru, Rh-arsenide-sulfide systems above 1020 °C

NASA Astrophysics Data System (ADS)

Helmy, Hassan M.; Bragagni, Alessandro

2017-11-01

The platinum-group element (PGE) contents in magmatic ores and rocks are normally in the low μg/g (even in the ng/g) level, yet they form discrete platinum-group mineral (PGM) phases. IPGE (Os, Ir, Ru) + Rh form alloys, sulfides, and sulfarsenides while Pt and Pd form arsenides, tellurides, bismuthoids and antimonides. We experimentally investigate the behavior of Os, Ru, Ir and Rh in As-bearing sulfide system between 1300 and 1020 °C and show that the prominent mineralogical difference between IPGE (+Rh) and Pt and Pd reflects different chemical preference in the sulfide melt. At temperatures above 1200 °C, Os shows a tendency to form alloys. Ruthenium forms a sulfide (laurite RuS2) while Ir and Rh form sulfarsenides (irarsite IrAsS and hollingworthite RhAsS, respectively). The chemical preference of PGE is selective: IPGE + Rh form metal-metal, metal-S and metal-AsS complexes while Pt and Pd form semimetal complexes. Selective complexing followed by mechanical separation of IPGE (and Rh)-ligand from Pt- and Pd-ligand associations lead to PGE fractionation.

High aspect ratio template and method for producing same

NASA Technical Reports Server (NTRS)

Sakamoto, Jeff S. (Inventor); Weiss, James R. (Inventor); Fleurial, Jean-Pierre (Inventor); Kisor, Adam (Inventor); Tuszynski, Mark (Inventor); Stokols, Shula (Inventor); Holt, Todd Edward (Inventor); Welker, David James (Inventor); Breckon, Christopher David (Inventor)

2010-01-01

Millimeter to nano-scale structures manufactured using a multi-component polymer fiber matrix are disclosed. The use of dissimilar polymers allows the selective dissolution of the polymers at various stages of the manufacturing process. In one application, biocompatible matrixes may be formed with long pore length and small pore size. The manufacturing process begins with a first polymer fiber arranged in a matrix formed by a second polymer fiber. End caps may be attached to provide structural support and the polymer fiber matrix selectively dissolved away leaving only the long polymer fibers. These may be exposed to another product, such as a biocompatible gel to form a biocompatible matrix. The polymer fibers may then be selectively dissolved leaving only a biocompatible gel scaffold with the pores formed by the dissolved polymer fibers.

47 CFR 2.303 - Other forms of identification of stations.

Code of Federal Regulations, 2010 CFR

2010-10-01

... whose signals are being relayed, or by network identification. Broadcasting (television booster.... (b) Digital selective calls will be authorized by the Commission and will be formed by groups of... identification number: 4 digits. (2) Ship station selective call number: 5 digits. (3) Predetermined group of...

DOE Office of Scientific and Technical Information (OSTI.GOV)

French, Jarrod B.; Yates, Phillip A.; Soysa, D.Radika

The final two steps of de novo uridine 5'-monophosphate (UMP) biosynthesis are catalyzed by orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC). In most prokaryotes and simple eukaryotes these two enzymes are encoded by separate genes, whereas in mammals they are expressed as a bifunctional gene product called UMP synthase (UMPS), with OPRT at the N terminus and OMPDC at the C terminus. Leishmania and some closely related organisms also express a bifunctional enzyme for these two steps, but the domain order is reversed relative to mammalian UMPS. In this work we demonstrate that L. donovani UMPS (LdUMPS) is anmore » essential enzyme in promastigotes and that it is sequestered in the parasite glycosome. We also present the crystal structure of the LdUMPS in complex with its product, UMP. This structure reveals an unusual tetramer with two head to head and two tail to tail interactions, resulting in two dimeric OMPDC and two dimeric OPRT functional domains. In addition, we provide structural and biochemical evidence that oligomerization of LdUMPS is controlled by product binding at the OPRT active site. We propose a model for the assembly of the catalytically relevant LdUMPS tetramer and discuss the implications for the structure of mammalian UMPS.« less

Meiosis and Haploid Gametes in the Pathogen Trypanosoma brucei

PubMed Central

Peacock, Lori; Bailey, Mick; Carrington, Mark; Gibson, Wendy

2014-01-01

Summary In eukaryote pathogens, sex is an important driving force in spreading genes for drug resistance, pathogenicity, and virulence [1]. For the parasitic trypanosomes that cause African sleeping sickness, mating occurs during transmission by the tsetse vector [2, 3] and involves meiosis [4], but haploid gametes have not yet been identified. Here, we show that meiosis is a normal part of development in the insect salivary glands for all subspecies of Trypanosoma brucei, including the human pathogens. By observing insect-derived trypanosomes during the window of peak expression of meiosis-specific genes, we identified promastigote-like (PL) cells that interacted with each other via their flagella and underwent fusion, as visualized by the mixing of cytoplasmic red and green fluorescent proteins. PL cells had a short, wide body, a very long anterior flagellum, and either one or two kinetoplasts, but only the anterior kinetoplast was associated with the flagellum. Measurement of nuclear DNA contents showed that PL cells were haploid relative to diploid metacyclics. Trypanosomes are among the earliest diverging eukaryotes, and our results support the hypothesis that meiosis and sexual reproduction are ubiquitous in eukaryotes and likely to have been early innovations [5]. PMID:24388851

Meiosis and haploid gametes in the pathogen Trypanosoma brucei.

PubMed

Peacock, Lori; Bailey, Mick; Carrington, Mark; Gibson, Wendy

2014-01-20

In eukaryote pathogens, sex is an important driving force in spreading genes for drug resistance, pathogenicity, and virulence. For the parasitic trypanosomes that cause African sleeping sickness, mating occurs during transmission by the tsetse vector and involves meiosis, but haploid gametes have not yet been identified. Here, we show that meiosis is a normal part of development in the insect salivary glands for all subspecies of Trypanosoma brucei, including the human pathogens. By observing insect-derived trypanosomes during the window of peak expression of meiosis-specific genes, we identified promastigote-like (PL) cells that interacted with each other via their flagella and underwent fusion, as visualized by the mixing of cytoplasmic red and green fluorescent proteins. PL cells had a short, wide body, a very long anterior flagellum, and either one or two kinetoplasts, but only the anterior kinetoplast was associated with the flagellum. Measurement of nuclear DNA contents showed that PL cells were haploid relative to diploid metacyclics. Trypanosomes are among the earliest diverging eukaryotes, and our results support the hypothesis that meiosis and sexual reproduction are ubiquitous in eukaryotes and likely to have been early innovations. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Target oriented drugs against leishmania. Annual summary report no. 2, 1 May 1980-30 April 1981

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zehavi, U.; El-On, J.

1981-01-31

Excreted Factor (EF) is a carbohydrate-rich material released by different strains of Leishmania during growth. It has antigenic properties similar to those of the intact parasite and plays a role in the infective process. Isolation and purification of EF is necessary for study of its biological function, its use for diagnostic purposes, its use in immunization experiments, the study of its biosynthesis, and the preparation of inhibitors of particular biosynthetic steps. Purification of EF by affinity chromatography was markedly improved by introducing Ricinus lectin (specific for galactose) column. This enabled us to obtain more reliable amino acid and sugar analysismore » and will be instrumental in more advanced physical, chemical, and immunological studies. We have developed a radioimmunoassay for leishmaniasis utilizing purified EF. The assay can distinguish between Leishmania strains and once further developed, should prove most valuable for the diagnosis of the disease. EF plays a role in the infective process of Leishmania. We have now shown that surface carbohydrate, related to EF, plays a role in the initial attachment of Leishmania promastigots to macrophages - a stage that is a prelude to their engulfment by the macrophages followed by multiplication in their cells.« less

Monoterpenic aldehydes as potential anti-Leishmania agents: activity of Cymbopogon citratus and citral on L. infantum, L. tropica and L. major.

PubMed

Machado, M; Pires, P; Dinis, A M; Santos-Rosa, M; Alves, V; Salgueiro, L; Cavaleiro, C; Sousa, M C

2012-03-01

In order to contribute for the search of new drugs for leishmaniasis, we study the susceptibility of Leishmania infantum, Leishmania tropica and Leishmania major to Cymbopogon citratus essential oil and major compounds, mrycene and citral. C. citratus and citral were the most active inhibiting L. infantum, L. tropica and L. major growth at IC(50) concentrations ranging from 25 to 52 μg/ml and from 34 to 42 μg/ml, respectively. L. infantum promastigotes exposed to essential oil and citral underwent considerable ultrastructural alterations, namely mitochondrial and kinetoplast swelling, autophagosomal structures, disruption of nuclear membrane and nuclear chromatin condensation. C. citratus essential oil and citral promoted the leishmanicidal effect by triggering a programmed cell death. In fact, the leishmanicidal activity was mediated via apoptosis as evidenced by externalization of phosphatidylserine, loss of mitochondrial membrane potential, and cell-cycle arrest at the G(0)/G(1) phase. Taken together, ours findings lead us to propose that citral was responsible for anti-Leishmania activity of the C. citratus and both may represent a valuable source for therapeutic control of leishmaniasis. Copyright © 2011 Elsevier Inc. All rights reserved.

Development of a Murine Infection Model with Leishmania killicki, Responsible for Cutaneous Leishmaniosis in Algeria: Application in Pharmacology

PubMed Central

Eddaikra, Naouel; Kherachi Djenad, Ihcene; Benbetka, Sihem; Benikhlef, Razika; Aït-Oudhia, Khatima; Moulti-Mati, Farida; Oury, Bruno; Sereno, Denis; Harrat, Zoubir

2016-01-01

In Algeria, Leishmania infantum, Leishmania major, and Leishmania killicki (Leishmania tropica) are responsible for cutaneous leishmaniosis. We established a murine model of L. killicki infection to investigate its infective capacity, some immunophysiopathological aspects, and its suitability for pharmacological purposes. Following the injection of L. major or L. killicki metacyclic promastigotes in the ear dermis of BALB/c mice, the course of infection was followed. The infection with L. killicki caused slower lesion formation than with L. major. The presence of L. killicki or L. major DNA and parasites was detected in the ear dermis and in lymph nodes, spleen, and liver. Lesions induced by L. killicki were nonulcerative in their aspect, whereas those caused by L. major were highly ulcerative and necrotic, which matches well with the lesion phenotype reported in humans for L. killicki and L. major, respectively. The treatment of L. killicki lesions by injection of Glucantime® significantly reduced the lesion thickness and parasite burden. Ear dermal injection of BALB/c mice constitutes a model to study lesions physiopathology caused by L. killicki and presents interest for in vivo screening of new compounds against this pathogen, emerging in Algeria. PMID:26949705

Development of a Murine Infection Model with Leishmania killicki, Responsible for Cutaneous Leishmaniosis in Algeria: Application in Pharmacology.

PubMed

Eddaikra, Naouel; Kherachi Djenad, Ihcene; Benbetka, Sihem; Benikhlef, Razika; Aït-Oudhia, Khatima; Moulti-Mati, Farida; Oury, Bruno; Sereno, Denis; Harrat, Zoubir

2016-01-01

In Algeria, Leishmania infantum, Leishmania major, and Leishmania killicki (Leishmania tropica) are responsible for cutaneous leishmaniosis. We established a murine model of L. killicki infection to investigate its infective capacity, some immunophysiopathological aspects, and its suitability for pharmacological purposes. Following the injection of L. major or L. killicki metacyclic promastigotes in the ear dermis of BALB/c mice, the course of infection was followed. The infection with L. killicki caused slower lesion formation than with L. major. The presence of L. killicki or L. major DNA and parasites was detected in the ear dermis and in lymph nodes, spleen, and liver. Lesions induced by L. killicki were nonulcerative in their aspect, whereas those caused by L. major were highly ulcerative and necrotic, which matches well with the lesion phenotype reported in humans for L. killicki and L. major, respectively. The treatment of L. killicki lesions by injection of Glucantime® significantly reduced the lesion thickness and parasite burden. Ear dermal injection of BALB/c mice constitutes a model to study lesions physiopathology caused by L. killicki and presents interest for in vivo screening of new compounds against this pathogen, emerging in Algeria.

A randomised, double-blind, controlled trial of a killed L. major vaccine plus BCG against zoonotic cutaneous leishmaniasis in Iran.

PubMed

Momeni, A Z; Jalayer, T; Emamjomeh, M; Khamesipour, A; Zicker, F; Ghassemi, R L; Dowlati, Y; Sharifi, I; Aminjavaheri, M; Shafiei, A; Alimohammadian, M H; Hashemi-Fesharki, R; Nasseri, K; Godal, T; Smith, P G; Modabber, F

1999-02-05

Safety and efficacy of killed (autoclaved) L. major promastigotes, ALM, mixed with BCG against zoonotic cutaneous leishmaniasis was tested in healthy volunteers (n = 2453) in a randomized double blind trial vs. BCG as control. Side-effects were similar in both groups but tended to be slightly more frequent and prolonged in the ALM + BCG group. Leishmanin skin test conversion (induration > or =5 mm) was significantly greater in the ALM + BCG than in the BCG group (36.2% vs. 7.9% on day-80 and 33% vs. 19%, after 1 year, respectively). Cumulative incidence rates for 2 years, were similar in both groups (18.0% vs. 18.5%). However, LST responders on day 80 (> or =5 mm) had a significantly lower incidence (35%) of CL during the first year than non-responders. A single dose of ALM + BCG is not sufficiently immunogenic to provide a measurable response when compared to BCG alone. A single dose of this vaccine has been shown to be safe with no evidence of an exacerbating response following natural infection; hence, multiple doses or other adjuvants should be considered to increase its immunogenicity.

Structure-activity relationships of the antimalarial agent artemisinin. 8. design, synthesis, and CoMFA studies toward the development of artemisinin-based drugs against leishmaniasis and malaria.

PubMed

Avery, Mitchell A; Muraleedharan, Kannoth M; Desai, Prashant V; Bandyopadhyaya, Achintya K; Furtado, Marise M; Tekwani, Babu L

2003-09-25

Artemisinin (1) and its analogues have been well studied for their antimalarial activity. Here we present the antimalarial activity of some novel C-9-modified artemisinin analogues synthesized using artemisitene as the key intermediate. Further, antileishmanial activity of more than 70 artemisinin derivatives against Leishmania donovani promastigotes is described for the first time. A comprehensive structure-activity relationship study using CoMFA is discussed. These analogues exhibited leishmanicidal activity in micromolar concentrations, and the overall activity profile appears to be similar to that against malaria. Substitution at the C-9beta position was shown to improve the activity in both cases. The 10-deoxo derivatives showed better activity compared to the corresponding lactones. In general, compounds with C-9alpha substitution exhibited lower antimalarial as well as antileishmanial activities compared to the corresponding C-9beta analogues. The importance of the peroxide group for the observed activity of these analogues against leishmania was evident from the fact that 1-deoxyartemisinin analogues did not exhibit antileishmanial activity. The study suggests the possibility of developing artemisinin analogues as potential drug candidates against both malaria and leishmaniasis.

Rotenone-sensitive mitochondrial potential in Phytomonas serpens: electrophoretic Ca(2+) accumulation.

PubMed

Moysés, Danuza Nogueira; Barrabin, Hector

2004-06-07

Phytomonas sp. are flagellated trypanosomatid plant parasites that cause diseases of economic importance in plantations of coffee, oil palm, cassava and coconuts. Here we investigated Ca(2+) uptake by the vanadate-insensitive compartments using permeabilized Phytomonas serpens promastigotes. This uptake occurs at a rate of 1.13+/-0.23 nmol Ca(2+) mg x protein(-1) min(-1). It is completely abolished by the H(+) ionophore FCCP and by valinomycin and nigericin. It is also inhibited by 2 microM ruthenium red, which, at this low concentration, is known to inhibit the mitochondrial calcium uniport. Furthermore, salicylhydroxamic acid (SHAM) and propylgallate, specific inhibitors of the alternative oxidase in plant and parasite mitochondria, are also effective as inhibitors of the Ca(2+) transport. These compounds abolish the membrane potential that is monitored with safranine O. Rotenone, an inhibitor of NADH-CoQ oxidoreductase, can also dissipate 100% of the membrane potential. It is suggested that the mitochondria of P. serpens can be energized via oxidation of NADH in a pathway involving the NADH-CoQ oxidoreductase and the alternative oxidase to regenerate the ubiquinone. The electrochemical H(+) gradient can be used to promote Ca(2+) uptake by the mitochondria.

Differential serological testing by simultaneous indirect immunofluorescent antibody test in canine leishmaniosis and ehrlichiosis.

PubMed

Guillén Llera, J L; López García, M L; Martín Reinoso, E; De Vivar González, R

2002-11-11

A mixed indirect fluorescence antibody test (IFAT), based on cultured promastigotes Leishmania infantum and formol-inactivated suspension of cells infected with the bacteria Ehrlichia canis, was applied to make a differential diagnosis between canine ehrlichiosis and leishmaniosis. A titre greater than 80 was considered positive for antibodies to E. canis and suggestive of antibodies to L. infantum. Positive sera were titrated subsequently by serial dilutions to confirm antibodies positive to Leishmania and establishing the antibody titre of both pathogens. Fluorescence was absent with negative control sera and background staining was minimal. No serological cross-reactions between positive sera for L. infantum or E. canis were detected. Results obtained by mixed IFAT did not differ when the same serum IFAT standard was compared. The test showed equivalent sensitivity (100%). The specifities were 100% for L. infantum and 98.5% for E. canis. The equivalence in sensitivity was confirmed by calculating the correlation coefficient between IFAT standards and mixed IFAT (r>or=0.99 for both pathogens). The results of our investigations demonstrated that mixed IFAT is a specific means of establishing serological differential diagnosis of canine leishmaniosis and ehrlichiosis.

[Approaches and problems in vaccine development against leishmaniasis].

PubMed

Allahverdiyev, Adil; Bağirova, Melahat; Cakir Koç, Rabia; Oztel, Olga Nehir; Elçıçek, Serhat; Ateş, Sezen Canım; Karaca, Tuğçe Deniz

2010-01-01

Leishmaniasis is a major public health problem of the world and Turkey. Recently there has been increasing interest in vaccine studies among strategies for control of leishmaniasis. Recently the increase of interest in vaccine studies among leishmaniasis control strategies makes the subject more up to date. So the aim of this review is to present information about recent vaccine studies, problems and new strategies for vaccine development studies. There are 3 generations of vaccine against leishmaniasis. First-generation vaccines are killed or live attenuated parasites; second-generation vaccines are recombinant or native antigens and live genetically modified parasites (knock out and suicidal cassettes), third generation vaccines are DNA vaccines. Also vector salivary proteins, dendritic cells and non-pathogenic L. tarentolae have been used as vaccine candidates. However there is still no effective vaccine against leishmaniasis. Since polymer conjugates considerably increase immunogenicity, polymer based vaccine studies have gained importance in recent years. However, there has not been such a study for an antileishmanial vaccine yet. LPG, surface antigen of Leishmania promastigotes, and polymer conjugates may be promising in antileishmanial vaccine studies so we are carrying out a TUBITAK Project on this subject which has been given the number, 1085170SBAG-4007.

Mechanism of ascaridole activation in Leishmania.

PubMed

Geroldinger, Gerald; Tonner, Matthias; Hettegger, Hubert; Bacher, Markus; Monzote, Lianet; Walter, Martin; Staniek, Katrin; Rosenau, Thomas; Gille, Lars

2017-05-15

Endoperoxides (EP) are an emerging class of drugs which have potential in antiparasitic therapy, but also in other fields. For malaria therapy the EP artemisinin (Art) and its derivatives are successfully used. We have shown in the past that the EP ascaridole (Asc) is useful for the treatment of cutaneous leishmaniasis in a mouse model. Biomimetic experiments suggested that these drugs need activation in the respective target pathogens to exert their function. In spite of this idea, direct activation of EP to radicals inside cells has never been demonstrated. Therefore, this study was initiated to explore the activation of Asc in biomimetic systems and inside Leishmania in comparison to Art. Using electron paramagnetic resonance spectroscopy (EPR) in combination with spin-trapping we identified the secondary alkyl radical intermediates arising from reduction by Fe 2+ in cell-free systems. Combined GC/NMR analysis confirmed the loss of isopropyl residues from Asc during this process as intermediates. This activation of Asc was stimulated by low molecular Fe 2+ complexes or alternatively by hemin in conjunction with thiol reductants, such as cysteine (Cys). In Leishmania tarentolae promastigotes (LtP) as model for pathogenic forms of Leishmania carbon-centered radicals were identified in the presence of Asc by EPR spin-trapping. Both Asc and Art inhibited the viability in LtP with IC 50 values in the low micromolar range while IC 50 values for J774 macrophages were considerably higher. A similar structure without EP bridge (1,4-cineole) resulted in no detectable radicals and possessed much less cytotoxicity in LtP and no selectivity for LtP compared to J774 cells. The Asc-derived radical formation in LtP was inhibited by the iron chelator deferoxamine (DFO), and stimulated by Cys (a suitable reductant for hemin). The IC 50 values for LtP viability in the presence of Asc or Art were increased significantly by the spin trap DMPO, while Cys and DFO increased only IC 50 values for Art. In a heme association assay Asc demonstrated a lower binding affinity to heme than Art. ICP-OES measurements revealed that in LtP the total iron concentrations were twice as high as values in J774 macrophages. Since low molecular iron was important in Asc activation we studied the influence of Asc on the labile iron pool (LIP) in LtP. Low temperature EPR experiments demonstrated that Asc shifts the redox balance of iron in the LIP to its oxidized state. These data demonstrate that univalent cleavage of Asc/Art in LtP is an essential part of their pharmacological mechanism. The structure of the EP determines whether activation by low molecular iron or heme is favored and the availability of these intracellular activators modulates their cytotoxicity. These findings may be helpful for synthesis of new Asc derivatives and understanding the action of EP in other cell types. Copyright © 2017 Elsevier Inc. All rights reserved.

Spread of an Inactive Form of Caspase-12 in Humans Is Due to Recent Positive Selection

PubMed Central

Xue, Yali ; Daly, Allan ; Yngvadottir, Bryndis ; Liu, Mengning ; Coop, Graham ; Kim, Yuseob ; Sabeti, Pardis ; Chen, Yuan ; Stalker, Jim ; Huckle, Elizabeth ; Burton, John ; Leonard, Steven ; Rogers, Jane ; Tyler-Smith, Chris 

2006-01-01

The human caspase-12 gene is polymorphic for the presence or absence of a stop codon, which results in the occurrence of both active (ancestral) and inactive (derived) forms of the gene in the population. It has been shown elsewhere that carriers of the inactive gene are more resistant to severe sepsis. We have now investigated whether the inactive form has spread because of neutral drift or positive selection. We determined its distribution in a worldwide sample of 52 populations and resequenced the gene in 77 individuals from the HapMap Yoruba, Han Chinese, and European populations. There is strong evidence of positive selection from low diversity, skewed allele-frequency spectra, and the predominance of a single haplotype. We suggest that the inactive form of the gene arose in Africa ∼100–500 thousand years ago (KYA) and was initially neutral or almost neutral but that positive selection beginning ∼60–100 KYA drove it to near fixation. We further propose that its selective advantage was sepsis resistance in populations that experienced more infectious diseases as population sizes and densities increased. PMID:16532395

Keck Deep Fields. I. Observations, Reductions, and the Selection of Faint Star-forming Galaxies at Redshifts z ~ 4, 3, and 2

NASA Astrophysics Data System (ADS)

Sawicki, Marcin; Thompson, David

2005-12-01

We introduce a very deep, Rlim~27, multicolor imaging survey of very faint star-forming galaxies at z~4, 3, 2.2, and 1.7. This survey, carried out on the Keck I telescope, uses the very same UnGRI filter system that is employed by the Steidel team to select galaxies at these redshifts and thus allows us to construct identically selected but much fainter samples. However, our survey reaches ~1.5 mag deeper than the work of Steidel and his group, letting us probe substantially below the characteristic luminosity L* and thus study the properties and redshift evolution of the faint component of the high-z galaxy population. The survey covers 169 arcmin2 in three spatially independent patches on the sky and-to R<=27-contains 427 GRI-selected z~4 Lyman break galaxies, 1481 UnGR-selected z~3 Lyman break galaxies, 2417 UnGR-selected z~2.2 star-forming galaxies, and 2043 UnGR-selected z~1.7 star-forming galaxies. In this paper, the first in a series, we introduce the survey, describe our observing and data reduction strategies, and outline the selection of our z~4, 3, 2.2, and 1.7 samples. Based on data obtained at the W. M. Keck Observatory, which is operated as a scientific partnership among the California Institute of Technology, the University of California, and the National Aeronautics and Space Administration. The Observatory was made possible by the generous financial support of the W. M. Keck Foundation.

Controlled placement and orientation of nanostructures

DOEpatents

Zettl, Alex K; Yuzvinsky, Thomas D; Fennimore, Adam M

2014-04-08

A method for controlled deposition and orientation of molecular sized nanoelectromechanical systems (NEMS) on substrates is disclosed. The method comprised: forming a thin layer of polymer coating on a substrate; exposing a selected portion of the thin layer of polymer to alter a selected portion of the thin layer of polymer; forming a suspension of nanostructures in a solvent, wherein the solvent suspends the nanostructures and activates the nanostructures in the solvent for deposition; and flowing a suspension of nanostructures across the layer of polymer in a flow direction; thereby: depositing a nanostructure in the suspension of nanostructures only to the selected portion of the thin layer of polymer coating on the substrate to form a deposited nanostructure oriented in the flow direction. By selectively employing portions of the method above, complex NEMS may be built of simpler NEMSs components.

Identifying Luminous AGN in Deep Surveys: Revised IRAC Selection Criteria

NASA Astrophysics Data System (ADS)

Donley, Jennifer; Koekemoer, A. M.; Brusa, M.; Capak, P.; Cardamone, C. N.; Civano, F.; Ilbert, O.; Impey, C. D.; Kartaltepe, J.; Miyaji, T.; Salvato, M.; Sanders, D. B.; Trump, J. R.; Zamorani, G.

2012-01-01

Spitzer IRAC selection is a powerful tool for identifying luminous AGN. The AGN selection wedges currently in use, however, are heavily contaminated by star-forming galaxies, especially at high redshift. Using the large samples of luminous AGN and high-redshift star-forming galaxies in COSMOS, we redefine the AGN selection criteria for use in deep IRAC surveys. The new IRAC criteria are designed to be both highly complete and reliable, and incorporate the best aspects of the current AGN selection wedges and of infrared power-law selection while excluding high redshift star-forming galaxies selected via the BzK, DRG, LBG, and SMG criteria. At QSO-luminosities of log L(2-10 keV)>44, the new IRAC criteria recover 75% of the hard X-ray and IRAC-detected XMM-COSMOS sample, yet only 37% of the IRAC AGN candidates have X-ray counterparts, a fraction that rises to 51% in regions with Chandra exposures of 50-160 ks. X-ray stacking of the individually X-ray non-detected AGN candidates leads to a hard X-ray signal indicative of heavily obscured to mildly Compton-thick obscuration (log NH >= 23.7). While IRAC selection recovers a substantial fraction of luminous unobscured and obscured AGN, it is incomplete to low-luminosity and host-dominated AGN.

Male-male competition, female mate choice and their interaction: determining total sexual selection.

PubMed

Hunt, John; Breuker, Casper J; Sadowski, Jennifer A; Moore, Allen J

2009-01-01

Empirical studies of sexual selection typically focus on one of the two mechanisms of sexual selection without integrating these into a description of total sexual selection, or study total sexual selection without quantifying the contributions of all of the mechanisms of sexual selection. However, this can provide an incomplete or misleading view of how sexually selected traits evolve if the mechanisms of sexual selection are opposing or differ in form. Here, we take a two-fold approach to advocate a direction for future studies of sexual selection. We first show how a quantitative partitioning and examination of sexual selection mechanisms can inform by identifying illustrative studies that describe both male-male competition and female mate choice acting on the same trait. In our sample, the most common trait where this occurred was body size, and selection was typically linear. We found that male-male competition and female mate choice can be reinforcing or opposing, although the former is most common in the literature. The mechanisms of sexual selection can occur simultaneously or sequentially, and we found they were more likely to be opposing when the mechanisms operated sequentially. The degree and timing that these mechanisms interact have important implications for the operation of sexual selection and needs to be considered in designing studies. Our examples highlight where empirical data are needed. We especially lack standardized measures of the form and strength of selection imposed by each mechanism of sexual selection and how they combine to determine total sexual selection. Secondly, using quantitative genetic principles, we outline how the selection imposed by individual mechanisms can be measured and combined to estimate the total strength and form of sexual selection. We discuss the evolutionary consequences of combining the mechanisms of sexual selection and interpreting total sexual selection. We suggest how this approach may result in empirical progress in the field of sexual selection.

Network Interactions Explain Sensitivity to Dynamic Faces in the Superior Temporal Sulcus.

PubMed

Furl, Nicholas; Henson, Richard N; Friston, Karl J; Calder, Andrew J

2015-09-01

The superior temporal sulcus (STS) in the human and monkey is sensitive to the motion of complex forms such as facial and bodily actions. We used functional magnetic resonance imaging (fMRI) to explore network-level explanations for how the form and motion information in dynamic facial expressions might be combined in the human STS. Ventral occipitotemporal areas selective for facial form were localized in occipital and fusiform face areas (OFA and FFA), and motion sensitivity was localized in the more dorsal temporal area V5. We then tested various connectivity models that modeled communication between the ventral form and dorsal motion pathways. We show that facial form information modulated transmission of motion information from V5 to the STS, and that this face-selective modulation likely originated in OFA. This finding shows that form-selective motion sensitivity in the STS can be explained in terms of modulation of gain control on information flow in the motion pathway, and provides a substantial constraint for theories of the perception of faces and biological motion. © The Author 2014. Published by Oxford University Press.

Shape Coding for Daymarks

DOT National Transportation Integrated Search

1974-03-01

Three experiments were conducted on form discrimination to select and evaluate forms for shape coding of daymarks. The discriminability of the forms was measured by the frequency with which each form was identified correctly and the frequency with wh...

Selection Skills and Collection Development in School Libraries.

ERIC Educational Resources Information Center

Graef, Robert; And Others

1990-01-01

Presents 11 articles that deal with selection skills and collection development in junior and senior high school libraries. Highlights include censorship concerns, selecting corporate-sponsored materials, written selection policies, budget considerations, sample selection and evaluation forms for print and nonprint materials, reference books, and…

Process for preparing tows from composite fiber blends

NASA Technical Reports Server (NTRS)

McMahon, Paul (Inventor); Chung, Tai-Shung (Inventor); Ying, Lincoln (Inventor)

1989-01-01

A continuous, substantially uniform tow useful in forming composite molded articles is prepared by forming a continuous tow of continuous carbon fibers, forming a continuous tow of thermoplastic polymer fibers to a selected width, uniformly and continuously spreading the carbon fiber two to a width that is essentially the same as the selected width for the thermoplastic polymer fiber tow, intermixing the tows intimately, uniformly and continuously, in a relatively tension-free state, and continuosuly withdrawing the intermixed tow.

Genetic Validation of Leishmania donovani Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity

PubMed Central

Chadha, Sanya; Mallampudi, N. Arjunreddy; Mohapatra, Debendra K.

2017-01-01

ABSTRACT Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis. Increasing resistance and severe side effects of existing drugs have led to the need to identify new chemotherapeutic targets. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and are required for protein synthesis. aaRSs are known drug targets for bacterial and fungal pathogens. Here, we have characterized and evaluated the essentiality of L. donovani lysyl-tRNA synthetase (LdLysRS). Two different coding sequences for lysyl-tRNA synthetases are annotated in the Leishmania genome database. LdLysRS-1 (LdBPK_150270.1), located on chromosome 15, is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 (LdBPK_300130.1), present on chromosome 30, is closer to bacteria. In the present study, we have characterized LdLysRS-1. Recombinant LdLysRS-1 displayed aminoacylation activity, and the protein localized to the cytosol. The LdLysRS-1 heterozygous mutants had a restrictive growth phenotype and attenuated infectivity. LdLysRS-1 appears to be an essential gene, as a chromosomal knockout of LdLysRS-1 could be generated when the gene was provided on a rescuing plasmid. Cladosporin, a fungal secondary metabolite and a known inhibitor of LysRS, was more potent against promastigotes (50% inhibitory concentration [IC50], 4.19 µM) and intracellular amastigotes (IC50, 1.09 µM) than were isomers of cladosporin (3-epi-isocladosporin and isocladosporin). These compounds exhibited low toxicity to mammalian cells. The specificity of inhibition of parasite growth caused by these inhibitors was further assessed using LdLysRS-1 heterozygous mutant strains and rescue mutant promastigotes. These inhibitors inhibited the aminoacylation activity of recombinant LdLysRS. Our data provide a framework for the development of a new class of drugs against this parasite. IMPORTANCE Aminoacyl-tRNA synthetases are housekeeping enzymes essential for protein translation, providing charged tRNAs for the proper construction of peptide chains. These enzymes provide raw materials for protein translation and also ensure fidelity of translation. L. donovani is a protozoan parasite that causes visceral leishmaniasis. It is a continuously proliferating parasite that depends heavily on efficient protein translation. Lysyl-tRNA synthetase is one of the aaRSs which charges lysine to its cognate tRNA. Two different coding sequences for lysyl-tRNA synthetases (LdLysRS) are present in this parasite. LdLysRS-1 is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 is closer to bacteria. Here, we have characterized LdLysRS-1 of L. donovani. LdLysRS-1 appears to be an essential gene, as the chromosomal null mutants did not survive. The heterozygous mutants showed slower growth kinetics and exhibited attenuated virulence. This study also provides a platform to explore LdLysRS-1 as a potential drug target. PMID:28875178

Genetic Validation of Leishmania donovani Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity.

PubMed

Chadha, Sanya; Mallampudi, N Arjunreddy; Mohapatra, Debendra K; Madhubala, Rentala

2017-01-01

Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis. Increasing resistance and severe side effects of existing drugs have led to the need to identify new chemotherapeutic targets. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and are required for protein synthesis. aaRSs are known drug targets for bacterial and fungal pathogens. Here, we have characterized and evaluated the essentiality of L. donovani lysyl-tRNA synthetase ( Ld LysRS). Two different coding sequences for lysyl-tRNA synthetases are annotated in the Leishmania genome database. Ld LysRS-1 (LdBPK_150270.1), located on chromosome 15, is closer to apicomplexans and eukaryotes, whereas Ld LysRS-2 (LdBPK_300130.1), present on chromosome 30, is closer to bacteria. In the present study, we have characterized Ld LysRS-1. Recombinant Ld LysRS-1 displayed aminoacylation activity, and the protein localized to the cytosol. The Ld LysRS-1 heterozygous mutants had a restrictive growth phenotype and attenuated infectivity. Ld LysRS-1 appears to be an essential gene, as a chromosomal knockout of Ld LysRS-1 could be generated when the gene was provided on a rescuing plasmid. Cladosporin, a fungal secondary metabolite and a known inhibitor of LysRS, was more potent against promastigotes (50% inhibitory concentration [IC 50 ], 4.19 µM) and intracellular amastigotes (IC 50 , 1.09 µM) than were isomers of cladosporin (3-epi-isocladosporin and isocladosporin). These compounds exhibited low toxicity to mammalian cells. The specificity of inhibition of parasite growth caused by these inhibitors was further assessed using Ld LysRS-1 heterozygous mutant strains and rescue mutant promastigotes. These inhibitors inhibited the aminoacylation activity of recombinant Ld LysRS. Our data provide a framework for the development of a new class of drugs against this parasite. IMPORTANCE Aminoacyl-tRNA synthetases are housekeeping enzymes essential for protein translation, providing charged tRNAs for the proper construction of peptide chains. These enzymes provide raw materials for protein translation and also ensure fidelity of translation. L. donovani is a protozoan parasite that causes visceral leishmaniasis. It is a continuously proliferating parasite that depends heavily on efficient protein translation. Lysyl-tRNA synthetase is one of the aaRSs which charges lysine to its cognate tRNA. Two different coding sequences for lysyl-tRNA synthetases ( Ld LysRS) are present in this parasite. Ld LysRS-1 is closer to apicomplexans and eukaryotes, whereas Ld LysRS-2 is closer to bacteria. Here, we have characterized Ld LysRS-1 of L. donovani . Ld LysRS-1 appears to be an essential gene, as the chromosomal null mutants did not survive. The heterozygous mutants showed slower growth kinetics and exhibited attenuated virulence. This study also provides a platform to explore Ld LysRS-1 as a potential drug target.

42 CFR 73.5 - Exemptions for HHS select agents and toxins.

Code of Federal Regulations, 2010 CFR

2010-10-01

... the select agent or toxin is reported to CDC or APHIS and to other appropriate authorities when... minor (Alastrim), or Yersinia pestis. This report must be followed by submission of APHIS/CDC Form 4.../CDC Form 4 must be submitted within seven calendar days after identification. (iii) Less stringent...

42 CFR 73.6 - Exemptions for overlap select agents and toxins.

Code of Federal Regulations, 2011 CFR

2011-10-01

... reported to CDC or APHIS and to other appropriate authorities when required by Federal, State, or local law... submission of APHIS/CDC Form 4 within seven calendar days after identification. (ii) For all other overlap select agents or toxins, APHIS/CDC Form 4 must be submitted within seven calendar days after...

42 CFR 73.6 - Exemptions for overlap select agents and toxins.

Code of Federal Regulations, 2010 CFR

2010-10-01

... reported to CDC or APHIS and to other appropriate authorities when required by Federal, State, or local law... submission of APHIS/CDC Form 4 within seven calendar days after identification. (ii) For all other overlap select agents or toxins, APHIS/CDC Form 4 must be submitted within seven calendar days after...

42 CFR 73.6 - Exemptions for overlap select agents and toxins.

Code of Federal Regulations, 2012 CFR

2012-10-01

... reported to CDC or APHIS and to other appropriate authorities when required by Federal, State, or local law... submission of APHIS/CDC Form 4 within seven calendar days after identification. (ii) For all other overlap select agents or toxins, APHIS/CDC Form 4 must be submitted within seven calendar days after...

42 CFR 73.5 - Exemptions for HHS select agents and toxins.

Code of Federal Regulations, 2011 CFR

2011-10-01

... the select agent or toxin is reported to CDC or APHIS and to other appropriate authorities when... minor (Alastrim), or Yersinia pestis. This report must be followed by submission of APHIS/CDC Form 4.../CDC Form 4 must be submitted within seven calendar days after identification. (iii) Less stringent...

42 CFR 73.5 - Exemptions for HHS select agents and toxins.

Code of Federal Regulations, 2012 CFR

2012-10-01

... the select agent or toxin is reported to CDC or APHIS and to other appropriate authorities when... minor (Alastrim), or Yersinia pestis. This report must be followed by submission of APHIS/CDC Form 4.../CDC Form 4 must be submitted within seven calendar days after identification. (iii) Less stringent...

Selective photooxidation of hydrocarbons in zeolites by oxygen

DOEpatents

Frei, Heinz; Blatter, Fritz; Sun, Hai

1998-01-01

A selective photooxidation process for the conversion of hydrocarbon molecules to partially oxygenated derivatives, which comprises the steps of adsorbing a hydrocarbon and oxygen onto a dehydrated zeolite support matrix to form a hydrocarbon-oxygen contact pair, and subsequently exposing the hydrocarbon-oxygen contact pair to visible light, thereby forming a partially oxygenated derivative.

Composite pipe to metal joint

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leslie, James C.; Leslie, II, James C.; Heard, James

A method for making a metal to composite tube joint including selecting an elongated interior fitting constructed with an exterior barrel, reduced in exterior diameter to form a distally facing annular shoulder and then projecting still further distally to form an interior sleeve having a radially outwardly facing bonding surface. Selecting an elongated metal outer sleeve formed proximally with a collar constructed for receipt over the barrel and increased in interior diameter and projecting distally to form an exterior sleeve having a radially inwardly facing bonding surface cooperating with the first bonding surface to form an annulus receiving an extremitymore » of a composite tube and a bond bonding the extremity of the tube to the bonding surfaces.« less

Sexual selection is a form of social selection.

PubMed

Lyon, Bruce E; Montgomerie, Robert

2012-08-19

Social selection influences the evolution of weapons, ornaments and behaviour in both males and females. Thus, social interactions in both sexual and non-sexual contexts can have a powerful influence on the evolution of traits that would otherwise appear to be detrimental to survival. Although clearly outlined by West-Eberhard in the early 1980s, the idea that social selection is a comprehensive framework for the study of ornaments and weapons has largely been ignored. In West-Eberhard's view, sexual selection is a form of social selection-a concept supported by several lines of evidence. Darwin's distinction between natural and sexual selection has been useful, but recent confusion about the limits of sexual selection suggests that some traits are not easily categorized as naturally or sexually selected. Because social selection theory has much to offer the current debates about both sexual selection and reproductive competition in females, it is sometimes viewed, narrowly, to be most useful when considering female roles. However, social selection theory encompasses much more than female reproductive competition. Our goal here was to provide that broader perspective.

FUSED REACTOR FUELS

DOEpatents

Mayer, S.W.

1962-11-13

This invention relates to a nuciear reactor fuel composition comprising (1) from about 0.01 to about 50 wt.% based on the total weight of said composition of at least one element selected from the class consisting of uranium, thorium, and plutonium, wherein said eiement is present in the form of at least one component selected from the class consisting of oxides, halides, and salts of oxygenated anions, with components comprising (2) at least one member selected from the class consisting of (a) sulfur, wherein the sulfur is in the form of at least one entity selected irom the class consisting of oxides of sulfur, metal sulfates, metal sulfites, metal halosulfonates, and acids of sulfur, (b) halogen, wherein said halogen is in the form of at least one compound selected from the class of metal halides, metal halosulfonates, and metal halophosphates, (c) phosphorus, wherein said phosphorus is in the form of at least one constituent selected from the class consisting of oxides of phosphorus, metal phosphates, metal phosphites, and metal halophosphates, (d) at least one oxide of a member selected from the class consisting of a metal and a metalloid wherein said oxide is free from an oxide of said element in (1); wherein the amount of at least one member selected from the class consisting of halogen and sulfur is at least about one at.% based on the amount of the sum of said sulfur, halogen, and phosphorus atom in said composition; and wherein the amount of said 2(a), 2(b) and 2(c) components in said composition which are free from said elements of uranium, thorium, arid plutonium, is at least about 60 wt.% based on the combined weight of the components of said composition which are free from said elements of uranium, thorium, and plutonium. (AEC)

Sensitive, Selective Test For Hydrazines

NASA Technical Reports Server (NTRS)

Roundbehler, David; Macdonald, Stephen

1993-01-01

Derivatives of hydrazines formed, then subjected to gas chromatography and detected via chemiluminescence. In method of detecting and quantifying hydrazine vapors, vapors reacted with dinitro compound to enhance sensitivity and selectivity. Hydrazine (HZ), monomethyl hydrazine, (MMH), and unsymmetrical dimethylhydrazine (UDMH) analyzed quantitatively and qualitatively, either alone or in mixtures. Vapors collected and reacted with 2,4-dinitrobenzaldehyde, (DNB), making it possible to concentrate hydrazine in derivative form, thereby increasing sensitivity to low initial concentrations. Increases selectivity because only those constituents of sample reacting with DNB concentrated for analysis.

Shuttle plate braiding machine

NASA Technical Reports Server (NTRS)

Huey, Jr., Cecil O. (Inventor)

1994-01-01

A method and apparatus for moving yarn in a selected pattern to form a braided article. The apparatus includes a segmented grid of stationary support elements and a plurality of shuttles configured to carry yarn. The shuttles are supported for movement on the grid assembly and each shuttle includes a retractable plunger for engaging a reciprocating shuttle plate that moves below the grid assembly. Such engagement at selected times causes the shuttles to move about the grid assembly in a selected pattern to form a braided article of a particular geometry.

Detecting evolutionary forces in language change.

PubMed

Newberry, Mitchell G; Ahern, Christopher A; Clark, Robin; Plotkin, Joshua B

2017-11-09

Both language and genes evolve by transmission over generations with opportunity for differential replication of forms. The understanding that gene frequencies change at random by genetic drift, even in the absence of natural selection, was a seminal advance in evolutionary biology. Stochastic drift must also occur in language as a result of randomness in how linguistic forms are copied between speakers. Here we quantify the strength of selection relative to stochastic drift in language evolution. We use time series derived from large corpora of annotated texts dating from the 12th to 21st centuries to analyse three well-known grammatical changes in English: the regularization of past-tense verbs, the introduction of the periphrastic 'do', and variation in verbal negation. We reject stochastic drift in favour of selection in some cases but not in others. In particular, we infer selection towards the irregular forms of some past-tense verbs, which is likely driven by changing frequencies of rhyming patterns over time. We show that stochastic drift is stronger for rare words, which may explain why rare forms are more prone to replacement than common ones. This work provides a method for testing selective theories of language change against a null model and reveals an underappreciated role for stochasticity in language evolution.

Pre and Post-copulatory Selection Favor Similar Genital Phenotypes in the Male Broad Horned Beetle.

PubMed

House, Clarissa M; Sharma, M D; Okada, Kensuke; Hosken, David J

2016-10-01

Sexual selection can operate before and after copulation and the same or different trait(s) can be targeted during these episodes of selection. The direction and form of sexual selection imposed on characters prior to mating has been relatively well described, but the same is not true after copulation. In general, when male-male competition and female choice favor the same traits then there is the expectation of reinforcing selection on male sexual traits that improve competitiveness before and after copulation. However, when male-male competition overrides pre-copulatory choice then the opposite could be true. With respect to studies of selection on genitalia there is good evidence that male genital morphology influences mating and fertilization success. However, whether genital morphology affects reproductive success in more than one context (i.e., mating versus fertilization success) is largely unknown. Here we use multivariate analysis to estimate linear and nonlinear selection on male body size and genital morphology in the flour beetle Gnatocerus cornutus, simulated in a non-competitive (i.e., monogamous) setting. This analysis estimates the form of selection on multiple traits and typically, linear (directional) selection is easiest to detect, while nonlinear selection is more complex and can be stabilizing, disruptive, or correlational. We find that mating generates stabilizing selection on male body size and genitalia, and fertilization causes a blend of directional and stabilizing selection. Differences in the form of selection across these bouts of selection result from a significant alteration of nonlinear selection on body size and a marginally significant difference in nonlinear selection on a component of genital shape. This suggests that both bouts of selection favor similar genital phenotypes, whereas the strong stabilizing selection imposed on male body size during mate acquisition is weak during fertilization. © The Author 2016. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.

Functional forms and price elasticities in a discrete continuous choice model of the residential water demand

NASA Astrophysics Data System (ADS)

Vásquez Lavín, F. A.; Hernandez, J. I.; Ponce, R. D.; Orrego, S. A.

2017-07-01

During recent decades, water demand estimation has gained considerable attention from scholars. From an econometric perspective, the most used functional forms include log-log and linear specifications. Despite the advances in this field and the relevance for policymaking, little attention has been paid to the functional forms used in these estimations, and most authors have not provided justifications for their selection of functional forms. A discrete continuous choice model of the residential water demand is estimated using six functional forms (log-log, full-log, log-quadratic, semilog, linear, and Stone-Geary), and the expected consumption and price elasticity are evaluated. From a policy perspective, our results highlight the relevance of functional form selection for both the expected consumption and price elasticity.

Leishmania proteophosphoglycans regurgitated from infected sand flies accelerate dermal wound repair and exacerbate leishmaniasis via insulin-like growth factor 1-dependent signalling

PubMed Central

Giraud, Emilie; Martin, Oihane; Dillon, Rod J.; Műller, Ingrid

2018-01-01

Leishmania parasites are transmitted to vertebrate hosts by female phlebotomine sand flies as they bloodfeed by lacerating the upper capillaries of the dermis with their barbed mouthparts. In the sand fly midgut secreted proteophosphoglycans from Leishmania form a biological plug known as the promastigote secretory gel (PSG), which blocks the gut and facilitates the regurgitation of infective parasites. The interaction between the wound created by the sand fly bite and PSG is not known. Here we nanoinjected a sand fly egested dose of PSG into BALB/c mouse skin that lead to the differential expression of 7,907 transcripts. These transcripts were transiently up-regulated during the first 6 hours post-wound and enriched for pathways involved in inflammation, cell proliferation, fibrosis, epithelial cell differentiation and wound remodelling. We found that PSG significantly accelerated wound healing in vitro and in mice; which was associated with an early up-regulation of transcripts involved in inflammation (IL-1β, IL-6, IL-10, TNFα) and inflammatory cell recruitment (CCL2, CCL3, CCL4, CXCL2), followed 6 days later by enhanced expression of transcripts associated with epithelial cell proliferation, fibroplasia and fibrosis (FGFR2, EGF, EGFR, IGF1). Dermal expression of IGF1 was enhanced following an infected sand fly bite and was acutely responsive to the deposition of PSG but not the inoculation of parasites or sand fly saliva. Antibody blockade of IGF1 ablated the gel’s ability to promote wound closure in mouse ears and significantly reduced the virulence of Leishmania mexicana infection delivered by an individual sand fly bite. Dermal macrophages recruited to air-pouches on the backs of mice revealed that IGF1 was pivotal to the PSG’s ability to promote macrophage alternative activation and Leishmania infection. Our data demonstrate that through the regurgitation of PSG Leishmania exploit the wound healing response of the host to the vector bite by promoting the action of IGF1 to drive the alternative activation of macrophages. PMID:29352310

Structure, Antimicrobial Activities and Mode of Interaction with Membranes of Bovel Phylloseptins from the Painted-Belly Leaf Frog, Phyllomedusa sauvagii

PubMed Central

Raja, Zahid; André, Sonia; Piesse, Christophe; Sereno, Denis; Nicolas, Pierre; Foulon, Thierry

2013-01-01

Transcriptomic and peptidomic analysis of skin secretions from the Painted-belly leaf frog Phyllomedusa sauvagii led to the identification of 5 novel phylloseptins (PLS-S2 to -S6) and also of phylloseptin-1 (PSN-1, here renamed PLS-S1), the only member of this family previously isolated in this frog. Synthesis and characterization of these phylloseptins revealed differences in their antimicrobial activities. PLS-S1, -S2, and -S4 (79–95% amino acid sequence identity; net charge  = +2) were highly potent and cidal against Gram-positive bacteria, including multidrug resistant S. aureus strains, and killed the promastigote stage of Leishmania infantum, L. braziliensis and L. major. By contrast, PLS-S3 (95% amino acid identity with PLS-S2; net charge  = +1) and -S5 (net charge  = +2) were found to be almost inactive against bacteria and protozoa. PLS-S6 was not studied as this peptide was closely related to PLS-S1. Differential scanning calorimetry on anionic and zwitterionic multilamellar vesicles combined with circular dichroism spectroscopy and membrane permeabilization assays on bacterial cells indicated that PLS-S1, -S2, and -S4 are structured in an amphipathic α-helix that disrupts the acyl chain packing of anionic lipid bilayers. As a result, regions of two coexisting phases could be formed, one phase rich in peptide and the other lipid-rich. After reaching a threshold peptide concentration, the disruption of lipid packing within the bilayer may lead to local cracks and disintegration of the microbial membrane. Differences in the net charge, α-helical folding propensity, and/or degree of amphipathicity between PLS-S1, -S2 and -S4, and between PLS-S3 and -S5 appear to be responsible for their marked differences in their antimicrobial activities. In addition to the detailed characterization of novel phylloseptins from P. sauvagii, our study provides additional data on the previously isolated PLS-S1 and on the mechanism of action of phylloseptins. PMID:23967105

A comparative evaluation of efficacy of chemotherapy, immunotherapy and immunochemotherapy in visceral leishmaniasis-an experimental study.

PubMed

Joshi, Jyoti; Malla, Nancy; Kaur, Sukhbir

2014-08-01

Visceral leishmaniasis (VL) represents the second most challenging infectious disease worldwide, leading to nearly 500,000 new cases and 60,000 deaths annually. Ninety per cent of VL cases occur in five countries namely Bangladesh, India, Nepal, Sudan and Brazil. No licensed vaccine is available till date against any form of leishmaniasis. High toxicity and increasing resistance to the current chemotherapeutic regimens have further complicated the situation in VL endemic regions of the world. To combat this situation, immunochemotherapy can provide a solution. In the present study, an attempt has been made to assess the in vivo antileishmanial efficacy of chemotherapy, immunotherapy and immunochemotherapy with the use of a first generation antigen Killed Leishmania donovani (KLD) along with a standard drug sodium stibogluconate (SSG) and a newly tested antileishmanial cisplatin. Inbred BALB/c mice were infected with 10(7) promastigotes/0.1 ml of Leishmania donovani. A month after infection, these animals were given specific immunotherapy (KLD/KLD+MPL-A) or chemotherapy (SSG/cisplatin) or immunochemotherapy (SSG+KLD/SSG+KLD+MPL-A/cisplatin+KLD/cisplatin+KLD+MPL-A). Animals were sacrificed on 1, 15 and 30(th) day post treatment. The efficacy of these combinations was assessed in terms of parasite load and by immunological investigations. Infected mice and normal mice served as controls. Results showed that combination of drug and KLD significantly reduced the parasite burden, enhanced the DTH (Delayed Type Hypersensitivity) responses, showed increased levels of IgG2a and decreased levels of IgG1 as compared to mice given chemotherapy or immunotherapy alone. Further maximum protection was provided by SSG+KLD+MPL-A and it was most effective as depicted by 98.5% reduction in parasite load, a potent increase in IFN-γ levels and a significant decrease in IL-10 and IL-4 levels thus skewing the immune response towards Th1 type. Hence, immunochemotherapy is more effective in control of VL in comparison to chemotherapy or immunotherapy. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Spectrally-engineered solar thermal photovoltaic devices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lenert, Andrej; Bierman, David; Chan, Walker

A solar thermal photovoltaic device, and method of forming same, includes a solar absorber and a spectrally selective emitter formed on either side of a thermally conductive substrate. The solar absorber is configured to absorb incident solar radiation. The solar absorber and the spectrally selective emitter are configured with an optimized emitter-to-absorber area ratio. The solar thermal photovoltaic device also includes a photovoltaic cell in thermal communication with the spectrally selective emitter. The spectrally selective emitter is configured to permit high emittance for energies above a bandgap of the photovoltaic cell and configured to permit low emittance for energies belowmore » the bandgap.« less

Nevada High School Proficiency Examination: Reading. Form E Released, Part 1 [and] Part 2.

ERIC Educational Resources Information Center

Nevada State Dept. of Education, Carson City.

This document presents a sample test form for high school reading and comprehension proficiency. The first section of the exam consists of 27 questions taken from 7 selections, which included 4 literature and poetry selections and the following nonfiction titles: Procedures for Answering a Call, Just the Facts, Garlic, and Pot Roast with…

Development of Officer Selection Battery Forms 3 and 4. Technical Report 603.

ERIC Educational Resources Information Center

Fischl, M. A.; And Others

This report describes the development, standardization, and validation of two parallel forms of the Officer Selection Battery, a 2-hour, group administrable, paper and pencil test for assessing men and women applying for the Reserve Officers Training Corps (ROTC). Based on an extensive job analysis, 1,400 experimental items in 12 job areas were…

42 CFR 73.6 - Exemptions for overlap select agents and toxins.

Code of Federal Regulations, 2013 CFR

2013-10-01

... reported to CDC or APHIS and to other appropriate authorities when required by Federal, State, or local law... pseudomallei. This report must be followed by submission of APHIS/CDC Form 4 within seven calendar days after identification. (ii) For all other overlap select agents or toxins, APHIS/CDC Form 4 must be submitted within...

42 CFR 73.6 - Exemptions for overlap select agents and toxins.

Code of Federal Regulations, 2014 CFR

2014-10-01

... reported to CDC or APHIS and to other appropriate authorities when required by Federal, State, or local law... pseudomallei. This report must be followed by submission of APHIS/CDC Form 4 within seven calendar days after identification. (ii) For all other overlap select agents or toxins, APHIS/CDC Form 4 must be submitted within...

A Guideline of Selecting and Reporting Intraclass Correlation Coefficients for Reliability Research.

PubMed

Koo, Terry K; Li, Mae Y

2016-06-01

Intraclass correlation coefficient (ICC) is a widely used reliability index in test-retest, intrarater, and interrater reliability analyses. This article introduces the basic concept of ICC in the content of reliability analysis. There are 10 forms of ICCs. Because each form involves distinct assumptions in their calculation and will lead to different interpretations, researchers should explicitly specify the ICC form they used in their calculation. A thorough review of the research design is needed in selecting the appropriate form of ICC to evaluate reliability. The best practice of reporting ICC should include software information, "model," "type," and "definition" selections. When coming across an article that includes ICC, readers should first check whether information about the ICC form has been reported and if an appropriate ICC form was used. Based on the 95% confident interval of the ICC estimate, values less than 0.5, between 0.5 and 0.75, between 0.75 and 0.9, and greater than 0.90 are indicative of poor, moderate, good, and excellent reliability, respectively. This article provides a practical guideline for clinical researchers to choose the correct form of ICC and suggests the best practice of reporting ICC parameters in scientific publications. This article also gives readers an appreciation for what to look for when coming across ICC while reading an article.

47 CFR 1.913 - Application and notification forms; electronic and manual filing.

Code of Federal Regulations, 2012 CFR

2012-10-01

... PRACTICE AND PROCEDURE Grants by Random Selection Wireless Radio Services Applications and Proceedings... Form 601, Application for Authorization in the Wireless Radio Services. FCC Form 601 and associated... 602, Wireless Radio Services Ownership Form. FCC Form 602 is used by applicants and licensees in...

NHEXAS PHASE I ARIZONA STUDY--STANDARD OPERATING PROCEDURE FOR CODING: FIELD FORMS (UA-D-37.0)

EPA Science Inventory

The purpose of this SOP is to define the coding strategy for selected field forms. Forms addressed here will be scanned into databases; databases are created because the forms contain critical values needed to calculate pollutant concentrations. Other forms not addressed by thi...

Method of making a composite tube to metal joint

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leslie, James C.; Leslie, II, James C.; Heard, James

A method for making a metal to composite tube joint including selecting an elongated interior fitting constructed with an exterior barrel, reduced in exterior diameter to form a distally facing annular shoulder and then projecting still further distally to form an interior sleeve having a radially outwardly facing bonding surface. Selecting an elongated metal outer sleeve formed proximally with a collar constructed for receipt over the barrel and increased in interior diameter and projecting distally to form an exterior sleeve having a radially inwardly facing bonding surface cooperating with the first bonding surface to form an annulus receiving an extremitymore » of a composite tube and a bond bonding the extremity of the tube to the bonding surfaces.« less

Different Height Forms of Spartina alterniflora Might Select Their Own Rhizospheric Bacterial Communities in Southern Coast of China.

PubMed

Lin, Li'an; Liu, Wenwen; Zhang, Manping; Lin, Xiaolan; Zhang, Yihui; Tian, Yun

2018-06-13

In the southernmost part of coast of China, two height forms of Spartina alterniflora, tall and short, have invaded Leizhou Peninsula within the last decade. However, the effect of different height forms of Spartina alterniflora on plant-microbe interaction has not been clarified. Here, the community structures of rhizosphere bacteria and the abundance of N- and S-cycling functional genes associated with selected S. alterniflora were investigated in the field and a common garden. The community structure of tall-form S. alterniflora was distinct from short-form S. alterniflora at OTU level in the field, even after transplantation into a common garden. The abundance of bacterial amoA, nirS, and nosZ in tall S. alterniflora was significantly greater than those in short S. alterniflora in the field; however, this difference disappeared in a 1-year common garden experiment. These results suggested that compared with the tall-form S. alterniflora, the rhizosphere of short-form S. alterniflora harbored fewer nitrification-denitrification related microorganisms, which might benefit from conserving N in an N limited habitat. Together, our results suggested that tall- and short-form S. alterniflora can host their specific rhizosphere microbial communities and had different strategies of N usage via selecting the composition of rhizosphere bacterial assemblages, which in turn might determine the growth and invasiveness of S. alterniflora in its introduced range.

76 FR 62412 - Agency Forms Undergoing Paperwork Reduction Act Review

Federal Register 2010, 2011, 2012, 2013, 2014

2011-10-07

... for Registration, (2) Request to Transfer Select Agent or Toxin, (3) Report of Theft, Loss, or Release..., or Release of Select Agent and Toxin, (4) Report of Identification of Select Agent or Toxin. There... Release of Select Agent or Toxin; (4) Report of Identification of Select Agent or Toxin; and (5) Request...

42 CFR 73.3 - HHS select agents and toxins.

Code of Federal Regulations, 2010 CFR

2010-10-01

... been genetically modified. (d) HHS select agents or toxins that meet any of the following criteria are... Recombinant Organisms: (1) Nucleic acids that can produce infectious forms of any of the select agent viruses...

NEUTRONIC REACTOR FUEL ELEMENT

DOEpatents

Shackleford, M.H.

1958-12-16

A fuel element possessing good stability and heat conducting properties is described. The fuel element comprises an outer tube formed of material selected from the group consisting of stainhess steel, V, Ti. Mo. or Zr, a fuel tube concentrically fitting within the outer tube and containing an oxide of an isotope selected from the group consisting of U/sup 235/, U/sup 233/, and Pu/sup 239/, and a hollow, porous core concentrically fitting within the fuel tube and formed of an oxide of an element selected from the group consisting of Mg, Be, and Zr.