Macrophage sphingolipids are essential for the entry of mycobacteria.

Science.gov (United States)

Viswanathan, Gopinath; Jafurulla, Md; Kumar, G Aditya; Raghunand, Tirumalai R; Chattopadhyay, Amitabha

2018-07-01

Mycobacteria are intracellular pathogens that can invade and survive within host macrophages. Mycobacterial infections remain a major cause of mortality and morbidity worldwide, with serious concerns of emergence of multi and extensively drug-resistant tuberculosis. While significant advances have been made in identifying mycobacterial virulence determinants, the detailed molecular mechanism of internalization of mycobacteria into host cells remains poorly understood. Although several studies have highlighted the crucial role of sphingolipids in mycobacterial growth, persistence and establishment of infection, the role of sphingolipids in the entry of mycobacteria into host cells is not known. In this work, we explored the role of host membrane sphingolipids in the entry of Mycobacterium smegmatis into J774A.1 macrophages. Our results show that metabolic depletion of sphingolipids in host macrophages results in a significant reduction in the entry of M. smegmatis. Importantly, the entry of Escherichia coli into host macrophages under similar conditions remained invariant, implying the specificity of the requirement of sphingolipids in mycobacterial entry. To the best of our knowledge, our results constitute the first report demonstrating the role of host macrophage sphingolipids in the entry of mycobacteria. Our results could help in the development of novel therapeutic strategies targeting sphingolipid-mediated entry of mycobacteria into host cells. Copyright © 2018 Elsevier B.V. All rights reserved.

Sphingolipids and plant defense/disease: the "death" connection and beyond

Directory of Open Access Journals (Sweden)

Robert eBerkey

2012-04-01

Full Text Available Sphingolipids comprise a major class of structural materials and lipid signaling molecules in all eukaryotic cells. Over the past two decades, there has been a phenomenal growth in the study of sphingolipids (i.e. sphingobiology at an average rate of >1000 research articles per year. Sphingolipid studies in plants, though accounting for only a small fraction (~6% of the total number of publications, have also enjoyed proportionally rapid growth in the past decade. Concomitant with the growth of sphingobiology, there has also been tremendous progress in our understanding of the molecular mechanisms of plant innate immunity. In this review, we (i cross examine and analyze the major findings that establish and strengthen the intimate connections between sphingolipid metabolism and plant programmed cell death (PCD associated with plant defense or disease; (ii highlight and compare key bioactive sphingolipids involved in the regulation of plant PCD and possibly defense; (iii discuss the potential role of sphingolipids in polarized membrane/protein trafficking and formation of lipid rafts as subdomains of cell membranes in relation to plant defense; and (iv where possible, attempt to identify potential parallels for immunity-related mechanisms involving sphingolipids across kingdoms.

Experimental treatment with sodium stibogluconate of hamsters infected with Leishmania (Leishmania) chagasi and Leishmania (Leishmania) amazonensis Tratamento experimental com stibogluconato de sódio em hamsters infectados com Leishmania (Leishmania) chagasi e Leishmania (Leishmania) amazonensis

OpenAIRE

Elizabeth M. de Figueiredo; Jaime Costa e Silva; Reginaldo P. Brazil

1999-01-01

The present paper reports the experimental treatment of hamsters infected with Leishmania chagasi and Leishmania amazonensis with sodium stibogluconate (20mg/kg/day x 20 days). Only with L. chagasi did the treatment result in the complete elimination of parasites from the spleen. However, no parasitological cure was achieved in hamsters infected with L. amazonensis.O presente trabalho é um relato do tratamento experimental de hamsters infectado com Leishmania chagasi e Leishmania amazonensis ...

The Role of Sphingolipids on Innate Immunity to Intestinal Salmonella Infection.

Science.gov (United States)

Huang, Fu-Chen

2017-08-07

Salmonella spp. remains a major public health problem for the whole world. To reduce the use of antimicrobial agents and drug-resistant Salmonella , a better strategy is to explore alternative therapy rather than to discover another antibiotic. Sphingolipid- and cholesterol-enriched lipid microdomains attract signaling proteins and orchestrate them toward cell signaling and membrane trafficking pathways. Recent studies have highlighted the crucial role of sphingolipids in the innate immunity against infecting pathogens. It is therefore mandatory to exploit the role of the membrane sphingolipids in the innate immunity of intestinal epithelia infected by this pathogen. In the present review, we focus on the role of sphingolipids in the innate immunity of intestinal epithelia against Salmonella infection, including adhesion, autophagy, bactericidal effect, barrier function, membrane trafficking, cytokine and antimicrobial peptide expression. The intervention of sphingolipid-enhanced foods to make our life healthy or pharmacological agents regulating sphingolipids is provided at the end.

The role of sphingolipids in neuronal plasticity of the brain.

Science.gov (United States)

Sonnino, Sandro; Prinetti, Alessandro

2016-05-01

This Editorial highlights a study by Müller et al. in which the authors suggest a new sphingolipid-dependent mechanism for behavioral extinction. Their study should be considered in the broad perspective of sphingolipid metabolic pathways and traffic (depicted in the graphic). Read the highlighted article 'A sphingolipid mechanism for behavioral extinction' on page 589. © 2016 International Society for Neurochemistry.

Sphingolipid metabolism diseases.

Science.gov (United States)

Kolter, Thomas; Sandhoff, Konrad

2006-12-01

Human diseases caused by alterations in the metabolism of sphingolipids or glycosphingolipids are mainly disorders of the degradation of these compounds. The sphingolipidoses are a group of monogenic inherited diseases caused by defects in the system of lysosomal sphingolipid degradation, with subsequent accumulation of non-degradable storage material in one or more organs. Most sphingolipidoses are associated with high mortality. Both, the ratio of substrate influx into the lysosomes and the reduced degradative capacity can be addressed by therapeutic approaches. In addition to symptomatic treatments, the current strategies for restoration of the reduced substrate degradation within the lysosome are enzyme replacement therapy (ERT), cell-mediated therapy (CMT) including bone marrow transplantation (BMT) and cell-mediated "cross correction", gene therapy, and enzyme-enhancement therapy with chemical chaperones. The reduction of substrate influx into the lysosomes can be achieved by substrate reduction therapy. Patients suffering from the attenuated form (type 1) of Gaucher disease and from Fabry disease have been successfully treated with ERT.

The GARP complex is required for cellular sphingolipid homeostasis

DEFF Research Database (Denmark)

Fröhlich, Florian; Petit, Constance; Kory, Nora

2015-01-01

(GARP) complex, which functions in endosome-to-Golgi retrograde vesicular transport, as a critical player in sphingolipid homeostasis. GARP deficiency leads to accumulation of sphingolipid synthesis intermediates, changes in sterol distribution, and lysosomal dysfunction. A GARP complex mutation...... analogous to a VPS53 allele causing progressive cerebello-cerebral atrophy type 2 (PCCA2) in humans exhibits similar, albeit weaker, phenotypes in yeast, providing mechanistic insights into disease pathogenesis. Inhibition of the first step of de novo sphingolipid synthesis is sufficient to mitigate many...

Polarized sphingolipid transport from the subapical compartment : Evidence for distinct sphingolipid domains

NARCIS (Netherlands)

van Ijzendoorn, SCD; Hoekstra, D

1999-01-01

In polarized HepG2 cells, the sphingolipids glucosylceramide and sphingomyelin (SM), transported along the reverse transcytotic pathway, are sorted in subapical compartments (SACs), and subsequently targeted to either apical or basolateral plasma membrane domains, respectively. In the present study,

Identification of Two Suppressors of CSG2 Calcium Sensitivity, SCS7 and SUR2, as Genes Encoding Hydroxylases of the Sphingolipid Biosynthetic Pathway of Saccharomyces cerevisiae

National Research Council Canada - National Science Library

Haak, Dale A

1997-01-01

.... The biochemical significance of much of this structural variability is not well understood. The sphingolipids of the yeast Saccharomyces cerevisiae undergo the a-hydroxylation of the very long chain fatty acid (VLCFA...

Altered Leukocyte Sphingolipid Pathway in Breast Cancer

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Larissa P. Maia

2017-11-01

Full Text Available Sphingolipid metabolism pathway is essential in membrane homeostasis, and its dysfunction has been associated with favorable tumor microenvironment, disease progression, and chemotherapy resistance. Its major components have key functions on survival and proliferation, with opposing effects. We have profiled the components of the sphingolipid pathway on leukocytes of breast cancer (BC patients undergoing chemotherapy treatment and without, including the five sphingosine 1-phosphate (S1P receptors, the major functional genes, and cytokines, in order to better understand the S1P signaling in the immune cells of these patients. To the best of our knowledge, this is the first characterization of the sphingolipid pathway in whole blood of BC patients. Skewed gene profiles favoring high SPHK1 expression toward S1P production during BC development was observed, which was reversed by chemotherapy treatment, and reached similar levels to those found in healthy donors. Such levels were also correlated with high levels of TNF-α. Our data revealed an important role of the sphingolipid pathway in immune cells in BC with skewed signaling of S1P receptors, which favored cancer development even under chemotherapy, and may probably be a trigger of cancer resistance. Thus, these molecules must be considered as a target pathway for combined BC therapeutics.

The activity of azithromycin against Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis in the golden hamster model

OpenAIRE

Sinagra,Ángel; Luna,Concepción; Abraham,David; Iannella,Maria del Carmen; Riarte,Adelina; Krolewiecki,Alejandro J.

2007-01-01

New therapeutic alternatives against leishmaniasis remain a priority. The activity of azithromycin against Leishmania (Leishmania) major has been previously demonstrated. Different responses among species of Leishmania make species-specific drug screening necessary. The activity of azithromycin against Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis was evaluated in golden hamsters infected through footpad injections of metacyclic promastigotes, and compared with unt...

Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It.

Science.gov (United States)

Kraft, Mary L

2016-01-01

Sphingolipids are structural components in the plasma membranes of eukaryotic cells. Their metabolism produces bioactive signaling molecules that modulate fundamental cellular processes. The segregation of sphingolipids into distinct membrane domains is likely essential for cellular function. This review presents the early studies of sphingolipid distribution in the plasma membranes of mammalian cells that shaped the most popular current model of plasma membrane organization. The results of traditional imaging studies of sphingolipid distribution in stimulated and resting cells are described. These data are compared with recent results obtained with advanced imaging techniques, including super-resolution fluorescence detection and high-resolution secondary ion mass spectrometry (SIMS). Emphasis is placed on the new insight into the sphingolipid organization within the plasma membrane that has resulted from the direct imaging of stable isotope-labeled lipids in actual cell membranes with high-resolution SIMS. Super-resolution fluorescence techniques have recently revealed the biophysical behaviors of sphingolipids and the unhindered diffusion of cholesterol analogs in the membranes of living cells are ultimately in contrast to the prevailing hypothetical model of plasma membrane organization. High-resolution SIMS studies also conflicted with the prevailing hypothesis, showing sphingolipids are concentrated in micrometer-scale membrane domains, but cholesterol is evenly distributed within the plasma membrane. Reductions in cellular cholesterol decreased the number of sphingolipid domains in the plasma membrane, whereas disruption of the cytoskeleton eliminated them. In addition, hemagglutinin, a transmembrane protein that is thought to be a putative raft marker, did not cluster within sphingolipid-enriched regions in the plasma membrane. Thus, sphingolipid distribution in the plasma membrane is dependent on the cytoskeleton, but not on favorable interactions with

Tumor PDT-associated immune response: relevance of sphingolipids

Science.gov (United States)

Korbelik, Mladen; Merchant, Soroush; Separovic, Duska M.

2010-02-01

Sphingolipids have become recognized as essential effector molecules in signal transduction with involvement in various aspects of cell function and death, immune response and cancer treatment response. Major representatives of sphingolipids family, ceramide, sphingosine and sphingosine-1-phosphate (S1P), have attracted interest in their relevance to tumor response to photodynamic therapy (PDT) because of their roles as enhancers of apoptosis, mediators of cell growth and vasculogenesis, and regulators of immune response. Our recent in vivo studies with mouse tumor models have confirmed that PDT treatment has a pronounced impact on sphingolipid profile in the targeted tumor and that significant advances in therapeutic gain with PDT can be attained by combining this modality with adjuvant treatment with ceramide analog LCL29.

Sphingolipid trafficking - Sorted out?

NARCIS (Netherlands)

van Meer, G.; Burger, K.N.J.

1992-01-01

Studies of intracellular membrane traffic have traditionally focused on the protein components of membranes, but what about lipids? Recent findings have drawn attention to the transport of one type of lipid, the sphingolipids. Their unique physical properties may allow them to aggregate into

A atividade da azitromicina contra a Leishmania (Viannia) braziliensis e a Leishmania (Leishmania) amazonensis no modelo golden hamster

OpenAIRE

Sinagra, Ángel; Luna, Concepción; Abraham, David; Iannella, Maria del Carmen; Riarte, Adelina; Krolewiecki, Alejandro J.

2007-01-01

New therapeutic alternatives against leishmaniasis remain a priority. The activity of azithromycin against Leishmania (Leishmania) major has been previously demonstrated. Different responses among species of Leishmania make species-specific drug screening necessary. The activity of azithromycin against Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis was evaluated in golden hamsters infected through footpad injections of metacyclic promastigotes, and compared with unt...

Genetic determinants of circulating sphingolipid concentrations in European populations.

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Andrew A Hicks

2009-10-01

Full Text Available Sphingolipids have essential roles as structural components of cell membranes and in cell signalling, and disruption of their metabolism causes several diseases, with diverse neurological, psychiatric, and metabolic consequences. Increasingly, variants within a few of the genes that encode enzymes involved in sphingolipid metabolism are being associated with complex disease phenotypes. Direct experimental evidence supports a role of specific sphingolipid species in several common complex chronic disease processes including atherosclerotic plaque formation, myocardial infarction (MI, cardiomyopathy, pancreatic beta-cell failure, insulin resistance, and type 2 diabetes mellitus. Therefore, sphingolipids represent novel and important intermediate phenotypes for genetic analysis, yet little is known about the major genetic variants that influence their circulating levels in the general population. We performed a genome-wide association study (GWAS between 318,237 single-nucleotide polymorphisms (SNPs and levels of circulating sphingomyelin (SM, dihydrosphingomyelin (Dih-SM, ceramide (Cer, and glucosylceramide (GluCer single lipid species (33 traits; and 43 matched metabolite ratios measured in 4,400 subjects from five diverse European populations. Associated variants (32 in five genomic regions were identified with genome-wide significant corrected p-values ranging down to 9.08x10(-66. The strongest associations were observed in or near 7 genes functionally involved in ceramide biosynthesis and trafficking: SPTLC3, LASS4, SGPP1, ATP10D, and FADS1-3. Variants in 3 loci (ATP10D, FADS3, and SPTLC3 associate with MI in a series of three German MI studies. An additional 70 variants across 23 candidate genes involved in sphingolipid-metabolizing pathways also demonstrate association (p = 10(-4 or less. Circulating concentrations of several key components in sphingolipid metabolism are thus under strong genetic control, and variants in these loci can be

Synthesis of a Panel of Carbon-13-Labelled (Glyco)Sphingolipids

NARCIS (Netherlands)

Wisse, Patrick; Gold, Henrik; Mirzaian, Mina; Ferraz, Maria J.; Lutteke, Ginger; van den Berg, Richard J. B. H. N.; van den Elst, Hans; Lugtenburg, Johan; van der Marel, Gijsbert A.; Aerts, Johannes M. F. G.; Codee, Jeroen D. C.; Overkleeft, Herman S.

2015-01-01

The synthesis of a focussed library of sphingolipids differing in the number and position of C-13 labels is described. The synthesised sphingolipids differ in substitution at both the sphingosine amine (either palmitoylated or unmodified) and the sphingosine primary hydroxyl (unmodified or

Preanalytical Biases in the Measurement of Human Blood Sphingolipids

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Robert Brunkhorst

2018-05-01

Full Text Available Dysregulation of blood sphingolipids is an emerging topic in clinical science. The objective of this study was to determine preanalytical biases that typically occur in clinical and translational studies and that influence measured blood sphingolipid levels. Therefore, we collected blood samples from four healthy male volunteers to investigate the effect of storage conditions (time, temperature, long-term storage, freeze–thaw cycles, blood drawing (venous or arterial sampling, prolonged venous compression, and sample preparation (centrifugation, freezing on sphingolipid levels measured by LC-MS/MS. Our data show that sphingosine 1-phosphate (S1P and sphinganine 1-phosphate (SA1P were upregulated in whole blood samples in a time- and temperature-dependent manner. Increased centrifugation at higher speeds led to lower amounts of S1P and SA1P. All other preanalytical biases did not significantly alter the amounts of S1P and SA1P. Further, in almost all settings, we did not detect differences in (dihydroceramide levels. In summary, besides time-, temperature-, and centrifugation-dependent changes in S1P and SA1P levels, sphingolipids in blood remained stable under practically relevant preanalytical conditions.

SPHINGOLIPID-DEPENDENT FUSION OF SEMLIKI FOREST VIRUS WITH CHOLESTEROL-CONTAINING LIPOSOMES REQUIRES BOTH THE 3-HYDROXYL GROUP AND THE DOUBLE-BOND OF THE SPHINGOLIPID BACKBONE

NARCIS (Netherlands)

CORVER, J; MOESBY, L; ERUKULLA, RK; REDDY, KC; BITTMAN, R; WILSCHUT, J

Low-pH-induced membrane fusion of Semliki Forest virus (SFV) in a model system is mediated by sphingolipids in the target membrane; ceramide is the sphingolipid minimally required (J. L. Nieva, R. Bron, J. Corver, and J. Wilschut, EMBO J. 13:2797-2804, 1994). Here, using various ceramide analogs, we

The way we view cellular (glyco)sphingolipids.

Science.gov (United States)

Hoetzl, Sandra; Sprong, Hein; van Meer, Gerrit

2007-11-01

Mammalian cells synthesize ceramide in the endoplasmic reticulum (ER) and convert this to sphingomyelin and complex glycosphingolipids on the inner, non-cytosolic surface of Golgi cisternae. From there, these lipids travel towards the outer, non-cytosolic surface of the plasma membrane and all membranes of the endocytic system, where they are eventually degraded. At the basis of the selective, anterograde traffic out of the Golgi lies the propensity of the sphingolipids to self-aggregate with cholesterol into microdomains termed 'lipid rafts'. At the plasma membrane surface these rafts are thought to function as the scaffold for various types of (glyco) signaling domains of different protein and lipid composition that can co-exist on one and the same cell. In the past decade, various unexpected findings on the sites where sphingolipid-mediated events occur have thrown a new light on the localization and transport mechanisms of sphingolipids. These findings are largely based on biochemical experiments. Further progress in the field is hampered by a lack of morphological techniques to localize lipids with nanometer resolution. In the present paper, we critically evaluate the published data and discuss techniques and potential improvements.

Sphingolipid topology and the dynamic organization and function of membrane proteins.

Science.gov (United States)

van Meer, Gerrit; Hoetzl, Sandra

2010-05-03

When acquiring internal membranes and vesicular transport, eukaryotic cells started to synthesize sphingolipids and sterols. The physical differences between these and the glycerophospholipids must have enabled the cells to segregate lipids in the membrane plane. Localizing this event to the Golgi then allowed them to create membranes of different lipid composition, notably a thin, flexible ER membrane, consisting of glycerolipids, and a sturdy plasma membrane containing at least 50% sphingolipids and sterols. Besides sorting membrane proteins, in the course of evolution the simple sphingolipids obtained key positions in cellular physiology by developing specific interactions with (membrane) proteins involved in the execution and control of signaling. The few signaling sphingolipids in mammals must provide basic transmission principles that evolution has built upon for organizing the specific regulatory pathways tuned to the needs of the different cell types in the body. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Sphingolipids in Congenital Diaphragmatic Hernia; Results from an International Multicenter Study.

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Kitty G Snoek

Full Text Available Congenital diaphragmatic hernia is a severe congenital anomaly with significant mortality and morbidity, for instance chronic lung disease. Sphingolipids have shown to be involved in lung injury, but their role in the pathophysiology of chronic lung disease has not been explored. We hypothesized that sphingolipid profiles in tracheal aspirates could play a role in predicting the mortality/ development of chronic lung disease in congenital diaphragmatic hernia patients. Furthermore, we hypothesized that sphingolipid profiles differ between ventilation modes; conventional mechanical ventilation versus high-frequency oscillation.Sphingolipid levels in tracheal aspirates were determined at days 1, 3, 7 and 14 in 72 neonates with congenital diaphragmatic hernia, born after > 34 weeks gestation at four high-volume congenital diaphragmatic hernia centers. Data were collected within a multicenter trial of initial ventilation strategy (NTR 1310.36 patients (50.0% died or developed chronic lung disease, 34 patients (47.2% by stratification were initially ventilated by conventional mechanical ventilation and 38 patients (52.8% by high-frequency oscillation. Multivariable logistic regression analysis with correction for side of the defect, liver position and observed-to-expected lung-to-head ratio, showed that none of the changes in sphingolipid levels were significantly associated with mortality /development of chronic lung disease. At day 14, long-chain ceramides 18:1 and 24:0 were significantly elevated in patients initially ventilated by conventional mechanical ventilation compared to high-frequency oscillation.We could not detect significant differences in temporal sphingolipid levels in congenital diaphragmatic hernia infants with mortality/development of chronic lung disease versus survivors without development of CLD. Elevated levels of ceramides 18:1 and 24:0 in the conventional mechanical ventilation group when compared to high

Increased transmission potential of Leishmania major/Leishmania infantum hybrids

OpenAIRE

Volf, Petr; Benkova, Ivana; Myskova, Jitka; Sadlova, Jovana; Campino, Lenea; Ravel, Christophe

2007-01-01

Development of Leishmania infantum/Leishmania major hybrids was studied in two sand fly species. In Phlebotomus papatasi, which supported development of L. major but not L. infantum, the hybrids produced heavy late-stage infections with high numbers of metacyclic promastigotes. In the permissive vector Lutzomyia longipalpis, all Leishmania strains included in this study developed well. Hybrids were found to express L. major lipophosphoglycan, apparently enabling them to survive in P. papatasi...

Autophagy in the light of sphingolipid metabolism

DEFF Research Database (Denmark)

Harvald, Eva Bang; Olsen, Anne Sofie Braun; Færgeman, Nils J.

2015-01-01

Maintenance of cellular homeostasis requires tight and coordinated control of numerous metabolic pathways, which are governed by interconnected networks of signaling pathways and energy-sensing regulators. Autophagy, a lysosomal degradation pathway by which the cell self-digests its own components......, has over the past decade been recognized as an essential part of metabolism. Autophagy not only rids the cell of excessive or damaged organelles, misfolded proteins, and invading microorganisms, it also provides nutrients to maintain crucial cellular functions. Besides serving as essential structural...... moieties of biomembranes, lipids including sphingolipids are increasingly being recognized as central regulators of a number of important cellular processes, including autophagy. In the present review we describe how sphingolipids, with special emphasis on ceramides and sphingosine-1-phosphate, can act...

Novel selective inhibitor of Leishmania (Leishmania) amazonensis arginase.

Science.gov (United States)

da Silva, Edson R; Boechat, Nubia; Pinheiro, Luiz C S; Bastos, Monica M; Costa, Carolina C P; Bartholomeu, Juliana C; da Costa, Talita H

2015-11-01

Arginase is a glycosomal enzyme in Leishmania that is involved in polyamine and trypanothione biosynthesis. The central role of arginase in Leishmania (Leishmania) amazonensis was demonstrated by the generation of two mutants: one with an arginase lacking the glycosomal addressing signal and one in which the arginase-coding gene was knocked out. Both of these mutants exhibited decreased infectivity. Thus, arginase seems to be a potential drug target for Leishmania treatment. In an attempt to search for arginase inhibitors, 29 derivatives of the [1,2,4]triazolo[1,5-a]pyrimidine system were tested against Leishmania (Leishmania) amazonensis arginase in vitro. The [1,2,4]triazolo[1,5-a]pyrimidine scaffold containing R1  = CF3 exhibited greater activity against the arginase rather than when the substituent R1  = CH3 in the 2-position. The novel compound 2-(5-methyl-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)hydrazinecarbothioamide (30) was the most potent, inhibiting arginase by a non-competitive mechanism, with the Ki and IC50 values for arginase inhibition estimated to be 17 ± 1 μm and 16.5 ± 0.5 μm, respectively. These results can guide the development of new drugs against leishmaniasis based on [1,2,4]triazolo[1,5-a]pyrimidine derivatives targeting the arginase enzyme. © 2015 John Wiley & Sons A/S.

Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

Directory of Open Access Journals (Sweden)

Yuichi Hirata

Full Text Available Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2 encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

Science.gov (United States)

Hirata, Yuichi; Ikeda, Kazutaka; Sudoh, Masayuki; Tokunaga, Yuko; Suzuki, Akemi; Weng, Leiyun; Ohta, Masatoshi; Tobita, Yoshimi; Okano, Ken; Ozeki, Kazuhisa; Kawasaki, Kenichi; Tsukuda, Takuo; Katsume, Asao; Aoki, Yuko; Umehara, Takuya; Sekiguchi, Satoshi; Toyoda, Tetsuya; Shimotohno, Kunitada; Soga, Tomoyoshi; Nishijima, Masahiro; Taguchi, Ryo; Kohara, Michinori

2012-01-01

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

Prevalence and Distribution of Leishmania RNA Virus 1 in Leishmania Parasites from French Guiana.

Science.gov (United States)

Ginouvès, Marine; Simon, Stéphane; Bourreau, Eliane; Lacoste, Vincent; Ronet, Catherine; Couppié, Pierre; Nacher, Mathieu; Demar, Magalie; Prévot, Ghislaine

2016-01-01

In South America, the presence of the Leishmania RNA virus type 1 (LRV1) was described in Leishmania guyanensis and Leishmania braziliensis strains. The aim of this study was to determine the prevalence distribution of LRV1 in Leishmania isolates in French Guiana given that, in this French overseas department, most Leishmania infections are due to these parasite species. The presence of the virus was observed in 74% of Leishmania spp. isolates, with a highest presence in the internal areas of the country. © The American Society of Tropical Medicine and Hygiene.

Low serum sphingolipids in children with attention deficit-hyperactivity disorder

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Marcela Patricia Henríquez-Henríquez

2015-08-01

Full Text Available Background: Attention deficit-hyperactivity disorder (ADHD is the most prevalent neuropsychiatric condition in childhood. ADHD is a multifactorial trait with a strong genetic component. One neurodevelopmental hypothesis is that ADHD is associated with a lag in brain maturation. Sphingolipids are essential for brain development and neuronal functioning, but their role in ADHD pathogenesis is unexplored. We hypothesized that serum sphingolipid levels distinguish ADHD patients from unaffected subjects. Methods: We characterized serum sphingolipid profiles of ADHD patients and two control groups: non-affected relatives and non-affected subjects without a family history of ADHD. Sphingolipids were measured by LC-MS/MS in 77 participants (28 ADHD patients, 28 related controls and 21 unrelated controls. ADHD diagnosis was based on the Diagnostic and Statistical Manual of Mental Disorders (DSM IV-TR. Diagnostic criteria were assessed by 2 independent observers. Groups were compared by parametrical statistics. Results: Serum sphingomyelins C16:0, C18:0, C18:1, C24:1, ceramide C24:0 and deoxy-ceramide C24:1 were significantly decreased in ADHD patients at 20-30% relative reductions. In our sample, decreased serum sphingomyelin levels distinguished ADHD patients with 79% sensitivity and 78% specificity. Conclusions: Our results showed lower levels of all major serum sphingomyelins in ADHD. These findings may reflect brain maturation and affect neuro-functional pathways characteristic for ADHD.

Establishment of HeLa cell mutants deficient in sphingolipid-related genes using TALENs.

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Toshiyuki Yamaji

Full Text Available Sphingolipids are essential components in eukaryotes and have various cellular functions. Recent developments in genome-editing technologies have facilitated gene disruption in various organisms and cell lines. We here show the disruption of various sphingolipid metabolic genes in human cervical carcinoma HeLa cells by using transcription activator-like effector nucleases (TALENs. A TALEN pair targeting the human CERT gene (alternative name COL4A3BP encoding a ceramide transport protein induced a loss-of-function phenotype in more than 60% of HeLa cells even though the cell line has a pseudo-triploid karyotype. We have isolated several loss-of-function mutant clones for CERT, UGCG (encoding glucosylceramide synthase, and B4GalT5 (encoding the major lactosylceramide synthase, and also a CERT/UGCG double-deficient clone. Characterization of these clones supported previous proposals that CERT primarily contributes to the synthesis of SM but not GlcCer, and that B4GalT5 is the major LacCer synthase. These newly established sphingolipid-deficient HeLa cell mutants together with our previously established stable transfectants provide a 'sphingolipid-modified HeLa cell panel,' which will be useful to elucidate the functions of various sphingolipid species against essentially the same genomic background.

Detection and molecular identification of leishmania RNA virus (LRV) in Iranian Leishmania species.

Science.gov (United States)

Hajjaran, Homa; Mahdi, Maryam; Mohebali, Mehdi; Samimi-Rad, Katayoun; Ataei-Pirkooh, Angila; Kazemi-Rad, Elham; Naddaf, Saied Reza; Raoofian, Reza

2016-12-01

Leishmania RNA virus (LRV) was first detected in members of the subgenus Leishmania (Viannia), and later, the virulence and metastasis of the New World species were attributed to this virus. The data on the presence of LRV in Old World species are confined to Leishmania major and a few Leishmania aethiopica isolates. The aim of this study was to survey the presence of LRV in various Iranian Leishmania species originating from patients and animal reservoir hosts. Genomic nucleic acids were extracted from 50 cultured isolates belonging to the species Leishmania major, Leishmania tropica, and Leishmania infantum. A partial sequence of the viral RNA-dependent RNA polymerase (RdRp) gene was amplified, sequenced and compared with appropriate sequences from the GenBank database. We detected the virus in two parasite specimens: an isolate of L. infantum derived from a visceral leishmaniasis (VL) patient who was unresponsive to meglumine antimoniate treatment, and an L. major isolate originating from a great gerbil, Rhombomys opimus. The Iranian LRV sequences showed the highest similarities to an Old World L. major LRV2 and were genetically distant from LRV1 isolates detected in New World Leishmania parasites. We could not attribute treatment failure in VL patient to the presence of LRV due to the limited number of specimens analyzed. Further studies with inclusion of more clinical samples are required to elucidate the potential role of LRVs in pathogenesis or treatment failure of Old World leishmaniasis.

High density of Leishmania major and rarity of other mammals' Leishmania in zoonotic cutaneous leishmaniasis foci, Iran.

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Bordbar, Ali; Parvizi, Parviz

2014-03-01

Only Leishmania major is well known as a causative agent of zoonotic cutaneous leishmaniasis (ZCL) in Iran. Our objective was to find Leishmania parasites circulating in reservoir hosts, sand flies and human simultaneously. Sand flies, rodents and prepared smears of humans were sampled. DNA of Leishmania parasites was extracted, and two fragments of ITS-rDNA gene amplified by PCR. RFLP and sequencing were employed to identify Leishmania parasites. Leishmania major and L. turanica were identified unequivocally by targeting and sequencing ITS-rDNA from humans, rodents and sand flies. The new Leishmania species close to gerbilli (GenBank Accession Nos. EF413076; EF413087) was discovered only in sand flies. Based on parasite detection of ITS-rDNA in main and potential reservoir hosts and vectors and humans, we conclude that at least two Leishmania species are common in the Turkmen Sahra ZCL focus. Phylogenetic analysis proved that the new Leishmania is closely related to Leishmania mammal parasites (Leishmania major, Leishmania turanica, Leishmania gerbilli). Its role as a principal agent of ZCL is unknown because it was found only in sand flies. Our findings shed new light on the transmission cycles of several Leishmania parasites in sand flies, reservoir hosts and humans. © 2014 John Wiley & Sons Ltd.

Membrane domains and polarized trafficking of sphingolipids

NARCIS (Netherlands)

Maier, O; Slimane, TA; Hoekstra, D

The plasma membrane of polarized cells consists of distinct domains, the apical and basolateral membrane that are characterized by a distinct lipid and protein content. Apical protein transport is largely mediated by (glyco)sphingolipid-cholesterol enriched membrane microdomains, so called rafts. In

Sphingolipids in human synovial fluid--a lipidomic study.

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Marta Krystyna Kosinska

Full Text Available Articular synovial fluid (SF is a complex mixture of components that regulate nutrition, communication, shock absorption, and lubrication. Alterations in its composition can be pathogenic. This lipidomic investigation aims to quantify the composition of sphingolipids (sphingomyelins, ceramides, and hexosyl- and dihexosylceramides and minor glycerophospholipid species, including (lysophosphatidic acid, (lysophosphatidylglycerol, and bis(monoacylglycerophosphate species, in the SF of knee joints from unaffected controls and from patients with early (eOA and late (lOA stages of osteoarthritis (OA, and rheumatoid arthritis (RA. SF without cells and cellular debris from 9 postmortem donors (control, 18 RA, 17 eOA, and 13 lOA patients were extracted to measure lipid species using electrospray ionization tandem mass spectrometry--directly or coupled with hydrophilic interaction liquid chromatography. We provide a novel, detailed overview of sphingolipid and minor glycerophospholipid species in human SF. A total of 41, 48, and 50 lipid species were significantly increased in eOA, lOA, and RA SF, respectively when compared with normal SF. The level of 21 lipid species differed in eOA SF versus SF from lOA, an observation that can be used to develop biomarkers. Sphingolipids can alter synovial inflammation and the repair responses of damaged joints. Thus, our lipidomic study provides the foundation for studying the biosynthesis and function of lipid species in health and most prevalent joint diseases.

Inhibition of Sphingolipid Metabolism Enhances Resveratrol Chemotherapy in Human Gastric Cancer Cells

OpenAIRE

Shin, Kyong-Oh; Park, Nam-Young; Seo, Cho-Hee; Hong, Seon-Pyo; Oh, Ki-Wan; Hong, Jin-Tae; Han, Sang-Kil; Lee, Yong-Moon

2012-01-01

Resveratrol, a chemopreventive agent, is rapidly metabolized in the intestine and liver via glucuronidation. Thus, the pharmacokinetics of resveratrol limits its efficacy. To improve efficacy, the activity of resveratrol was investigated in the context of sphingolipid metabolism in human gastric cancer cells. Diverse sphingolipid metabolites, including dihydroceramides (DHCer), were tested for their ability to induce resveratrol cytotoxicity. Exposure to resveratrol (100 ?M) for 24 hr induced...

Temporal changes in sphingolipids and systemic insulin sensitivity during the transition from gestation to lactation.

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J Eduardo Rico

Full Text Available Reduced insulin action develops naturally during the peripartum to ensure maternal nutrient delivery to the fetus and neonate. However, increased insulin resistance can facilitate excessive lipolysis which in turn promotes metabolic disease in overweight dairy cattle. Increased fatty acid availability favors the accumulation of the sphingolipid ceramide and is implicated in the pathogenesis of insulin resistance, however, the relationship between sphingolipid metabolism and insulin resistance during the peripartum remains largely unknown. Our objectives were to characterize temporal responses in plasma and tissue sphingolipids in lean and overweight peripartal cows and to establish the relationships between sphingolipid supply and lipolysis, hepatic lipid deposition, and systemic insulin action. Twenty-one multiparous lean and overweight Holstein cows were enrolled in a longitudinal study spanning the transition from gestation to lactation (d -21 to 21, relative to parturition. Plasma, liver, and skeletal muscle samples were obtained, and sphingolipids were profiled using LC/MS/MS. Insulin sensitivity was assessed utilizing intravenous insulin and glucose challenges. Our results demonstrated the following: first, insulin resistance develops postpartum concurrently with increased lipolysis and hepatic lipid accumulation; second, ceramides and glycosylated ceramides accumulate during the transition from gestation to lactation and are further elevated in overweight cows; third, ceramide accrual is associated with lipolysis and liver lipid accumulation, and C16:0- and C24:0-ceramide are inversely associated with systemic insulin sensitivity postpartum; fourth, plasma sphingomyelin, a potential source of ceramides reaches a nadir at parturition and is closely associated with feed intake; fifth, select sphingomyelins are lower in the plasma of overweight cows during the peripartal period. Our results demonstrate that dynamic changes occur in

Temporal changes in sphingolipids and systemic insulin sensitivity during the transition from gestation to lactation

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Rico, J. Eduardo; Saed Samii, Sina; Mathews, Alice T.; Lovett, Jacqueline; Haughey, Norman J.; McFadden, Joseph W.

2017-01-01

Reduced insulin action develops naturally during the peripartum to ensure maternal nutrient delivery to the fetus and neonate. However, increased insulin resistance can facilitate excessive lipolysis which in turn promotes metabolic disease in overweight dairy cattle. Increased fatty acid availability favors the accumulation of the sphingolipid ceramide and is implicated in the pathogenesis of insulin resistance, however, the relationship between sphingolipid metabolism and insulin resistance during the peripartum remains largely unknown. Our objectives were to characterize temporal responses in plasma and tissue sphingolipids in lean and overweight peripartal cows and to establish the relationships between sphingolipid supply and lipolysis, hepatic lipid deposition, and systemic insulin action. Twenty-one multiparous lean and overweight Holstein cows were enrolled in a longitudinal study spanning the transition from gestation to lactation (d -21 to 21, relative to parturition). Plasma, liver, and skeletal muscle samples were obtained, and sphingolipids were profiled using LC/MS/MS. Insulin sensitivity was assessed utilizing intravenous insulin and glucose challenges. Our results demonstrated the following: first, insulin resistance develops postpartum concurrently with increased lipolysis and hepatic lipid accumulation; second, ceramides and glycosylated ceramides accumulate during the transition from gestation to lactation and are further elevated in overweight cows; third, ceramide accrual is associated with lipolysis and liver lipid accumulation, and C16:0- and C24:0-ceramide are inversely associated with systemic insulin sensitivity postpartum; fourth, plasma sphingomyelin, a potential source of ceramides reaches a nadir at parturition and is closely associated with feed intake; fifth, select sphingomyelins are lower in the plasma of overweight cows during the peripartal period. Our results demonstrate that dynamic changes occur in peripartal sphingolipids

Effects of nitro-heterocyclic derivatives against Leishmania (Leishmania) infantum promastigotes and intracellular amastigotes.

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Petri e Silva, Simone Carolina Soares; Palace-Berl, Fanny; Tavares, Leoberto Costa; Soares, Sandra Regina Castro; Lindoso, José Angelo Lauletta

2016-04-01

Leishmaniasis is an overlooked tropical disease affecting approximately 1 million people in several countries. Clinical manifestation depends on the interaction between Leishmania and the host's immune response. Currently available treatment options for leishmaniasis are limited and induce severe side effects. In this research, we tested nitro-heterocyclic compounds (BSF series) as a new alternative against Leishmania. Its activity was measured in Leishmania (Leishmania) infantum promastigotes and intracellular amastigotes using MTT colorimetric assay. Additionally, we assessed the phosphatidylserine exposure by promastigotes, measured by flow cytometry, as well as nitric oxide production, measured by Griess' method. The nitro-heterocyclic compounds (BSF series) showed activity against L. (L.) infantum promastigotes, inducting the phosphatidylserine exposition by promastigotes, decreasing intracellular amastigotes and increasing oxide nitric production. The selectivity index was more prominent to Leishmania than to macrophages. Compared to amphotericin b, our compounds presented higher IC50, however the selectivity index was more specific to parasite than to amphotericin b. In conclusion, these nitro-heterocyclic compounds showed to be promising as an anti-Leishmania drug, in in vitro studies. Copyright © 2016 Elsevier Inc. All rights reserved.

Fiber Specific Changes in Sphingolipid Metabolism in Skeletal Muscles of Hyperthyroid Rats

OpenAIRE

Chabowski, A.; ?endzian-Piotrowska, M.; Mik?osz, A.; ?ukaszuk, B.; Kurek, K.; G?rski, J.

2013-01-01

Thyroid hormones (T3, T4) are well known modulators of different cellular signals including the sphingomyelin pathway. However, studies regarding downstream effects of T3 on sphingolipid metabolism in skeletal muscle are scarce. In the present work we sought to investigate the effects of hyperthyroidism on the activity of the key enzymes of ceramide metabolism as well as the content of fundamental sphingolipids. Based on fiber/metabolic differences, we chose three different skeletal muscles, ...

Diphtheria toxin translocation across cellular membranes is regulated by sphingolipids

International Nuclear Information System (INIS)

Spilsberg, Bjorn; Hanada, Kentaro; Sandvig, Kirsten

2005-01-01

Diphtheria toxin is translocated across cellular membranes when receptor-bound toxin is exposed to low pH. To study the role of sphingolipids for toxin translocation, both a mutant cell line lacking the first enzyme in de novo sphingolipid synthesis, serine palmitoyltransferase, and a specific inhibitor of the same enzyme, myriocin, were used. The serine palmitoyltransferase-deficient cell line (LY-B) was found to be 10-15 times more sensitive to diphtheria toxin than the genetically complemented cell line (LY-B/cLCB1) and the wild-type cell line (CHO-K1), both when toxin translocation directly across the plasma membrane was induced by exposing cells with surface-bound toxin to low pH, and when the toxin followed its normal route via acidified endosomes into the cytosol. Toxin binding was similar in these three cell lines. Furthermore, inhibition of serine palmitoyltransferase activity by addition of myriocin sensitized the two control cell lines (LY-B/cLCB1 and CHO-K1) to diphtheria toxin, whereas, as expected, no effect was observed in cells lacking serine palmitoyltransferase (LY-B). In conclusion, diphtheria toxin translocation is facilitated by depletion of membrane sphingolipids

Detection and characterization of Leishmania (Leishmania and Leishmania (Viannia by SYBR green-based real-time PCR and high resolution melt analysis targeting kinetoplast minicircle DNA.

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Marcello Ceccarelli

Full Text Available Leishmaniasis is a neglected disease with a broad clinical spectrum which includes asymptomatic infection. A thorough diagnosis, able to distinguish and quantify Leishmania parasites in a clinical sample, constitutes a key step in choosing an appropriate therapy, making an accurate prognosis and performing epidemiological studies. Several molecular techniques have been shown to be effective in the diagnosis of leishmaniasis. In particular, a number of PCR methods have been developed on various target DNA sequences including kinetoplast minicircle constant regions. The first aim of this study was to develop a SYBR green-based qPCR assay for Leishmania (Leishmania infantum detection and quantification, using kinetoplast minicircle constant region as target. To this end, two assays were compared: the first used previously published primer pairs (qPCR1, whereas the second used a nested primer pairs generating a shorter PCR product (qPCR2. The second aim of this study was to evaluate the possibility to discriminate among subgenera Leishmania (Leishmania and Leishmania (Viannia using the qPCR2 assay followed by melting or High Resolution Melt (HRM analysis. Both assays used in this study showed good sensitivity and specificity, and a good correlation with standard IFAT methods in 62 canine clinical samples. However, the qPCR2 assay allowed to discriminate between Leishmania (Leishmania and Leishmania (Viannia subgenera through melting or HRM analysis. In addition to developing assays, we investigated the number and genetic variability of kinetoplast minicircles in the Leishmania (L. infantum WHO international reference strain (MHOM/TN/80/IPT1, highlighting the presence of minicircle subclasses and sequence heterogeneity. Specifically, the kinetoplast minicircle number per cell was estimated to be 26,566±1,192, while the subclass of minicircles amplifiable by qPCR2 was estimated to be 1,263±115. This heterogeneity, also observed in canine clinical

Sphingolipid trafficking and protein sorting in epithelial cells

NARCIS (Netherlands)

Slimane, TA; Hoekstra, D

2002-01-01

Sphingolipids represent a minor, but highly dynamic subclass of lipids in all eukaryotic cells. They are involved in functions that range from structural protection to signal transduction and protein sorting, and participate in lipid raft assembly. In polarized epithelial cells, which display an

Sphingolipids as a new factor in the pathomechanism of preeclampsia - Mass spectrometry analysis.

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Karol Charkiewicz

Full Text Available The aim of the study was to analyse a panel of 11 sphingolipids in plasma and three blood fractions (platelet-poor plasma, platelets and red blood cells of women with mild preeclampsia.We recruited 21 women between 25-40 weeks gestation with diagnosed mild preeclampsia to the study group and 36 healthy women with uncomplicated pregnancies, who corresponded with the study group according to gestational age, to the control group. To assess the concentration of 11 sphingolipids in the blood plasma and blood fractions, we used ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC/MS/MS.We showed a significant increase in the concentration of eight sphingolipids in the plasma of women with preeclampsia in comparison to the control group: Sph (p = 0.0032, S1P (p = 0.0289, C20-Cer (p < 0.0001, C18-Cer (p < 0.0001, C16-Cer (p = 0.012, C18:1-Cer (p = 0.003, C22-Cer (p = 0.0071, and C24:1-Cer (p = 0.0085.We showed that selected sphingolipids, especially C20-Cer and C18-Cer, are totally new factors in the pathomechanism of PE and that these bioactive lipids may play an important role in apoptosis and autophagy.

Inhibition of growth of Leishmania mexicana mexicana by Leishmania mexicana amazonensis during "in vitro" co-cultivation Inibição do crescimento de Leishmania mexicana mexicana por Leishmania mexicana amazonensis durante o co-cultivo "in vitro"

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Raquel S. Pacheco

1987-12-01

Full Text Available Inhibition of one Leishmania subspecies by exometabolites of another subspecies, a phenomenon not previously reported, is suggested by our recent observations in cell cloning experiments with Leishmania mexicana mexicana and Leishmania mexicana amazonensis. Clones were identified using the technique of schizodeme analysis. The phenomenon observed is clearly relevant to studies of parasite isolation, leishmanial metabolism, cross-immunity and chemotherapy.Inhibição do crescimento de um subespécie de Leishmania por exometabólitos de outra subespécie, um fenômeno ainda não notificado, é sugerido em nossas recentes observações em experimentos de clonagem celular com Leishmania mexicana mexicana e Leishmania mexicana amazonensis. Os clones foram identificados usando a técnica de análise de esquizodemas. O fenômeno observado é claramente relevante em estudos de isolamento parasitário, metabolismo, imunidade cruzada e quimioterapia.

Nitric oxide-sphingolipid interplays in plant signalling: a new enigma from the Sphinx?

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Isabelle eGuillas

2013-09-01

Full Text Available Nitric oxide (NO emerged as one of the major signalling molecules operating during plant development and plant responses to its environment. Beyond the identification of the direct molecular targets of NO, a series of studies considered its interplay with other actors of signal transduction and the integration of nitric oxide into complex signalling networks. Beside the close relationships between NO and calcium or phosphatidic acid signalling pathways that are now well-established, recent reports paved the way for interplays between NO and sphingolipids. This mini-review summarises our current knowledge of the influence NO and sphingolipids might exert on each other in plant physiology. Based on comparisons with examples from the animal field, it further indicates that, although sphingolipid-NO interplays are common features in signalling networks of eukaryotic cells, the underlying mechanisms and molecular targets significantly differ.

Leishmania (Leishmania infantum chagasi em canídeos silvestres mantidos em cativeiro, no Estado de Mato Grosso Leishmania (Leishmania infantum chagasi in wild canids kept in captivity in the State of Mato Grosso

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Nely Pinheiro Souza

2010-06-01

Full Text Available INTRODUÇÃO: Leishmaniose visceral é uma zoonose que acomete diversos mamíferos tendo os canídeos domésticos como principais reservatórios em ambiente urbano. A presente nota descreve a infecção de canídeos silvestres por Leishmania (Leishmania infantum chagasi mantidos em cativeiro no Estado de Mato Grosso, Brasil. MÉTODOS: De seis raposas (Cerdocyon thous e um cachorro vinagre (Spheotos venaticus, foram coletadas amostras de pele, medula óssea e linfonodo para detecção e caracterização de Leishmania sp pela técnica de PCR-RFLP. RESULTADOS: Todos as animais pesquisados apresentaram-se positivos para Leishmania (L. infantum chagasi. CONCLUSÕES: Destaca-se a importância de monitoramento adequado dos mesmos, além do maior controle desta enfermidade já que estes animais estão em ambientes de recreação pública.INTRODUCTION: Visceral leishmaniasis is a zoonosis that affects many mammals, and domestic canids are the main reservoirs in urban environments. This note describes infection by Leishmania (Leishmania infantum chagasi among wild canids kept in captivity in the State of Mato Grosso, Brazil. METHODS: Skin, bone marrow and lymph node samples were collected from six crab-eating foxes (Cerdocyon thous and one bush dog (Spheotos venaticus, in order to detect and characterize Leishmania using the PCR-RFLP technique. RESULTS: All the animals studied were positive for Leishmania (L. infantum chagasi. CONCLUSIONS: This study highlights the importance of adequate monitoring of these animals, as well as greater control of this disease, given that these animals are in a public recreation environment.

Newly identified essential amino acid residues affecting ^8-sphingolipid desaturase activity revealed by site-directed mutagenesis

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In order to identify amino acid residues crucial for the enzymatic activity of ^8-sphingolipid desaturases, a sequence comparison was performed among ^8-sphingolipid desaturases and ^6-fatty acid desaturase from various plants. In addition to the known conserved cytb5 (cytochrome b5) HPGG motif and...

Plasma and ovarian tissue sphingolipids profiling in patients with advanced ovarian cancer.

Science.gov (United States)

Knapp, Paweł; Bodnar, Lubomir; Błachnio-Zabielska, Agnieszka; Świderska, Magdalena; Chabowski, Adrian

2017-10-01

The role of lipids in carcinogenesis through induction of abnormal cell lines in the human body is currently undisputable. Based on the literature, bioactive sphingolipids play an essential role in the development and progression of cancer and are involved in the metastatic process. The aim of this study was to determine the concentration of selected sphingolipids in patients with advanced ovarian cancer (AOC, FIGO III/IV, high grade ovarian cancer). Seventy-four patients with ovarian cancer were enrolled. Plasma concentrations of C16-Cer, C18:1-Cer and C18-Cer were assessed by LC/MS/MS. The content of tissue sphingolipids was measured using a UHPLC/MS/MS. Plasma concentration of 3 ceramides: C16-Cer, C18:1-Cer and C18-Cer was significantly elevated in women with advanced ovarian cancer compared to control group (P=0.031; 0.022; 0.020; respectively). There were increases in concentration of 5 ceramides: C16-Cer, C18:1-Cer, C18-Cer, C24:1-Cer, C24-Cer (P=0.025; 0.049; 0.032; 0.005; 0.013, respectively) and S1P (P=0.004) in ovarian tissue of women with advanced ovarian cancer compared to healthy individuals. Importantly, significantly higher risk of ovarian cancer when the plasma concentration of C16-Cer>311.88ng/100μl (AUC: 0.76, P=0.0261); C18:1-Cer>4.75ng/100μl (AUC: 0.77, P=0.0160) and C18-Cer>100.76ng/100μl (AUC:0.77, P=0.0136) was noticed. Bioactive sphingolipids play an essential role in the development and progression of cancer and they also take part in the process of metastasizing. This study suggests that some sphingolipids can be used as potential biomarkers of advanced ovarian cancer and that they can play an important role in the pathogenesis of this disease. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Fiber specific changes in sphingolipid metabolism in skeletal muscles of hyperthyroid rats.

Science.gov (United States)

Chabowski, A; Zendzian-Piotrowska, M; Mikłosz, A; Łukaszuk, B; Kurek, K; Górski, J

2013-07-01

Thyroid hormones (T3, T4) are well known modulators of different cellular signals including the sphingomyelin pathway. However, studies regarding downstream effects of T3 on sphingolipid metabolism in skeletal muscle are scarce. In the present work we sought to investigate the effects of hyperthyroidism on the activity of the key enzymes of ceramide metabolism as well as the content of fundamental sphingolipids. Based on fiber/metabolic differences, we chose three different skeletal muscles, with diverse fiber compositions: soleus (slow-twitch oxidative), red (fast-twitch oxidative-glycolytic) and white (fast-twitch glycolytic) section of gastrocnemius. We demonstrated that T3 induced accumulation of sphinganine, ceramide, sphingosine, as well as sphingomyelin, mostly in soleus and in red, but not white section of gastrocnemius. Concomitantly, the activity of serine palmitoyltransferase and acid/neutral ceramidase was increased in more oxidative muscles. In conclusion, hyperthyroidism induced fiber specific changes in the content of sphingolipids that were relatively more related to de novo synthesis of ceramide rather than to its generation via hydrolysis of sphingomyelin.

First evidence of Leishmania infection in European brown hare (Lepus europaeus) in Greece: GIS analysis and phylogenetic position within the Leishmania spp.

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Tsokana, C N; Sokos, C; Giannakopoulos, A; Mamuris, Z; Birtsas, P; Papaspyropoulos, K; Valiakos, G; Spyrou, V; Lefkaditis, M; Chatzopoulos, D C; Kantere, M; Manolakou, K; Touloudi, A; Burriel, A Rodi; Ferroglio, E; Hadjichristodoulou, C; Billinis, C

2016-01-01

Although the existence of a sylvatic transmission cycle of Leishmania spp., independent from the domestic cycle, has been proposed, data are scarce on Leishmania infection in wild mammals in Greece. In this study, we aimed to investigate the presence of Leishmania infection in the European brown hare in Greece, to infer the phylogenetic position of the Leishmania parasites detected in hares in Greece, and to identify any possible correlation between Leishmania infection in hares with environmental parameters, using the geographical information system (GIS). Spleen samples from 166 hares were tested by internal transcribed spacer-1 (ITS-1)-nested PCR for the detection of Leishmania DNA. Phylogenetic analysis was performed on Leishmania sequences from hares in Greece in conjunction with Leishmania sequences from dogs in Greece and 46 Leishmania sequences retrieved from GenBank. The Leishmania DNA prevalence in hares was found to be 23.49 % (95 % confidence interval (CI) 17.27-30.69). The phylogenetic analysis confirmed that the Leishmania sequences from hares in Greece belong in the Leishmania donovani complex. The widespread Leishmania infection in hares should be taken into consideration because under specific circumstances, this species can act as a reservoir host. This study suggests that the role of wild animals, including hares, in the epidemiology of Leishmania spp. in Greece deserves further elucidation.

Direct detection of Leishmania from clinical samples.

Science.gov (United States)

Waitumbi, John N; Bast, Joshua; Nyakoe, Nancy; Magiri, Charles; Quintana, Miguel; Takhampunya, Ratree; Schuster, Anthony L; Van de Wyngaerde, Marshall T; McAvin, James C; Coleman, Russell E

2017-01-01

The ability to rapidly and accurately diagnose leishmaniasis is a military priority. Testing was conducted to evaluate diagnostic sensitivity and specificity of field-expedient Leishmania genus and visceral Leishmania specific dual-fluorogenic, hydrolysis probe (TaqMan), polymerase chain reaction assays previously established for use in vector surveillance. Blood samples of patients with confirmed visceral leishmaniasis and controls without the disease from Baringo District, Kenya, were tested. Leishmania genus assay sensitivity was 100% (14/14) and specificity was 84% (16/19). Visceral Leishmania assay sensitivity was 93% (13/14) and specificity 80% (4/5). Cutaneous leishmaniasis (CL) skin scrapes of patients from Honduras were also evaluated. Leishmania genus assay sensitivity was 100% (10/10). Visceral Leishmania assay specificity was 100% (10/10) from cutaneous leishmaniasis samples; no fluorescence above background was reported. These results show promise in a rapid, sensitive, and specific method for Leishmania direct detection from clinical samples.

Canine leishmaniosis caused by Leishmania major and Leishmania tropica: comparative findings and serology.

Science.gov (United States)

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

First evidence of autochthonous cases of Leishmania (Leishmania) infantum in horse (Equus caballus) in the Americas and mixed infection of Leishmania infantum and Leishmania (Viannia) braziliensis.

Science.gov (United States)

Soares, Isabel R; Silva, Soraia O; Moreira, Filipe Moraghi; Prado, Luan Gavião; Fantini, Priscila; Maranhão, Renata de Pino Albuquerque; da Silva Filho, José Monteiro; Melo, Maria Norma; Palhares, Maristela S

2013-11-08

This study reports the first evidence of infection by Leishmania infantum in Equus caballus in Americas and the first mixed infection of L. infantum/Leishmania braziliensis on this mammalian species in the world. The diagnoses was based on presence of parasites in lesions and bone marrow aspirates, their identification by using specific primers for L. infantum and L. braziliensis complexes and also serological methods IFAT and ELISA. The analysis of the PCR products suggested mixed infection in three animals. Further studies involving equine leishmaniasis are carrying out in order to clarify the dynamic of Leishmania sp. in this mammalian specie and their role in the transmission of those parasites in urban endemic area of Belo Horizonte, Minas Gerais State, Brazil. Copyright © 2013 Elsevier B.V. All rights reserved.

Role of sphingolipids in murine radiation-induced lung injury: protection by sphingosine 1-phosphate analogs

OpenAIRE

Mathew, Biji; Jacobson, Jeffrey R.; Berdyshev, Evgeny; Huang, Yong; Sun, Xiaoguang; Zhao, Yutong; Gerhold, Lynnette M.; Siegler, Jessica; Evenoski, Carrie; Wang, Ting; Zhou, Tong; Zaidi, Rafe; Moreno-Vinasco, Liliana; Bittman, Robert; Chen, Chin Tu

2011-01-01

Clinically significant radiation-induced lung injury (RILI) is a common toxicity in patients administered thoracic radiotherapy. Although the molecular etiology is poorly understood, we previously characterized a murine model of RILI in which alterations in lung barrier integrity surfaced as a potentially important pathobiological event and genome-wide lung gene mRNA levels identified dysregulation of sphingolipid metabolic pathway genes. We hypothesized that sphingolipid signaling components...

Evaluation of sphingolipids in Wistar rats treated to prolonged and single oral doses of fumonisin b₁.

Science.gov (United States)

Direito, Glória M; Almeida, Adriana P; Aquino, Simone; dos Reis, Tatiana Alves; Pozzi, Claudia Rodrigues; Corrêa, Benedito

2009-01-01

The objective of the present study was to evaluate sphingolipid levels (sphingosine-So and sphinganine-Sa) and to compare the Sa/So ratio in liver, serum and urine of Wistar rats after prolonged administration (21 days) of fumonisin B(1) (FB(1)). In parallel, the kinetics of sphingolipid elimination in urine was studied in animals receiving a single dose of FB(1). Prolonged exposure to FB(1) caused an increase in Sa levels in urine, serum and liver. The most marked effect on sphingolipid biosynthesis was observed in animals treated with the highest dose of FB(1). Animals receiving a single dose of FB(1) presented variations in Sa and So levels and in the Sa/So ratio.

Critical Role of the Sphingolipid Pathway in Stroke: a Review of Current Utility and Potential Therapeutic Targets.

Science.gov (United States)

Sun, Na; Keep, Richard F; Hua, Ya; Xi, Guohua

2016-10-01

Sphingolipids are a series of cell membrane-derived lipids which act as signaling molecules and play a critical role in cell death and survival, proliferation, recognition, and migration. Sphingosine-1-phosphate acts as a key signaling molecule and regulates lymphocyte trafficking, glial cell activation, vasoconstriction, endothelial barrier function, and neuronal death pathways which plays a critical role in numerous neurological conditions. Stroke is a second leading cause of death all over the world and effective therapies are still in great demand, including ischemic stroke and hemorrhagic stroke as well as poststroke repair. Significantly, sphingolipid activities change after stroke and correlate with stroke outcome, which has promoted efforts to testify whether the sphingolipid pathway could be a novel therapeutic target in stroke. The sphingolipid metabolic pathway, the connection between the pathway and stroke, as well as therapeutic interventions to manipulate the pathway to reduce stroke-induced brain injury are discussed in this review.

Genome-wide association study identifies novel loci associated with circulating phospho- and sphingolipid concentrations.

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Ayşe Demirkan

Full Text Available Phospho- and sphingolipids are crucial cellular and intracellular compounds. These lipids are required for active transport, a number of enzymatic processes, membrane formation, and cell signalling. Disruption of their metabolism leads to several diseases, with diverse neurological, psychiatric, and metabolic consequences. A large number of phospholipid and sphingolipid species can be detected and measured in human plasma. We conducted a meta-analysis of five European family-based genome-wide association studies (N = 4034 on plasma levels of 24 sphingomyelins (SPM, 9 ceramides (CER, 57 phosphatidylcholines (PC, 20 lysophosphatidylcholines (LPC, 27 phosphatidylethanolamines (PE, and 16 PE-based plasmalogens (PLPE, as well as their proportions in each major class. This effort yielded 25 genome-wide significant loci for phospholipids (smallest P-value = 9.88×10(-204 and 10 loci for sphingolipids (smallest P-value = 3.10×10(-57. After a correction for multiple comparisons (P-value<2.2×10(-9, we observed four novel loci significantly associated with phospholipids (PAQR9, AGPAT1, PKD2L1, PDXDC1 and two with sphingolipids (PLD2 and APOE explaining up to 3.1% of the variance. Further analysis of the top findings with respect to within class molar proportions uncovered three additional loci for phospholipids (PNLIPRP2, PCDH20, and ABDH3 suggesting their involvement in either fatty acid elongation/saturation processes or fatty acid specific turnover mechanisms. Among those, 14 loci (KCNH7, AGPAT1, PNLIPRP2, SYT9, FADS1-2-3, DLG2, APOA1, ELOVL2, CDK17, LIPC, PDXDC1, PLD2, LASS4, and APOE mapped into the glycerophospholipid and 12 loci (ILKAP, ITGA9, AGPAT1, FADS1-2-3, APOA1, PCDH20, LIPC, PDXDC1, SGPP1, APOE, LASS4, and PLD2 to the sphingolipid pathways. In large meta-analyses, associations between FADS1-2-3 and carotid intima media thickness, AGPAT1 and type 2 diabetes, and APOA1 and coronary artery disease were observed. In conclusion, our

The sphingolipid long-chain base-Pkh1/2-Ypk1/2 signaling pathway regulates eisosome assembly and turnover

DEFF Research Database (Denmark)

Luo, Guangzuo; Gruhler, Albrecht; Liu, Ying

2008-01-01

Eisosomes are recently described fungal structures that play roles in the organization of the plasma membrane and endocytosis. Their major protein components are Pil1 and Lsp1, and previous studies showed that these proteins are phosphorylated by the sphingolipid long-chain base-activated Pkh1...... that phosphorylation is critical for eisosome organization. We also found that eisosomes are dynamic structures and disassemble when the Ypk protein kinases, which are activated by the sphingolipid-Pkh signaling pathway, are inactivated or when the sphingolipid signal is pharmacologically blocked with myriocin. We...

Regulation of stem-cell mediated host immunity by the sphingolipid ...

International Development Research Centre (IDRC) Digital Library (Canada)

Regulation of stem-cell mediated host immunity by the sphingolipid pathway ... in the generation of mature immune cells and the functioning of the surrounding ... methods with human cells and genetically engineered mice to examine how the ...

Gluconeogenesis in Leishmania mexicana

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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

Nitric oxide production by Peromyscus yucatanicus (Rodentia infected with Leishmania (Leishmania mexicana

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Elsy Nalleli Loría-Cervera

2013-04-01

Full Text Available Peromyscus yucatanicus (Rodentia: Cricetidae is a primary reservoir of Leishmania (Leishmania mexicana (Kinetoplastida: Trypanosomatidae. Nitric oxide (NO generally plays a crucial role in the containment and elimination of Leishmania. The aim of this study was to determine the amount of NO produced by P. yucatanicus infected with L. (L. mexicana. Subclinical and clinical infections were established in P. yucatanicus through inoculation with 1 x 10 2 and 2.5 x 10 6 promastigotes, respectively. Peritoneal macrophages were cultured alone or co-cultured with lymphocytes with or without soluble Leishmania antigen. The level of NO production was determined using the Griess reaction. The amount of NO produced was significantly higher (p ≤ 0.0001 in co-cultured macrophages and lymphocytes than in macrophages cultured alone. No differences in NO production were found between P. yucatanicus with subclinical L. (L. mexicana infections and animals with clinical infections. These results support the hypothesis that the immunological mechanisms of NO production in P. yucatanicus are similar to those described in mouse models of leishmaniasis and, despite NO production, P. yucatanicus is unable to clear the parasite infection.

Detection of Leishmania RNA virus in Leishmania parasites.

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Haroun Zangger

Full Text Available Patients suffering from cutaneous leishmaniasis (CL caused by New World Leishmania (Viannia species are at high risk of developing mucosal (ML or disseminated cutaneous leishmaniasis (DCL. After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence.This study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2 stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice.We propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis.

Sphingolipid metabolism correlates with cerebrospinal fluid Beta amyloid levels in Alzheimer's disease.

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Alfred N Fonteh

Full Text Available Sphingolipids are important in many brain functions but their role in Alzheimer's disease (AD is not completely defined. A major limit is availability of fresh brain tissue with defined AD pathology. The discovery that cerebrospinal fluid (CSF contains abundant nanoparticles that include synaptic vesicles and large dense core vesicles offer an accessible sample to study these organelles, while the supernatant fluid allows study of brain interstitial metabolism. Our objective was to characterize sphingolipids in nanoparticles representative of membrane vesicle metabolism, and in supernatant fluid representative of interstitial metabolism from study participants with varying levels of cognitive dysfunction. We recently described the recruitment, diagnosis, and CSF collection from cognitively normal or impaired study participants. Using liquid chromatography tandem mass spectrometry, we report that cognitively normal participants had measureable levels of sphingomyelin, ceramide, and dihydroceramide species, but that their distribution differed between nanoparticles and supernatant fluid, and further differed in those with cognitive impairment. In CSF from AD compared with cognitively normal participants: a total sphingomyelin levels were lower in nanoparticles and supernatant fluid; b levels of ceramide species were lower in nanoparticles and higher in supernatant fluid; c three sphingomyelin species were reduced in the nanoparticle fraction. Moreover, three sphingomyelin species in the nanoparticle fraction were lower in mild cognitive impairment compared with cognitively normal participants. The activity of acid, but not neutral sphingomyelinase was significantly reduced in the CSF from AD participants. The reduction in acid sphingomylinase in CSF from AD participants was independent of depression and psychotropic medications. Acid sphingomyelinase activity positively correlated with amyloid β42 concentration in CSF from cognitively normal but

Phylogenetic analysis of HSP70 and cyt b gene sequences for Chinese Leishmania isolates and ultrastructural characteristics of Chinese Leishmania sp.

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Yuan, Dongmei; Qin, Hanxiao; Zhang, Jianguo; Liao, Lin; Chen, Qiwei; Chen, Dali; Chen, Jianping

2017-02-01

Leishmaniasis is a worldwide epidemic disease caused by the genus Leishmania, which is still endemic in the west and northwest areas of China. Some viewpoints of the traditional taxonomy of Chinese Leishmania have been challenged by recent phylogenetic researches based on different molecular markers. However, the taxonomic positions and phylogenetic relationships of Chinese Leishmania isolates remain controversial, which need for more data and further analysis. In this study, the heat shock protein 70 (HSP70) gene and cytochrome b (cyt b) gene were used for phylogenetic analysis of Chinese Leishmania isolates from patients, dogs, gerbils, and sand flies in different geographic origins. Besides, for the interesting Leishmania sp. in China, the ultrastructure of three Chinese Leishmania sp. strains (MHOM/CN/90/SC10H2, SD, GL) were observed by transmission electron microscopy. Bayesian trees from HSP70 and cyt b congruently indicated that the 14 Chinese Leishmania isolates belong to three Leishmania species including L. donovani complex, L. gerbilli, and L. (Sauroleishmania) sp. Their identity further confirmed that the undescribed Leishmania species causing visceral Leishmaniasis (VL) in China is closely related to L. tarentolae. The phylogenetic results from HSP70 also suggested the classification of subspecies within L. donovani complex: KXG-918, KXG-927, KXG-Liu, KXG-Xu, 9044, SC6, and KXG-65 belong to L. donovani; Cy, WenChuan, and 801 were proposed to be L. infantum. Through transmission electron microscopy, unexpectedly, the Golgi apparatus were not observed in SC10H2, SD, and GL, which was similar to previous reports of reptilian Leishmania. The statistical analysis of microtubule counts separated SC10H2, SD, and GL as one group from any other reference strain (L. donovani MHOM/IN/80/DD8; L. tropica MHOM/SU/74/K27; L. gerbilli MRHO/CN/60/GERBILLI). The ultrastructural characteristics of Leishmania sp. partly lend support to the phylogenetic inference that

In vitro anti-leishmania evaluation of nickel complexes with a triazolopyrimidine derivative against Leishmania infantum and Leishmania braziliensis.

Science.gov (United States)

Ramírez-Macías, Inmaculada; Maldonado, Carmen R; Marín, Clotilde; Olmo, Francisco; Gutiérrez-Sánchez, Ramón; Rosales, María J; Quirós, Miguel; Salas, Juan M; Sánchez-Moreno, Manuel

2012-07-01

Studies on the anti-proliferative activity in vitro of seven ternary nickel (II) complexes with a triazolopyrimidine derivative and different aliphatic or aromatic amines as auxiliary ligands against promastigote and amastigote forms of Leishmania infantum and Leishmania braziliensis have been carried out. These compounds are not toxic for the host cells and two of them are effective at lower concentrations than the reference drug used in the present study (Glucantime). In general, the in vitro growth rate of Leishmania spp. was reduced, its capacity to infect cells was negatively affected and the multiplication of the amastigotes decreased. Ultrastructural analysis and metabolism excretion studies were executed in order to propose a possible mechanism for the action of the assayed compounds. Our results show that the potential mechanism is at the level of organelles membranes, either by direct action on the microtubules or by their disorganization, leading to vacuolization, degradation and ultimately cell death. Copyright © 2012 Elsevier Inc. All rights reserved.

Natural infection of bats with Leishmania in Ethiopia.

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Kassahun, Aysheshm; Sadlova, Jovana; Benda, Petr; Kostalova, Tatiana; Warburg, Alon; Hailu, Asrat; Baneth, Gad; Volf, Petr; Votypka, Jan

2015-10-01

The leishmaniases, a group of diseases with a worldwide-distribution, are caused by different species of Leishmania parasites. Both cutaneous and visceral leishmaniasis remain important public health problems in Ethiopia. Epidemiological cycles of these protozoans involve various sand fly (Diptera: Psychodidae) vectors and mammalian hosts, including humans. In recent years, Leishmania infections in bats have been reported in the New World countries endemic to leishmaniasis. The aim of this study was to survey natural Leishmania infection in bats collected from various regions of Ethiopia. Total DNA was isolated from spleens of 163 bats belonging to 23 species and 18 genera. Leishmania infection was detected by real-time (RT) PCR targeting a kinetoplast (k) DNA and internal transcribed spacer one (ITS1) gene of the parasite. Detection was confirmed by sequencing of the PCR products. Leishmania kDNA was detected in eight (4.9%) bats; four of them had been captured in the Aba-Roba and Awash-Methara regions that are endemic for leishmaniasis, while the other four specimens originated from non-endemic localities of Metu, Bedele and Masha. Leishmania isolates from two bats were confirmed by ITS1 PCR to be Leishmania tropica and Leishmania major, isolated from two individual bats, Cardioderma cor and Nycteris hispida, respectively. These results represent the first confirmed observation of natural infection of bats with the Old World Leishmania. Hence, bats should be considered putative hosts of Leishmania spp. affecting humans with a significant role in the transmission. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Leishmania hijacking of the macrophage intracellular compartments.

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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.

Evaluation of Sphingolipids in Wistar Rats Treated to Prolonged and Single Oral Doses of Fumonisin B1

Science.gov (United States)

Direito, Glória M.; Almeida, Adriana P.; Aquino, Simone; dos Reis, Tatiana Alves; Pozzi, Claudia Rodrigues; Corrêa, Benedito

2009-01-01

The objective of the present study was to evaluate sphingolipid levels (sphingosine-So and sphinganine-Sa) and to compare the Sa/So ratio in liver, serum and urine of Wistar rats after prolonged administration (21 days) of fumonisin B1 (FB1). In parallel, the kinetics of sphingolipid elimination in urine was studied in animals receiving a single dose of FB1. Prolonged exposure to FB1 caused an increase in Sa levels in urine, serum and liver. The most marked effect on sphingolipid biosynthesis was observed in animals treated with the highest dose of FB1. Animals receiving a single dose of FB1 presented variations in Sa and So levels and in the Sa/So ratio. PMID:19333435

Experimental mixed infection of Leishmania (Leishmania) amazonensis and Leishmania (L.) infantum in hamsters (Mesocricetus auratus).

Science.gov (United States)

DE Lima Celeste, Jordanna Luíza; Venuto Moura, Ana Paula; França-Silva, João Carlos; Matos DE Sousa, Gabriela; Oliveira Silva, Soraia; Norma Melo, Maria; Luiz Tafuri, Wagner; Carvalho Souza, Carolina; Monteiro DE Andrade, Hélida

2017-08-01

In South America, visceral leishmaniasis is frequently caused by Leishmania infantum and, at an unknown frequency, by Leishmania amazonensis. Therefore, mixed infections with these organisms are possible. Mixed infections might affect the clinical course, immune response, diagnosis, treatment and epidemiology of the disease. Here we describe the clinical course of mixed infections with L. amazonensis and L. infantum in a hamster model. We show that mixed infections are associated with more severe clinical disease than infection with L. amazonensis or L. infantum alone. In spleens with mixed infections, L. infantum outcompeted L. amazonensis in the tissue, but not in culture from tissue. We found increased levels of IgG in animals infected with L. infantum. Although more than 30 bands were revealed in a Western blot, the highest immunogenicity was observed with proteins having molecular masses of 95 and 90 kDa, whereas proteins with molecular masses of lower than 50 kDa were reactive frequently with serum from hamsters infected with L. amazonensis, and proteins with molecular masses of 80 and 70 kDa were reactive only with serum from hamsters infected with L. infantum. This finding has important implications regarding the biology of Leishmania and humoral immune responses to infections with these organisms.

Dysregulations in circulating sphingolipids associate with disease activity indices in female patients with systemic lupus erythematosus: a cross-sectional study.

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Checa, A; Idborg, H; Zandian, A; Sar, D Garcia; Surowiec, I; Trygg, J; Svenungsson, E; Jakobsson, P-J; Nilsson, P; Gunnarsson, I; Wheelock, C E

2017-09-01

Objective The objective of this study was to investigate the association of clinical and renal disease activity with circulating sphingolipids in patients with systemic lupus erythematosus. Methods We used liquid chromatography tandem mass spectrometry to measure the levels of 27 sphingolipids in plasma from 107 female systemic lupus erythematosus patients and 23 controls selected using a design of experiment approach. We investigated the associations between sphingolipids and two disease activity indices, the Systemic Lupus Activity Measurement and the Systemic Lupus Erythematosus Disease Activity Index. Damage was scored according to the Systemic Lupus International Collaborating Clinics damage index. Renal activity was evaluated with the British Island Lupus Activity Group index. The effects of immunosuppressive treatment on sphingolipid levels were evaluated before and after treatment in 22 female systemic lupus erythematosus patients with active disease. Results Circulating sphingolipids from the ceramide and hexosylceramide families were increased, and sphingoid bases were decreased, in systemic lupus erythematosus patients compared to controls. The ratio of C 16:0 -ceramide to sphingosine-1-phosphate was the best discriminator between patients and controls, with an area under the receiver-operating curve of 0.77. The C 16:0 -ceramide to sphingosine-1-phosphate ratio was associated with ongoing disease activity according to the Systemic Lupus Activity Measurement and the Systemic Lupus Erythematosus Disease Activity Index, but not with accumulated damage according to the Systemic Lupus International Collaborating Clinics Damage Index. Levels of C 16:0 - and C 24:1 -hexosylceramides were able to discriminate patients with current versus inactive/no renal involvement. All dysregulated sphingolipids were normalized after immunosuppressive treatment. Conclusion We provide evidence that sphingolipids are dysregulated in systemic lupus erythematosus and associated

Cholesterol, sphingolipids, and glycolipids: What do we know about their role in raft-like membranes?

DEFF Research Database (Denmark)

Rog, T.; Vattulainen, I.

2014-01-01

Lipids rafts are considered to be functional nanoscale membrane domains enriched in cholesterol and sphingolipids, characteristic in particular of the external leaflet of cell membranes. Lipids, together with membrane-associated proteins, are therefore considered to form nanoscale units with pote......Lipids rafts are considered to be functional nanoscale membrane domains enriched in cholesterol and sphingolipids, characteristic in particular of the external leaflet of cell membranes. Lipids, together with membrane-associated proteins, are therefore considered to form nanoscale units...... with potential specific functions. Although the understanding of the structure of rafts in living cells is quite limited, the possible functions of rafts are widely discussed in the literature, highlighting their importance in cellular functions. In this review, we discuss the understanding of rafts that has...... emerged based on recent atomistic and coarse-grained molecular dynamics simulation studies on the key lipid raft components, which include cholesterol, sphingolipids, glycolipids, and the proteins interacting with these classes of lipids. The simulation results are compared to experiments when possible...

TOR Complex 2-Ypk1 Signaling Maintains Sphingolipid Homeostasis by Sensing and Regulating ROS Accumulation

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Brad J. Niles

2014-02-01

Full Text Available Reactive oxygen species (ROS are produced during normal metabolism and can function as signaling molecules. However, ROS at elevated levels can damage cells. Here, we identify the conserved target of rapamycin complex 2 (TORC2/Ypk1 signaling module as an important regulator of ROS in the model eukaryotic organism, S. cerevisiae. We show that TORC2/Ypk1 suppresses ROS produced both by mitochondria as well as by nonmitochondrial sources, including changes in acidification of the vacuole. Furthermore, we link vacuole-related ROS to sphingolipids, essential components of cellular membranes, whose synthesis is also controlled by TORC2/Ypk1 signaling. In total, our data reveal that TORC2/Ypk1 act within a homeostatic feedback loop to maintain sphingolipid levels and that ROS are a critical regulatory signal within this system. Thus, ROS sensing and signaling by TORC2/Ypk1 play a central physiological role in sphingolipid biosynthesis and in the maintenance of cell growth and viability.

Leishmania infantum and Leishmania braziliensis: differences and similarities to evade the innate immune system

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Sarah Athayde Couto Falcão

2016-08-01

Full Text Available Visceral Leishmaniasis is a severe form of the disease, caused by Leishmania infantum in the New World. Patients present an anergic immune response that favors parasite establishment and spreading through tissues like bone marrow and liver. On the other hand, Leishmania braziliensis causes localized cutaneous lesions, which can be self healing in some individuals. Interactions between host and parasite are essential to understand disease pathogenesis and progression. In this context, dendritic cells (DCs act as essential bridges that connect innate and adaptive immune responses. In this way, the aim of this study was to compare the effects of these two Leishmania species, in some aspects of human dendritic cells biology to better understanding of the evasion mechanisms of Leishmania from host innate immune response. To do so, DCs were obtained from monocytes from whole peripheral blood’s healthy volunteers donors and infected with L. infantum or L. braziliensis for 24 hours. We observed similar rates of infection (around 40% as well as parasite burden for both Leishmania species. Concerning surface molecules, we observed that both parasites induced CD86 expression when DCs were infected for 24h. On the other hand, we detected a lower surface expression of CD209 in the presence of both L. braziliensis and L. infantum, but only the last one promoted the survival of dendritic cells after 24 hours. Therefore, DCs infected by both Leishmania species showed a higher expression of CD86 and a decrease of CD209 expression, suggesting that both enter DCs through CD209 molecule. However, only L. infantum had the ability to inhibit DC apoptotic death, as an evasion mechanism that enables its spreading to organs like bone marrow and liver. Lastly, L. braziliensis was more silent parasite, once it did not inhibit DC apoptosis in our in vitro model.

Targeting the GPI biosynthetic pathway.

Science.gov (United States)

Yadav, Usha; Khan, Mohd Ashraf

2018-02-27

The GPI (Glycosylphosphatidylinositol) biosynthetic pathway is a multistep conserved pathway in eukaryotes that culminates in the generation of GPI glycolipid which in turn anchors many proteins (GPI-APs) to the cell surface. In spite of the overall conservation of the pathway, there still exist subtle differences in the GPI pathway of mammals and other eukaryotes which holds a great promise so far as the development of drugs/inhibitors against specific targets in the GPI pathway of pathogens is concerned. Many of the GPI structures and their anchored proteins in pathogenic protozoans and fungi act as pathogenicity factors. Notable examples include GPI-anchored variant surface glycoprotein (VSG) in Trypanosoma brucei, GPI-anchored merozoite surface protein 1 (MSP1) and MSP2 in Plasmodium falciparum, protein-free GPI related molecules like lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) in Leishmania spp., GPI-anchored Gal/GalNAc lectin and proteophosphoglycans in Entamoeba histolytica or the GPI-anchored mannoproteins in pathogenic fungi like Candida albicans. Research in this active area has already yielded encouraging results in Trypanosoma brucei by the development of parasite-specific inhibitors of GlcNCONH 2 -β-PI, GlcNCONH 2 -(2-O-octyl)-PI and salicylic hydroxamic acid (SHAM) targeting trypanosomal GlcNAc-PI de-N-acetylase as well as the development of antifungal inhibitors like BIQ/E1210/gepinacin/G365/G884 and YW3548/M743/M720 targeting the GPI specific fungal inositol acyltransferase (Gwt1) and the phosphoethanolamine transferase-I (Mcd4), respectively. These confirm the fact that the GPI pathway continues to be the focus of researchers, given its implications for the betterment of human life.

Is autophagy the key mechanism by which the sphingolipid rheostat controls the cell fate decision?

Science.gov (United States)

Lavieu, Gregory; Scarlatti, Francesca; Sala, Giusy; Levade, Thierry; Ghidoni, Riccardo; Botti, Joëlle; Codogno, Patrice

2007-01-01

Sphingolipids are major constituents of biological membrane and some of them behave as second messengers involved in the cell fate decision. Ceramide and sphingosine 1-phosphate (S1P) constitute a rheostat system in which ceramide promotes cell death and S1P increases cell survival. We have shown that both sphingolipids are able to trigger autophagy with opposing outcomes on cell survival. Here we discuss and speculate on the diverging functions of the autophagic pathways induced by ceramide and S1P, respectively.

Complex modulation of peptidolytic activity of cathepsin D by sphingolipids

Czech Academy of Sciences Publication Activity Database

Žebrakovská, Iva; Máša, Martin; Srp, Jaroslav; Horn, Martin; Vávrová, K.; Mareš, Michael

2011-01-01

Roč. 1811, č. 12 (2011), s. 1097-1104 ISSN 1388-1981 R&D Projects: GA AV ČR IAA400550705 Institutional research plan: CEZ:AV0Z40550506 Keywords : sphingolipid * phospholipid * inhibition * activation * cathepsin D * enzyme regulation Subject RIV: CE - Biochemistry Impact factor: 5.269, year: 2011

Sphingolipid topology and the dynamic organization and function of membrane proteins

NARCIS (Netherlands)

van Meer, G.|info:eu-repo/dai/nl/068570368; Hoetzl, S.

2010-01-01

When acquiring internal membranes and vesicular transport, eukaryotic cells started to synthesize sphingolipids and sterols. The physical differences between these and the glycerophospholipids must have enabled the cells to segregate lipids in the membrane plane. Localizing this event to the Golgi

Sphingolipid Metabolism Correlates with Cerebrospinal Fluid Beta Amyloid Levels in Alzheimer’s Disease

Science.gov (United States)

Fonteh, Alfred N.; Ormseth, Cora; Chiang, Jiarong; Cipolla, Matthew; Arakaki, Xianghong; Harrington, Michael G.

2015-01-01

Sphingolipids are important in many brain functions but their role in Alzheimer’s disease (AD) is not completely defined. A major limit is availability of fresh brain tissue with defined AD pathology. The discovery that cerebrospinal fluid (CSF) contains abundant nanoparticles that include synaptic vesicles and large dense core vesicles offer an accessible sample to study these organelles, while the supernatant fluid allows study of brain interstitial metabolism. Our objective was to characterize sphingolipids in nanoparticles representative of membrane vesicle metabolism, and in supernatant fluid representative of interstitial metabolism from study participants with varying levels of cognitive dysfunction. We recently described the recruitment, diagnosis, and CSF collection from cognitively normal or impaired study participants. Using liquid chromatography tandem mass spectrometry, we report that cognitively normal participants had measureable levels of sphingomyelin, ceramide, and dihydroceramide species, but that their distribution differed between nanoparticles and supernatant fluid, and further differed in those with cognitive impairment. In CSF from AD compared with cognitively normal participants: a) total sphingomyelin levels were lower in nanoparticles and supernatant fluid; b) levels of ceramide species were lower in nanoparticles and higher in supernatant fluid; c) three sphingomyelin species were reduced in the nanoparticle fraction. Moreover, three sphingomyelin species in the nanoparticle fraction were lower in mild cognitive impairment compared with cognitively normal participants. The activity of acid, but not neutral sphingomyelinase was significantly reduced in the CSF from AD participants. The reduction in acid sphingomylinase in CSF from AD participants was independent of depression and psychotropic medications. Acid sphingomyelinase activity positively correlated with amyloid β42 concentration in CSF from cognitively normal but not impaired

Molecular identification of Leishmania species in Taybad district, Iran

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Salehi Ghodratollah

2014-09-01

Full Text Available Objective: To identify Leishmania species in patients with cutaneous leishmaniasis in the city of Taybad in Razavi Khorasan Province from April 2012 to March 2013. Methods: Among 52 persons who referred to Health Center of Taybad with suspected skin lesions, stained slide smears of 35 patients showed positive result for Leishmania. Also polymerase chain reaction assay performed using specific kDNA primers. Data of patients were analyzed with SPSS. Results: Of 35 positive smears for Leishmania, 21 (60% belonged to males and 14 (40% belonged to females. Polymerase chain reaction bands were observed in all 35 samples of which 31 (88.6% samples showed Leishmania tropica and 4 (11.4% showed Leishmania major. The highest infected age group was 11-20 years old. Conclusions: Both anthroponotic cutaneous leishmaniasis and zoonotic cutaneous leishmaniasis are present in Taybad. Leishmania tropica is the dominant causative species for anthroponotic cutaneous leishmaniasis. Further study is recommended to discover probable reservoir and vector for Leishmania major in Taybad.

Innate Immunity against Leishmania Infections

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Gurung, Prajwal; Kanneganti, Thirumala-Devi

2015-01-01

Leishmaniasis is a major health problem that affects more than 300 million people throughout the world. The morbidity associated with the disease causes serious economic burden in Leishmania endemic regions. Despite the morbidity and economic burden associated with Leishmaniasis, this disease rarely gets noticed and is still categorized under neglected tropical diseases. The lack of research combined with the ability of Leishmania to evade immune recognition has rendered our efforts to design therapeutic treatments or vaccines challenging. Herein, we review the literature on Leishmania from innate immune perspective and discuss potential problems as well as solutions and future directions that could aid in identifying novel therapeutic targets to eliminate this parasite. PMID:26249747

Vanillin biosynthetic pathways in plants.

Science.gov (United States)

Kundu, Anish

2017-06-01

The present review compiles the up-to-date knowledge on vanillin biosynthesis in plant systems to focus principally on the enzymatic reactions of in planta vanillin biosynthetic pathway and to find out its impact and prospect in future research in this field. Vanillin, a very popular flavouring compound, is widely used throughout the world. The principal natural resource of vanillin is the cured vanilla pods. Due to the high demand of vanillin as a flavouring agent, it is necessary to explore its biosynthetic enzymes and genes, so that improvement in its commercial production can be achieved through metabolic engineering. In spite of significant advancement in elucidating vanillin biosynthetic pathway in the last two decades, no conclusive demonstration had been reported yet for plant system. Several biosynthetic enzymes have been worked upon but divergences in published reports, particularly in characterizing the crucial biochemical steps of vanillin biosynthesis, such as side-chain shortening, methylation, and glucoside formation and have created a space for discussion. Recently, published reviews on vanillin biosynthesis have focused mainly on the biotechnological approaches and bioconversion in microbial systems. This review, however, aims to compile in brief the overall vanillin biosynthetic route and present a comparative as well as comprehensive description of enzymes involved in the pathway in Vanilla planifolia and other plants. Special emphasis has been given on the key enzymatic biochemical reactions that have been investigated extensively. Finally, the present standpoint and future prospects have been highlighted.

Two New Sphingolipids from the Leaves of Piper betle L.

OpenAIRE

Chen, Duo-Zhi; Xiong, Hua-Bin; Tian, Kai; Guo, Jun-Ming; Huang, Xiang-Zhong; Jiang, Zhi-Yong

2013-01-01

Two new sphingolipids, pipercerebrosides A (1) and B (2), were isolated from the leaves of Piper betle L. Their structures, including absolute configurations, were determined by spectroscopic analysis and chemical degradation. These two compounds did not show significant cytotoxic activity against the cancer cell lines K562 and HL-60 in a MTT assay.

Two new sphingolipids from the leaves of Piper betle L.

Science.gov (United States)

Chen, Duo-Zhi; Xiong, Hua-Bin; Tian, Kai; Guo, Jun-Ming; Huang, Xiang-Zhong; Jiang, Zhi-Yong

2013-09-12

Two new sphingolipids, pipercerebrosides A (1) and B (2), were isolated from the leaves of Piper betle L. Their structures, including absolute configurations, were determined by spectroscopic analysis and chemical degradation. These two compounds did not show significant cytotoxic activity against the cancer cell lines K562 and HL-60 in a MTT assay.

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

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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.

Persistence of Leishmania antigen in C57Bl/6j inbred mice infected with Leishmania (Leishmania amazonensis Persistência do antígeno da Leishmania no camundongo isogênico C57Bl/6j infectado com a Leishmania (Leishmania amazonensis

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C. Vasconcellos

1999-07-01

Full Text Available PURPOSE. To develop an animal model for studying mucocutaneous leishmaniasis. METHODS. The hind footpad of C57Bl/6j inbred mice was experimentally infected with 10(7 Leishmania (Leishmania amazonensis promastigote and the skin was studied through light and electron transmission microscopy and immunohistochemistry (PAP techniques. RESULTS. There were morphological evidences of cellular immune mechanisms and hypersensitivity reaction after eight weeks of infection and metastasis and well shaped parasites at ultrastructural level by fifty-one weeks post infection. Relapse of infection with mucosa lesions occurred around the 50th week after inoculation. CONCLUSION. The use of this animal model in long term follow up could be an useful experimental model for human mucocutaneous leishmaniasis.OBJETIVO. Desenvolver um modelo experimental para o estudo da leishmaniose cutâneo-mucosa. MÉTODOS. O coxim plantar traseiro de camundongos isogênicos C57Bl/6j foi inoculado com 10(7 formas promastigotas da Leishmania (Leishmania amazonensis e a pele foi estudada através da microscopia óptica e eletrônica e de técnica imunohistoquímica (PAP. RESULTADOS. Ocorreram evidências morfológicas de mecanismos imunes mediados por células, concomitantemente ao de reação de hipersensibilidade, após a oitava semana de infecção e a presença de parasitas com ultraestrutura preservada na quinquagésima primeira semana após a infecção. Houve recidiva da infecção com surgimento de lesões mucosas por volta da 50a semana pós inoculação. CONCLUSÃO. Este modelo animal, com um período de tempo de seguimento prolongado, poderia ser empregado como modelo para o estudo experimental da leishmaniose cutâneo-mucosa.

Two New Sphingolipids from the Leaves of Piper betle L.

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Zhi-Yong Jiang

2013-09-01

Full Text Available Two new sphingolipids, pipercerebrosides A (1 and B (2, were isolated from the leaves of Piper betle L. Their structures, including absolute configurations, were determined by spectroscopic analysis and chemical degradation. These two compounds did not show significant cytotoxic activity against the cancer cell lines K562 and HL-60 in a MTT assay.

Phylogenomic reconstruction supports supercontinent origins for Leishmania.

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Harkins, Kelly M; Schwartz, Rachel S; Cartwright, Reed A; Stone, Anne C

2016-03-01

Leishmania, a genus of parasites transmitted to human hosts and mammalian/reptilian reservoirs by an insect vector, is the causative agent of the human disease complex leishmaniasis. The evolutionary relationships within the genus Leishmania and its origins are the source of ongoing debate, reflected in conflicting phylogenetic and biogeographic reconstructions. This study employs a recently described bioinformatics method, SISRS, to identify over 200,000 informative sites across the genome from newly sequenced and publicly available Leishmania data. This dataset is used to reconstruct the evolutionary relationships of this genus. Additionally, we constructed a large multi-gene dataset, using it to reconstruct the phylogeny and estimate divergence dates for species. We conclude that the genus Leishmania evolved at least 90-100 million years ago, supporting a modified version of the Multiple Origins hypothesis that we call the Supercontinent hypothesis. According to this scenario, separate Leishmania clades emerged prior to, and during, the breakup of Gondwana. Additionally, we confirm that reptile-infecting Leishmania are derived from mammalian forms and that the species that infect porcupines and sloths form a clade long separated from other species. Finally, we firmly place the guinea-pig infecting species, Leishmaniaenriettii, the globally dispersed Leishmaniasiamensis, and the newly identified Australian species from a kangaroo, as sibling species whose distribution arises from the ancient connection between Australia, Antarctica, and South America. Copyright © 2015 Elsevier B.V. All rights reserved.

Screening for Inhibitors of Essential Leishmania Glucose Transporters

Science.gov (United States)

2013-07-01

Leishmania Glucose Transporters PRINCIPAL INVESTIGATOR: Scott M. Landfear, Ph.D. CONTRACTING ORGANIZATION: Oregon Health & Science...COVERED 1 July 2009- 30 June 2013 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Screening for Inhibitors of Essential Leishmania Glucose Transporters 5b...The objective of this project was to identify compounds that selectively inhibit the essential Leishmania glucose transporters and could hence serve

[Molecular typing of Leishmania (Leishmania) amazonensis and species of the subgenus Viannia associated with cutaneous and mucosal leishmaniasis in Colombia: A concordance study].

Science.gov (United States)

Ovalle-Bracho, Clemencia; Camargo, Carolina; Díaz-Toro, Yira; Parra-Muñoz, Marcela

2018-03-15

Multilocus enzyme electrophoresis (MLEE) is the reference standard for the characterization of Leishmania species. The test is restricted to specialized laboratories due to its technical complexity, cost, and time required to obtain results. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is used to identify Leishmania species. To establish the concordance between the two tests as identifying methods for circulating species in Colombia. A total of 96 isolates from patients with cutaneous or mucosal leishmaniasis were selected and identified by MLEE and PCR-RFLP with miniexon and hsp70 as the molecular targets, which were used sequentially. Restriction enzymes HaeIII and BccI were similarly applied. Cohen's kappa coefficient and the 95% confidence interval (CI) were calculated. The kappa coefficient and the 95% CI between MLEE and PCR-RFLP displayed "very good" concordance with a coefficient of 0.98 (CI95%: 0.98 to 1.00). The identified species were Leishmania Viannia braziliensis, Leishmania Viannia panamensis, Leishmania Viannia guyanensis and Leishmania Leishmania amazonensis. A total of 80 of the 96 isolates were sequenced and the results obtained by PCR-RFLP were confirmed. Due to the concordance obtained between tests results with the amplification of the genes miniexon and hsp70, PCR-RFLP is proposed as an alternative for identifying circulating Leishmania species in Colombia.

Leishmania infections: Molecular targets and diagnosis.

Science.gov (United States)

Akhoundi, Mohammad; Downing, Tim; Votýpka, Jan; Kuhls, Katrin; Lukeš, Julius; Cannet, Arnaud; Ravel, Christophe; Marty, Pierre; Delaunay, Pascal; Kasbari, Mohamed; Granouillac, Bruno; Gradoni, Luigi; Sereno, Denis

2017-10-01

Progress in the diagnosis of leishmaniases depends on the development of effective methods and the discovery of suitable biomarkers. We propose firstly an update classification of Leishmania species and their synonymies. We demonstrate a global map highlighting the geography of known endemic Leishmania species pathogenic to humans. We summarize a complete list of techniques currently in use and discuss their advantages and limitations. The available data highlights the benefits of molecular markers in terms of their sensitivity and specificity to quantify variation from the subgeneric level to species complexes, (sub) species within complexes, and individual populations and infection foci. Each DNA-based detection method is supplied with a comprehensive description of markers and primers and proposal for a classification based on the role of each target and primer in the detection, identification and quantification of leishmaniasis infection. We outline a genome-wide map of genes informative for diagnosis that have been used for Leishmania genotyping. Furthermore, we propose a classification method based on the suitability of well-studied molecular markers for typing the 21 known Leishmania species pathogenic to humans. This can be applied to newly discovered species and to hybrid strains originating from inter-species crosses. Developing more effective and sensitive diagnostic methods and biomarkers is vital for enhancing Leishmania infection control programs. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

(Glyco)sphingolipids are sorted in sub-apical compartments in HepG2 cells : A role for non-Golgi-related intracellular sites in the polarized distribution of (glyco)sphingolipids

NARCIS (Netherlands)

van IJzendoorn, SCD; Hoekstra, D

1998-01-01

In polarized HepG2 cells, the fluorescent sphingolipid analogues of glucosylceramide (C-6-NBD-GlcCer) and sphingomyelin (C-6-NBD-SM) display a preferential localization at the apical and basolateral domain, respectively, which is expressed during apical to basolateral transcytosis of the lipids (van

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

Science.gov (United States)

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.

Quantification of anti-Leishmania antibodies in saliva of dogs.

Science.gov (United States)

Cantos-Barreda, Ana; Escribano, Damián; Bernal, Luis J; Cerón, José J; Martínez-Subiela, Silvia

2017-08-15

Detection of serum anti-Leishmania antibodies by quantitative or qualitative techniques has been the most used method to diagnose Canine Leishmaniosis (CanL). Nevertheless, saliva may represent an alternative to blood because it is easy to collect, painless and non-invasive in comparison with serum. In this study, two time-resolved immunofluorometric assays (TR-IFMAs) for quantification of anti-Leishmania IgG2 and IgA antibodies in saliva were developed and validated and their ability to distinguish Leishmania-seronegative from seropositive dogs was evaluated. The analytical study was performed by evaluation of assay precision, sensitivity and accuracy. In addition, serum from 48 dogs (21 Leishmania-seropositive and 27 Leishmania-seronegative) were analyzed by TR-IFMAs. The assays were precise, with an intra- and inter-assay coefficients of variation lower than 11%, and showed high level of accuracy, as determined by linearity under dilution (R 2 =0.99) and recovery tests (>88.60%). Anti-Leishmania IgG2 antibodies in saliva were significantly higher in the seropositive group compared with the seronegative (pLeishmania IgA antibodies between both groups were observed. Furthermore, TR-IFMA for quantification of anti-Leishmania IgG2 antibodies in saliva showed higher differences between seropositive and seronegative dogs than the commercial assay used in serum. In conclusion, TR-IFMAs developed may be used to quantify anti-Leishmania IgG2 and IgA antibodies in canine saliva with an adequate precision, analytical sensitivity and accuracy. Quantification of anti-Leishmania IgG2 antibodies in saliva could be potentially used to evaluate the humoral response in CanL. However, IgA in saliva seemed not to have diagnostic value for this disease. For future studies, it would be desirable to evaluate the ability of the IgG2 assay to detect dogs with subclinical disease or with low antibody titers in serum and also to study the antibodies behaviour in saliva during the

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

Science.gov (United States)

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.

The Genome Sequence of Leishmania (Leishmania) amazonensis: Functional Annotation and Extended Analysis of Gene Models

Science.gov (United States)

Real, Fernando; Vidal, Ramon Oliveira; Carazzolle, Marcelo Falsarella; Mondego, Jorge Maurício Costa; Costa, Gustavo Gilson Lacerda; Herai, Roberto Hirochi; Würtele, Martin; de Carvalho, Lucas Miguel; e Ferreira, Renata Carmona; Mortara, Renato Arruda; Barbiéri, Clara Lucia; Mieczkowski, Piotr; da Silveira, José Franco; Briones, Marcelo Ribeiro da Silva; Pereira, Gonçalo Amarante Guimarães; Bahia, Diana

2013-01-01

We present the sequencing and annotation of the Leishmania (Leishmania) amazonensis genome, an etiological agent of human cutaneous leishmaniasis in the Amazon region of Brazil. L. (L.) amazonensis shares features with Leishmania (L.) mexicana but also exhibits unique characteristics regarding geographical distribution and clinical manifestations of cutaneous lesions (e.g. borderline disseminated cutaneous leishmaniasis). Predicted genes were scored for orthologous gene families and conserved domains in comparison with other human pathogenic Leishmania spp. Carboxypeptidase, aminotransferase, and 3′-nucleotidase genes and ATPase, thioredoxin, and chaperone-related domains were represented more abundantly in L. (L.) amazonensis and L. (L.) mexicana species. Phylogenetic analysis revealed that these two species share groups of amastin surface proteins unique to the genus that could be related to specific features of disease outcomes and host cell interactions. Additionally, we describe a hypothetical hybrid interactome of potentially secreted L. (L.) amazonensis proteins and host proteins under the assumption that parasite factors mimic their mammalian counterparts. The model predicts an interaction between an L. (L.) amazonensis heat-shock protein and mammalian Toll-like receptor 9, which is implicated in important immune responses such as cytokine and nitric oxide production. The analysis presented here represents valuable information for future studies of leishmaniasis pathogenicity and treatment. PMID:23857904

Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions

Science.gov (United States)

Kovacs, Erika; Harmat, Veronika; Tóth, Judit; Vértessy, Beáta G.; Módos, Károly; Kardos, József; Liliom, Károly

2010-01-01

Lipid-protein interactions are rarely characterized at a structural molecular level due to technical difficulties; however, the biological significance of understanding the mechanism of these interactions is outstanding. In this report, we provide mechanistic insight into the inhibitory complex formation of the lipid mediator sphingosylphosphorylcholine with calmodulin, the most central and ubiquitous regulator protein in calcium signaling. We applied crystallographic, thermodynamic, kinetic, and spectroscopic approaches using purified bovine calmodulin and bovine cerebral microsomal fraction to arrive at our conclusions. Here we present 1) a 1.6-Å resolution crystal structure of their complex, in which the sphingolipid occupies the conventional hydrophobic binding site on calmodulin; 2) a peculiar stoichiometry-dependent binding process: at low or high protein-to-lipid ratio calmodulin binds lipid micelles or a few lipid molecules in a compact globular conformation, respectively, and 3) evidence that the sphingolipid displaces calmodulin from its targets on cerebral microsomes. We have ascertained the specificity of the interaction using structurally related lipids as controls. Our observations reveal the structural basis of selective calmodulin inhibition by the sphingolipid. On the basis of the crystallographic and biophysical characterization of the calmodulin–sphingosylphosphorylcholine interaction, we propose a novel lipid-protein binding model, which might be applicable to other interactions as well.—Kovacs, E., Harmat, V., Tóth, J., Vértessy, B. G., Módos, K., Kardos, J., Liliom, K. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions. PMID:20522785

Cryopreservation of Leishmania Species in Manisa Province.

Science.gov (United States)

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.

Understanding serine proteases implications on Leishmania spp lifecycle.

Science.gov (United States)

Alves, Carlos Roberto; Souza, Raquel Santos de; Charret, Karen Dos Santos; Côrtes, Luzia Monteiro de Castro; Sá-Silva, Matheus Pereira de; Barral-Veloso, Laura; Oliveira, Luiz Filipe Gonçalves; da Silva, Franklin Souza

2018-01-01

Serine proteases have significant functions over a broad range of relevant biological processes to the Leishmania spp lifecycle. Data gathered here present an update on the Leishmania spp serine proteases and the status of these enzymes as part of the parasite degradome. The serine protease genes (n = 26 to 28) in Leishmania spp, which encode proteins with a wide range of molecular masses (35 kDa-115 kDa), are described along with their degrees of chromosomal and allelic synteny. Amid 17 putative Leishmania spp serine proteases, only ∼18% were experimentally demonstrated, as: signal peptidases that remove the signal peptide from secretory pre-proteins, maturases of other proteins and with metacaspase-like activity. These enzymes include those of clans SB, SC and SF. Classical inhibitors of serine proteases are used as tools for the characterization and investigation of Leishmania spp. Endogenous serine protease inhibitors, which are ecotin-like, can act modulating host actions. However, crude or synthetic based-natural serine protease inhibitors, such as potato tuber extract, Stichodactyla helianthus protease inhibitor I, fukugetin and epoxy-α-lapachone act on parasitic serine proteases and are promising leishmanicidal agents. The functional interrelationship between serine proteases and other Leishmania spp proteins demonstrate essential functions of these enzymes in parasite physiology and therefore their value as targets for leishmaniasis treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

Disruption of sphingolipid biosynthesis in hepatocyte nodules: selective proliferative stimulus induced by fumonisin B1

International Nuclear Information System (INIS)

Westhuizen, Liana van der; Gelderblom, Wentzel C.A.; Shephard, Gordon S.; Swanevelder, Sonja

2004-01-01

In order to investigate the role of sphingolipid disruption in the cancer promoting potential of fumonisin B 1 (FB 1 ) in the development of hepatocyte nodules, male Fischer 344 rats were subjected to cancer initiation (FB 1 containing diet or diethylnitrosamine (DEN) by i.p. injection) and promotion (2-acetylaminofluorene with partial hepatectomy, 2-AAF/PH) treatments followed by a secondary FB 1 dietary regimen. Sphinganine (Sa) and sphingosine (So) levels were measured by high performance liquid chromatography in control, surrounding and nodular liver tissues of the rats. The disruption of sphingolipid biosynthesis by the secondary FB 1 treatment in the control rats was significantly (P 1 initiation and 2-AAF/PH promotion. When comparing the groups subjected to the secondary FB 1 treatment, the initiation effected by FB 1 was less (P 1 initiation was marginally increased in the nodules compared to the surrounding liver after 2-AAF/PH promotion and significantly (P 1 treatment. Although, the FB 1 -induced hepatocyte nodules were not resistant to the disruption of sphingolipid biosynthesis, the nodular So levels were increased and might provide a selective growth stimulus possibly induced by bio-active sphingoid intermediates such as sphingosine 1-phosphate (S1P)

A conserved signaling network monitors delivery of sphingolipids to the plasma membrane in budding yeast.

Science.gov (United States)

Clarke, Jesse; Dephoure, Noah; Horecka, Ira; Gygi, Steven; Kellogg, Douglas

2017-10-01

In budding yeast, cell cycle progression and ribosome biogenesis are dependent on plasma membrane growth, which ensures that events of cell growth are coordinated with each other and with the cell cycle. However, the signals that link the cell cycle and ribosome biogenesis to membrane growth are poorly understood. Here we used proteome-wide mass spectrometry to systematically discover signals associated with membrane growth. The results suggest that membrane trafficking events required for membrane growth generate sphingolipid-dependent signals. A conserved signaling network appears to play an essential role in signaling by responding to delivery of sphingolipids to the plasma membrane. In addition, sphingolipid-dependent signals control phosphorylation of protein kinase C (Pkc1), which plays an essential role in the pathways that link the cell cycle and ribosome biogenesis to membrane growth. Together these discoveries provide new clues as to how growth--dependent signals control cell growth and the cell cycle. © 2017 Clarke et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Prevalence and risk factors associated with Leishmania infection in Trang Province, southern Thailand.

Science.gov (United States)

Manomat, Jipada; Leelayoova, Saovanee; Bualert, Lertwut; Tan-Ariya, Peerapan; Siripattanapipong, Suradej; Mungthin, Mathirut; Naaglor, Tawee; Piyaraj, Phunlerd

2017-11-01

Autochthonous cutaneous and visceral leishmaniasis (VL) caused by Leishmania martiniquensis and Leishmania siamensis have been considered emerging infectious diseases in Thailand. The disease burden is significantly underestimated, especially the prevalence of Leishmania infection among HIV-positive patients. A cross-sectional study was conducted to determine the prevalence and risk factors associated with Leishmania infection among patients with HIV/AIDS living in Trang province, southern Thailand, between 2015 and 2016. Antibodies against Leishmania infection were assayed using the direct agglutination test (DAT). DNA of Leishmania was detected by ITS1-PCR using the buffy coat. Species of Leishmania were also identified. Of 724 participants, the prevalence of Leishmania infection was 25.1% (182/724) using either DAT or PCR assays. Seroprevalence of Leishmania infection was 18.5% (134/724), while Leishmania DNA detected by the PCR method was 8.4% (61/724). Of these, 24.9% (180/724) were asymptomatic, whereas 0.3% (2/724) were symptomatic VL and VL/CL (cutaneous leishmaniasis). At least five species were identified: L. siamensis, L. martiniquensis, L. donovani complex, L. lainsoni, and L. major. Multivariate analysis showed that CD4+ levels Leishmania infection. Those who were PCR positive for Leishmania DNA were significantly associated with a detectable viral load, whereas non-injection drug use (NIDU) and CD4+ levels Leishmania seropositivity. A magnitude of the prevalence of underreporting Leishmania infection among Thai patients with HIV was revealed in this study. Effective public health policy to prevent and control disease transmission is urgently needed.

Actin filaments and microtubules are involved in different membrane traffic pathways that transport sphingolipids to the apical surface of polarized HepG2 cells

NARCIS (Netherlands)

Zegers, MMP; Zaal, KJM; van Ijzendoorn, SCD; Klappe, K; Hoekstra, D

In polarized HepG2 hepatoma cells, sphingolipids are transported to the apical, bile canalicular membrane by two different transport routes, as revealed with fluorescently tagged sphingolipid analogs. One route involves direct, transcytosis-independent transport of Golgi-derived glucosylceramide and

Human mixed infections of Leishmania spp. and Leishmania-Trypanosoma cruzi in a sub Andean Bolivian area: identification by polymerase chain reaction/hybridization and isoenzyme

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B Bastrenta

2003-03-01

Full Text Available Parasites belonging to Leishmania braziliensis, Leishmania donovani, Leishmania mexicana complexes and Trypanosoma cruzi (clones 20 and 39 were searched in blood, lesions and strains collected from 28 patients with active cutaneous leishmaniasis and one patient with visceral leishmaniasis. PCR-hybridization with specific probes of Leishmania complexes (L. braziliensis, L. donovani and L. mexicana and T. cruzi clones was applied to the different DNA samples. Over 29 patients, 8 (27.6% presented a mixed infection Leishmania complex species, 17 (58.6% a mixed infection Leishmania-T. cruzi, and 4 (13.8% a multi Leishmania-T. cruzi infection. Several patients were infected by the two Bolivian major clones 20 and 39 of T. cruzi (44.8%. The L. braziliensis complex was more frequently detected in lesions than in blood and a reverse result was observed for L. mexicana complex. The polymerase chain reaction-hybridization design offers new arguments supporting the idea of an underestimated rate of visceral leishmanisis in Bolivia. Parasites were isolated by culture from the blood of two patients and lesions of 10 patients. The UPGMA (unweighted pair-group method with arithmetic averages dendrogram computed from Jaccard's distances obtained from 11 isoenzyme loci data confirmed the presence of the three Leishmania complexes and undoubtedly identified human infections by L. (V. braziliensis, L. (L. chagasi and L. (L. mexicana species. Additional evidence of parasite mixtures was visualized through mixed isoenzyme profiles, L. (V. braziliensis-L. (L. mexicana and Leishmania spp.-T. cruzi.The epidemiological profile in the studied area appeared more complex than currently known. This is the first report of parasitological evidence of Bolivian patients with trypanosomatidae multi infections and consequences on the diseases' control and patient treatments are discussed.

Description of Leishmania (Leishmania forattinii sp. n., a new parasite infecting opossums and rodents in Brazil

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Elizaide L. A. Yoshida

1993-09-01

Full Text Available A new parasite species of Leishmania is described, L. (Leishmania forattinii sp. n., which was isolated from a pooled triturate of liver and spleen of a opossum (Didelphis marsupialis aurita and from skin samples from a rodent (Proechmys iheringi denigratus, captured in primary forest on the Atlantic Cost of Brazil. Our results on the basis of biological and molecular criteria indicate that this taxonomically distinct parasite ias a new species of the L. mexicana complex, but closely related to L. (L. aristidesi Laison & shaw, 1979, as revelated by phenetic and phylogenetic numerical analyses of the enzyme data. L. forattinii was clearly distinguishable from other Leishmania species of the genus usisng enzyme electrophoresis, monoclonal antibodies, molecular karyotypes, analysis of restriction enzyme digestion patterns of kinetoplast DNA (kDNA, as well as the use of kDNA hybridization procedures.

Role of pro-inflammatory cytokine IL-17 in Leishmania pathogenesis and in protective immunity by Leishmania vaccines.

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Banerjee, Antara; Bhattacharya, Parna; Joshi, Amritanshu B; Ismail, Nevien; Dey, Ranadhir; Nakhasi, Hira L

2016-11-01

The clinical outcome of Leishmania pathogenesis ranges from active skin lesions to fatal visceral dissemination and severely impaired T cell immunity. It is well established that a strong Th1 immune response is protective against cutaneous forms of the disease, however a mixed Th1/Th2 response is most commonly observed against visceral infections as evident from previous studies. Aside from Th1/Th2 cytokines, the pro-inflammatory IL-17 cytokine family plays an important role in the clearance of intracellular pathogens. In Leishmania induced skin lesions, IL-17 produced by Th17 cells is shown to exacerbate the disease, suggesting a role in pathogenesis. However, a protective role for IL-17 is indicated by the expansion of IL-17 producing cells in vaccine-induced immunity. In human visceral leishmaniasis (VL) it has been demonstrated that IL-17 and IL-22 are associated with protection against re-exposure to Leishmania, which further suggests the involvement of IL-17 in vaccine induced protective immunity. Although there is no vaccine against any form of leishmaniasis, the development of genetically modified live attenuated parasites as vaccine candidates prove to be promising, as they successfully induce a robust protective immune response in various animal models. However, the role of IL-17 producing cells and Th17 cells in response to these vaccine candidates remains unexplored. In this article, we review the role of IL-17 in Leishmania pathogenesis and the potential impact on vaccine induced immunity, with a special focus on live attenuated Leishmania parasites. Published by Elsevier Inc.

Leishmania Hijacks Myeloid Cells for Immune Escape

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María Martínez-López

2018-05-01

Full Text Available Protozoan parasites of the Leishmania genus are the causative agents of leishmaniasis, a group of neglected tropical diseases whose clinical manifestations vary depending on the infectious Leishmania species but also on host factors. Recognition of the parasite by host myeloid immune cells is a key to trigger an effective Leishmania-specific immunity. However, the parasite is able to persist in host myeloid cells by evading, delaying and manipulating host immunity in order to escape host resistance and ensure its transmission. Neutrophils are first in infiltrating infection sites and could act either favoring or protecting against infection, depending on factors such as the genetic background of the host or the parasite species. Macrophages are the main host cells where the parasites grow and divide. However, macrophages are also the main effector population involved in parasite clearance. Parasite elimination by macrophages requires the priming and development of an effector Th1 adaptive immunity driven by specific subtypes of dendritic cells. Herein, we will provide a comprehensive outline of how myeloid cells regulate innate and adaptive immunity against Leishmania, and the mechanisms used by the parasites to promote their evasion and sabotage. Understanding the interactions between Leishmania and the host myeloid cells may lead to the development of new therapeutic approaches and improved vaccination to leishmaniases, an important worldwide health problem in which current therapeutic or preventive approaches are limited.

Arginase activity of Leishmania isolated from patients with cutaneous leishmaniasis.

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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.

Leishmania Surveillance and Diagnostic Capability in Support of the Joint Biological Agent Identification and Diagnostic System (JBAIDS) and Leishmania Vector Surveillance

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2013-02-07

01-10-09 to 07-02-13 ’+. I II L~ J.\\NU :::OU~ Ill L~ :la. l-UI’I I 11J.\\l- I NUIVI~~I1 LEISHMANIA SURVEILLANCE AND DIAGNOSTIC CAPABILITY IN None...SUPPORT OF THE JOINT BIOLOGICAL AGENT IDENTIFICATION AND :lD. l:JI1J.\\NI NUIVI~~I1 DIAGNOSTIC SYSTEM (JBAIDS) None . ./ LEISHMANIA VECTOR...Field Station at Kisumu completed project activities through a resource sharing arrangement with the 59th MDW. Testing of the Leishmania epidemiology

Bioengineering natural product biosynthetic pathways for therapeutic applications.

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Wu, Ming-Cheng; Law, Brian; Wilkinson, Barrie; Micklefield, Jason

2012-12-01

With the advent of next-generation DNA sequencing technologies, the number of microbial genome sequences has increased dramatically, revealing a vast array of new biosynthetic gene clusters. Genomics data provide a tremendous opportunity to discover new natural products, and also to guide the bioengineering of new and existing natural product scaffolds for therapeutic applications. Notably, it is apparent that the vast majority of biosynthetic gene clusters are either silent or produce very low quantities of the corresponding natural products. It is imperative therefore to devise methods for activating unproductive biosynthetic pathways to provide the quantities of natural products needed for further development. Moreover, on the basis of our expanding mechanistic and structural knowledge of biosynthetic assembly-line enzymes, new strategies for re-programming biosynthetic pathways have emerged, resulting in focused libraries of modified products with potentially improved biological properties. In this review we will focus on the latest bioengineering approaches that have been utilised to optimise yields and increase the structural diversity of natural product scaffolds for future clinical applications. Copyright © 2012 Elsevier Ltd. All rights reserved.

Calcein+/PI- as an early apoptotic feature in Leishmania.

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Louise Basmaciyan

Full Text Available Although leishmaniases are responsible for high morbidity and mortality all over the world, no really satisfying treatment exists. Furthermore, the corresponding parasite Leishmania undergoes a very characteristic form of programmed cell death. Indeed, different stimuli can induce morphological and biochemical apoptotic-like features. However, the key proteins involved in mammal apoptosis, such as caspases and death receptors, are not encoded in the genome of this parasite. Currently, little is known about Leishmania apoptosis, notably owing to the lack of specific tools for programmed cell death analysis in these parasites. Furthermore, there is a need for a better understanding of Leishmania programmed cell death in order (i to better understand the role of apoptosis in unicellular organisms, (ii to better understand apoptosis in general through the study of an ancestral eukaryote, and (iii to identify new therapeutic targets against leishmaniases. To advance understanding of apoptosis in Leishmania, in this study we developed a new tool based on the quantification of calcein and propidium iodide by flow cytometry. This double labeling can be employed to distinguish early apoptosis, late apoptosis and necrosis in Leishmania live cells with a very simple and rapid assay. This paper should, therefore, be of interest for people working on Leishmania and related parasites.

Calcein+/PI- as an early apoptotic feature in Leishmania.

Science.gov (United States)

Basmaciyan, Louise; Azas, Nadine; Casanova, Magali

2017-01-01

Although leishmaniases are responsible for high morbidity and mortality all over the world, no really satisfying treatment exists. Furthermore, the corresponding parasite Leishmania undergoes a very characteristic form of programmed cell death. Indeed, different stimuli can induce morphological and biochemical apoptotic-like features. However, the key proteins involved in mammal apoptosis, such as caspases and death receptors, are not encoded in the genome of this parasite. Currently, little is known about Leishmania apoptosis, notably owing to the lack of specific tools for programmed cell death analysis in these parasites. Furthermore, there is a need for a better understanding of Leishmania programmed cell death in order (i) to better understand the role of apoptosis in unicellular organisms, (ii) to better understand apoptosis in general through the study of an ancestral eukaryote, and (iii) to identify new therapeutic targets against leishmaniases. To advance understanding of apoptosis in Leishmania, in this study we developed a new tool based on the quantification of calcein and propidium iodide by flow cytometry. This double labeling can be employed to distinguish early apoptosis, late apoptosis and necrosis in Leishmania live cells with a very simple and rapid assay. This paper should, therefore, be of interest for people working on Leishmania and related parasites.

FIRST REPORT OF CUTANEOUS LEISHMANIASIS CAUSED BY Leishmania (Leishmania infantum chagasi IN AN URBAN AREA OF RIO DE JANEIRO, BRAZIL

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Marcelo Rosandiski LYRA

2015-10-01

Full Text Available SUMMARY American tegumentary leishmaniasis (ATL is an infectious disease caused by protozoa of the genus Leishmania, and transmitted by sandflies. In the state of Rio de Janeiro, almost all of the cases of American tegumentary leishmaniasis (ATL are caused by Leishmania (Viannia braziliensis, while cases of visceral leishmaniasis (VL are caused by Leishmania (Leishmania infantum chagasi. The resurgence of autochthonous VL cases in Rio de Janeiro is related to the geographic expansion of the vector Lutzomyia longipalpis and its ability to adapt to urban areas. We report the first case of leishmaniasis with exclusively cutaneous manifestations caused by L. (L. infantum chagasi in an urban area of Rio de Janeiro. An eighty-one-year-old woman presented three pleomorphic skin lesions that were not associated with systemic symptoms or visceromegalies. Multilocus enzyme electrophoresis identified L. (L. infantum chagasi, but direct smear and PCR of bone narrow were negative for Leishmania sp. (suggesting exclusively cutaneous involvement. We discuss the different dermatological presentations of viscerotropic leishmaniasis of the New and Old World, and the clinical and epidemiological importance of the case. Etiologic diagnosis of ATL based upon exclusive clinical criteria may lead to incorrect conclusions. We should be aware of the constant changes in epidemiological patterns related to leishmaniases.

Glycosylation of inositol phosphorylceramide sphingolipids is required for normal growth and reproduction in Arabidopsis

Energy Technology Data Exchange (ETDEWEB)

Tartaglio, Virginia [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Rennie, Emilie A. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Univ. of Nebraska, Lincoln, NE (United States). Center for Plant Science Innovation and Dept. of Biochemistry; Cahoon, Rebecca [Univ. of Nebraska, Lincoln, NE (United States). Center for Plant Science Innovation and Dept. of Biochemistry; Wang, George [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Baidoo, Edward [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Mortimer, Jennifer C. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Cahoon, Edgar B. [Univ. of Nebraska, Lincoln, NE (United States). Center for Plant Science Innovation and Dept. of Biochemistry; Scheller, Henrik V. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology

2016-09-19

Sphingolipids are a major component of plant plasma membranes and endomembranes, and mediate a diverse range of biological processes. Study of the highly glycosylated glycosyl inositol phosphorylceramide (GIPC) sphingolipids has been slow as a result of challenges associated with the extractability of GIPCs, and their functions in the plant remain poorly characterized. We recently discovered an Arabidopsis GIPC glucuronosyltransferase, INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE 1 (IPUT1), which is the first enzyme in the GIPC glycosylation pathway. Plants homozygous for the iput1 loss-of-function mutation were unobtainable, and so the developmental effects of reduced GIPC glucuronosylation could not be analyzed in planta. Using a pollen-specific rescue construct, we have here isolated homozygous iput1 mutants. The iput1 mutants show severe dwarfism, compromised pollen tube guidance, and constitutive activation of salicyclic acid-mediated defense pathways. The mutants also possess reduced GIPCs, increased ceramides, and an increased incorporation of short-chain fatty acids and dihydroxylated bases into inositol phosphorylceramides and GIPCs. The assignment of a direct role for GIPC glycan head groups in the impaired processes in iput1 mutants is complicated by the vast compensatory changes in the sphingolipidome; however, our results reveal that the glycosylation steps of GIPC biosynthesis are important regulated components of sphingolipid metabolism. In conclusion, this study corroborates previously suggested roles for GIPC glycans in plant growth and defense, suggests important role s for them in reproduction and demonstrates that the entire sphingolipidome is sensitive to their status.

Genome-wide association study identifies novel loci associated with circulating phospho- and sphingolipid concentrations

NARCIS (Netherlands)

A. Demirkan (Ayşe); C.M. van Duijn (Cornelia); P. Ugocsai (Peter); A.J. Isaacs (Aaron); P.P. Pramstaller (Peter Paul); G. Liebisch (Gerhard); J.F. Wilson (James); A. Johansson (Åsa); I. Rudan (Igor); Y.S. Aulchenko (Yurii); A.V. Kirichenko (Anatoly); A.C.J.W. Janssens (Cécile); R.C. Jansen (Ritsert); C. Gnewuch (Carsten); I. Domingues (Inês); C. Pattaro (Cristian); S.H. Wild (Sarah); I. Jonasson (Inger); O. Polasek (Ozren); I.V. Zorkoltseva (Irina); A. Hofman (Albert); L.C. Karssen (Lennart); M.V. Struchalin (Maksim); J. Floyd (Jamie); W. Igl (Wilmar); Z. Biloglav (Zrinka); L. Broer (Linda); A. Pfeufer (Arne); I. Pichler (Irene); S. Campbell (Susan); G. Zaboli (Ghazal); I. Kolcic (Ivana); F. Rivadeneira Ramirez (Fernando); J.E. Huffman (Jennifer); N. Hastie (Nick); A.G. Uitterlinden (André); L. Franke (Lude); C.S. Franklin (Christopher); V. Vitart (Veronique); C.P. Nelson (Christopher P.); M. Preuss (Michael); J.C. Bis (Joshua); C.J. O'Donnell (Christopher); N. Franceschini (Nora); J.C.M. Witteman (Jacqueline); T.I. Axenovich (Tatiana); B.A. Oostra (Ben); T. Meitinger (Thomas); A.A. Hicks (Andrew); C. Hayward (Caroline); A.F. Wright (Alan); U. Gyllensten (Ulf); H. Campbell (Harry); G. Schmitz (Gerd)

2012-01-01

textabstractPhospho- and sphingolipids are crucial cellular and intracellular compounds. These lipids are required for active transport, a number of enzymatic processes, membrane formation, and cell signalling. Disruption of their metabolism leads to several diseases, with diverse neurological,

Leishmaniasis cutánea zosteriforme causada por Leishmania (Viannia panamensis y Leishmania (Viannia braziliensis: reporte de tres casos

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Camilo Andrés Morales

2014-09-01

Se presentan tres casos de leishmaniasis cutánea zosteriforme en los que se identificaron Leishmania panamensis y Leishmania braziliensis como especies infectantes. La sospecha epidemiológica derivada de la procedencia de los pacientes, así como la sospecha clínica a partir del reconocimiento de una presentación infrecuente de la enfermedad, permitieron hacer el diagnóstico.

TRANSCRIPTIONAL INHIBITION OF INTERLEUKIN-12 PROMOTER ACTIVITY IN LEISHMANIA SPP.-INFECTED MACROPHAGES

Science.gov (United States)

Jayakumar, Asha; Widenmaier, Robyn; Ma, Xiaojing; McDowell, Mary Ann

2009-01-01

To establish and persist within a host, Leishmania spp. parasites delay the onset of cell-mediated immunity by suppressing interleukin-12 (IL-12) production from host macrophages. Although it is established that Leishmania spp.-infected macrophages have impaired IL-12 production, the mechanisms that account for this suppression remain to be completely elucidated. Using a luciferase reporter assay assessing IL-12 transcription, we report here that Leishmania major, Leishmania donovani, and Leishmania chagasi inhibit IL-12 transcription in response to interferon-gamma, lipopolysaccharide, and CD40 ligand and that Leishmania spp. lipophosphoglycan, phosphoglycans, and major surface protein are not necessary for inhibition. In addition, all the Leishmania spp. strains and life-cycle stages tested inhibited IL-12 promoter activity. Our data further reveal that autocrine-acting host factors play no role in the inhibitory response and that phagocytosis signaling is necessary for inhibition of IL-12. PMID:18372625

Sand fly captures with Disney traps in area of occurrence of Leishmania (Leishmania) amazonensis in the state of Mato Grosso do Sul, mid-western Brazil.

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Dorval, Maria Elizabeth Cavalheiros; Alves, Tulia Peixoto; Cristaldo, Geucira; Rocha, Hilda Carlos da; Alves, Murilo Andrade; Oshiro, Elisa Teruya; Oliveira, Alessandra Gutierrez de; Brazil, Reginaldo Peçanha; Galati, Eunice Aparecida Bianchi; Cunha, Rivaldo Venancio da

2010-01-01

The work was conducted to study phlebotomine fauna (Diptera: Psychodidae) and aspects of American cutaneous leishmaniasis transmission in a forested area where Leishmania (Leishmania) amazonensis occurs, situated in the municipality of Bela Vista, State of Mato Grosso do Sul, Brazil. The captures were conducted with modified Disney traps, using hamster (Mesocricetus auratus) as bait, from May 2004 to January 2006. Ten species of phlebotomine sandflies were captured: Brumptomyia avellari, Brumptomyia brumpti, Bichromomyia flaviscutellata, Evandromyia bourrouli, Evandromyia lenti, Lutzomyia longipalpis, Psathyromyia campograndensis, Psathyromyia punctigeniculata, Psathyromyia shannoni and Sciopemyia sordellii. The two predominant species were Ev bourrouli (57.3%) and Bi flaviscutellata (41.4%), present at all sampling sites. Two of the 36 hamsters used as bait presented natural infection with Leishmania. The parasite was identified as Leishmania (Leishmania) amazonensis. Analysis of the results revealed the efficiency of Disney traps for capturing Bichromomyia flaviscutellata and the simultaneous presence of both vector and the Leishmania species transmitted by the same can be considered a predictive factor of the occurrence of leishmaniasis outbreaks for the human population that occupies the location.

Sphingolipids: Key Regulators of Apoptosis and Pivotal Players in Cancer Drug Resistance

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Paola Giussani

2014-03-01

Full Text Available Drug resistance elicited by cancer cells still constitutes a huge problem that frequently impairs the efficacy of both conventional and novel molecular therapies. Chemotherapy usually acts to induce apoptosis in cancer cells; therefore, the investigation of apoptosis control and of the mechanisms used by cancer cells to evade apoptosis could be translated in an improvement of therapies. Among many tools acquired by cancer cells to this end, the de-regulated synthesis and metabolism of sphingolipids have been well documented. Sphingolipids are known to play many structural and signalling roles in cells, as they are involved in the control of growth, survival, adhesion, and motility. In particular, in order to increase survival, cancer cells: (a counteract the accumulation of ceramide that is endowed with pro-apoptotic potential and is induced by many drugs; (b increase the synthesis of sphingosine-1-phosphate and glucosylceramide that are pro-survivals signals; (c modify the synthesis and the metabolism of complex glycosphingolipids, particularly increasing the levels of modified species of gangliosides such as 9-O acetylated GD3 (αNeu5Ac(2-8αNeu5Ac(2-3βGal(1-4βGlc(1-1Cer or N-glycolyl GM3 (αNeu5Ac (2-3βGal(1-4βGlc(1-1Cer and de-N-acetyl GM3 (NeuNH(2βGal(1-4βGlc(1-1Cer endowed with anti-apoptotic roles and of globoside Gb3 related to a higher expression of the multidrug resistance gene MDR1. In light of this evidence, the employment of chemical or genetic approaches specifically targeting sphingolipid dysregulations appears a promising tool for the improvement of current chemotherapy efficacy.

Histopatologia da forma localizada de leishmaniose cutânea por Leishmania (Leishmania amazonensis Histopathology of the localized form of cutaneous leishmaniasis due to Leishmania (Leishmania amazonensis

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Mário A. P. Moraes

1994-10-01

Full Text Available São descritas as alterações microscópicas presentes na forma localizada (ulcerada da Leishmaniose cutânea produzida por Leishmania (Leishmania amazonensis. Nesse tipo de manifestação, menos conhecido do que a forma anérgica ou difusa devida ao mesmo agente, as lesões são clinicamente idênticas às de leishmaniose cutânea causada por espécies outras de Leishmania, pertencentes ao subgênero Viannia. Na infecção localizada por L. (L. amazonensis, entretanto, há um aspecto peculiar, só recentemente conhecido, ou seja, cerca de 50% dos indivíduos atingidos não reagem ao teste de Montenegro. A principal característica histológica observada foi a acumulação na derme, quase sempre focal, de numerosos macrófagos contendo no citoplasma um grande vacúolo cheio de amastigotas. O quadro é semelhante ao da forma difusa, porém sem o aspecto histiocitomatóide, próprio da última. Afora esses grupos de macrófagos, vêem-se também, na forma localizada, muitas células mononucleares da inflamação, principalmente plasmócitos e macrófagos não parasitados. Os acúmulos de macrófagos com amastigotas, quando volumosos, podem sofrer necrose na parte central; os parasitos, contidos nas células, são destruídos com elas ou liberados, e sua eliminação através da úlcera deve contribuir para a cura do processo. Esse tipo de necrose nunca foi descrito em casos da forma difusa. Não houve grande diferença, no quadro histológico, entre pacientes Montenegro-negativos e positivos. Apenas em alguns casos, do grupo Montenegro-positivo, havia granulomas formados por histiócitos epitelióides sem parasitos. Quanto à persistência das células com parasitos nas lesões, observou-se que aos seis meses ou mais de evolução, em ambos os grupos, ainda estavam elas presentes. Tal achado não é comum na leishmaniose tegumentar por L. (V. braziliensis.The microscopic changes found in the localized form of the human cutaneous leishmaniasis due

The role of ORMDL proteins, guardians of cellular sphingolipids, in asthma

Czech Academy of Sciences Publication Activity Database

Paulenda, Tomáš; Dráber, Petr

2016-01-01

Roč. 71, č. 7 (2016), s. 918-930 ISSN 0105-4538 R&D Projects: GA ČR(CZ) GA14-00703S; GA ČR(CZ) GA14-09807S; GA ČR(CZ) GBP302/12/G101 Institutional support: RVO:68378050 Keywords : asthma * cellular membranes * endoplasmic * reticulum * ORMDL3 * sphingolipids Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.361, year: 2016

Phylogenetic position of Leishmania isolates from Khyber Pakhtunkhwa province of Pakistan.

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Khan, Nazma Habib; Messenger, Louisa A; Wahid, Sobia; Sutherland, Colin J

2016-08-01

Several species of the genus Leishmania are causative agents of cutaneous leishmaniasis in Pakistan. This study aimed to determine phylogenetic placement of Leishmania species causing cutaneous leishmaniasis in Khyber Pakhtunkhwa province, Pakistan (34 Leishmania tropica, 3 Leishmania infantum), in-relation to species from other geographical areas using gene sequences encoding cytochrome b (cytb) and internal transcribed spacer 2 (its2). Based on cytochrome b sequence analysis, L. tropica strains from Pakistan and other geographical regions were differentiated into two genotype groups, A and B. Within the province, five distinct L. tropica genotypes were recognized; two in group A, three in group B. Two L. infantum isolates from the province were closely associated with both Afro-Eurasian and American species of the Leishmania donovani complex, including Leishmania chagasi, L. infantum and L. donovani from Sudan and Ethiopia; while a third L. infantum isolate could not be differentiated from visceralizing Kenyan and Indian L. donovani. We observed apposite phylogenetic placement of CL-causing L. tropica and L. infantum from Khyber Pakhtunkhwa. Affinities ascribed to Leishmania spp. From the region are valuable in tracing potential importation of leishmaniasis. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Role of sphingolipids in murine radiation-induced lung injury: protection by sphingosine 1-phosphate analogs

Science.gov (United States)

Mathew, Biji; Jacobson, Jeffrey R.; Berdyshev, Evgeny; Huang, Yong; Sun, Xiaoguang; Zhao, Yutong; Gerhold, Lynnette M.; Siegler, Jessica; Evenoski, Carrie; Wang, Ting; Zhou, Tong; Zaidi, Rafe; Moreno-Vinasco, Liliana; Bittman, Robert; Chen, Chin Tu; LaRiviere, Patrick J.; Sammani, Saad; Lussier, Yves A.; Dudek, Steven M.; Natarajan, Viswanathan; Weichselbaum, Ralph R.; Garcia, Joe G. N.

2011-01-01

Clinically significant radiation-induced lung injury (RILI) is a common toxicity in patients administered thoracic radiotherapy. Although the molecular etiology is poorly understood, we previously characterized a murine model of RILI in which alterations in lung barrier integrity surfaced as a potentially important pathobiological event and genome-wide lung gene mRNA levels identified dysregulation of sphingolipid metabolic pathway genes. We hypothesized that sphingolipid signaling components serve as modulators and novel therapeutic targets of RILI. Sphingolipid involvement in murine RILI was confirmed by radiation-induced increases in lung expression of sphingosine kinase (SphK) isoforms 1 and 2 and increases in the ratio of ceramide to sphingosine 1-phosphate (S1P) and dihydro-S1P (DHS1P) levels in plasma, bronchoalveolar lavage fluid, and lung tissue. Mice with a targeted deletion of SphK1 (SphK1−/−) or with reduced expression of S1P receptors (S1PR1+/−, S1PR2−/−, and S1PR3−/−) exhibited marked RILI susceptibility. Finally, studies of 3 potent vascular barrier-protective S1P analogs, FTY720, (S)-FTY720-phosphonate (fTyS), and SEW-2871, identified significant RILI attenuation and radiation-induced gene dysregulation by the phosphonate analog, fTyS (0.1 and 1 mg/kg i.p., 2×/wk) and to a lesser degree by SEW-2871 (1 mg/kg i.p., 2×/wk), compared with those in controls. These results support the targeting of S1P signaling as a novel therapeutic strategy in RILI.—Mathew, B., Jacobson, J. R., Berdyshev, E., Huang, Y., Sun, X., Zhao, Y., Gerhold, L. M., Siegler, J., Evenoski, C., Wang, T., Zhou, T., Zaidi, R., Moreno-Vinasco, L., Bittman, R., Chen, C. T., LaRiviere, P. J., Sammani, S., Lussier, Y. A., Dudek, S. M., Natarajan, V., Weichselbaum, R. R., Garcia, J. G. N. Role of sphingolipids in murine radiation-induced lung injury: protection by sphingosine 1-phosphate analogs. PMID:21712494

Erythema exsudativum multiforme after a Leishmania skin test

NARCIS (Netherlands)

Wind, Bas S.; Guimarães, Luiz H.; Machado, Paulo R. L.

2014-01-01

A 45-year-old otherwise healthy male from an endemic region for Leishmania braziliensis infection in Bahia, Brazil, presented with three erosive hemorrhagic infiltrated plaques on the left shin accompanied with lymphadenopathy in the groin since one month. A Leishmania skin test performed on the

Molecular Identification of Leishmania spp. in Sand Flies (Diptera: Psychodidae, Phlebotominae) From Ecuador

Science.gov (United States)

Cevallos, Varsovia; Morales, Diego; Baldeón, Manuel E; Cárdenas, Paúl; Rojas-Silva, Patricio; Ponce, Patricio

2017-01-01

Abstract The detection and identification of natural infections in sand flies by Leishmania protozoan species in endemic areas is a key factor in assessing the risk of leishmaniasis and in designing prevention and control measures for this infectious disease. In this study, we analyzed the Leishmania DNA using nuclear ribosomal internal transcript spacer (ITS) sequences. Parasite DNA was extracted from naturally infected, blood-fed sand flies collected in nine localities considered leishmaniasis-endemic foci in Ecuador. The species of parasites identified in sand flies were Leishmania major-like, Leishmania naiffi, Leishmania mexicana, Leishmania lainsoni, and “Leishmania sp. siamensis”. Sand fly specimens of Brumptomyia leopoldoi, Mycropigomyia cayennensis, Nyssomyia yuilli yuilli, Nyssomyia trapidoi, Pressatia triacantha, Pressatia dysponeta, Psychodopygus carrerai carrerai, Psychodopygus panamensis, and Trichophoromyia ubiquitalis were found positive for Leishmania parasite. These findings contribute to a better understanding of the epidemiology and transmission dynamics of the disease in high-risk areas of Ecuador. PMID:28981860

Molecular Identification of Leishmania spp. in Sand Flies (Diptera: Psychodidae, Phlebotominae) From Ecuador.

Science.gov (United States)

Quiroga, Cristina; Cevallos, Varsovia; Morales, Diego; Baldeón, Manuel E; Cárdenas, Paúl; Rojas-Silva, Patricio; Ponce, Patricio

2017-11-07

The detection and identification of natural infections in sand flies by Leishmania protozoan species in endemic areas is a key factor in assessing the risk of leishmaniasis and in designing prevention and control measures for this infectious disease. In this study, we analyzed the Leishmania DNA using nuclear ribosomal internal transcript spacer (ITS) sequences. Parasite DNA was extracted from naturally infected, blood-fed sand flies collected in nine localities considered leishmaniasis-endemic foci in Ecuador.The species of parasites identified in sand flies were Leishmania major-like, Leishmania naiffi, Leishmania mexicana, Leishmania lainsoni, and "Leishmania sp. siamensis". Sand fly specimens of Brumptomyia leopoldoi, Mycropigomyia cayennensis, Nyssomyia yuilli yuilli, Nyssomyia trapidoi, Pressatia triacantha, Pressatia dysponeta, Psychodopygus carrerai carrerai, Psychodopygus panamensis, and Trichophoromyia ubiquitalis were found positive for Leishmania parasite. These findings contribute to a better understanding of the epidemiology and transmission dynamics of the disease in high-risk areas of Ecuador. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.

Monocyte-Derived Signals Activate Human Natural Killer Cells in Response to Leishmania Parasites

Science.gov (United States)

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

Monocyte-Derived Signals Activate Human Natural Killer Cells in Response to Leishmania Parasites

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Helena Messlinger

2018-01-01

Full Text Available 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

Analysis of kinetoplast cytochrome b gene of 16 Leishmania isolates from different foci of China: different species of Leishmania in China and their phylogenetic inference

Science.gov (United States)

2013-01-01

Background Leishmania species belong to the family Trypanosomatidae and cause leishmaniasis, a geographically widespread disease that infects humans and other vertebrates. This disease remains endemic in China. Due to the large geographic area and complex ecological environment, the taxonomic position and phylogenetic relationship of Chinese Leishmania isolates remain uncertain. A recent internal transcribed spacer 1 and cytochrome oxidase II phylogeny of Chinese Leishmania isolates has challenged some aspects of their traditional taxonomy as well as cladistics hypotheses of their phylogeny. The current study was designed to provide further disease background and sequence analysis. Methods We systematically analyzed 50 cytochrome b (cyt b) gene sequences of 19 isolates (16 from China, 3 from other countries) sequenced after polymerase chain reaction (PCR) using a special primer for cyt b as well as 31 sequences downloaded from GenBank. After alignment, the data were analyzed using the maximum parsimony, Bayesian and netwok methods. Results Sequences of six haplotypes representing 10 Chinese isolates formed a monophyletic group and clustered with Leishmania tarentolae. The isolates GS1, GS7, XJ771 of this study from China clustered with other isolates of Leishmania donovani complex. The isolate JS1 was a sister to Leishmania tropica, which represented an L. tropica complex instead of clustering with L. donovani complex or with the other 10 Chinese isolates. The isolates KXG-2 and GS-GER20 formed a monophyletic group with Leishmania turanica from central Asia. In the different phylogenetic trees, all of the Chinese isolates occurred in at least four groups regardless of geographic distribution. Conclusions The undescribed Leishmania species of China, which are clearly causative agents of canine leishmaniasis and human visceral leishmaniasis and are related to Sauroleishmania, may have evolved from a common ancestral parasite that came from the Americas and may have

Occurrence of Leishmania (Leishmania chagasi in a domestic cat (Felis catus in Andradina, São Paulo, Brazil: case report Ocorrência de Leishmania (Leishmania chagasi em gato doméstico (Felis catus em Andradina, São Paulo, Brasil: relato de caso

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Willian Marinho Dourado Coelho

2010-12-01

Full Text Available This work describes natural infection by Leishmania in a domestic cat where amastigote forms of the parasite were observed in the popliteal lymph node imprint. Positive and negative serological reactions were observed by enzyme-linked immunosorbent assay (ELISA and indirect immunofluorescence assay (IFA, respectively. Polymerase chain reaction (PCR revealed that the nucleotide sequence of the sample was identical to Leishmania (L. chagasi. This is the first report of the disease in felines of the city of Andradina, SP, an area considered endemic for canine and human visceral leishmaniasis.Neste trabalho, é relatada a infecção natural por Leishmania em um gato doméstico no qual, formas amastigotas do parasito foram observadas em imprint de linfonodo poplíteo. Reações sorológicas positivas e negativas foram observadas pelo teste de imunoadsorção enzimática (ELISA e reação de imunofluorescência indireta (RIFI, respectivamente. A reação em cadeia da polimerase (PCR revelou que a sequência de nucleotídeos foi idêntica à Leishmania (L. chagasi. Este é o primeiro relato da doença em felino da cidade de Andradina, Estado de São Paulo, Brasil, área considerada endêmica para leishmaniose visceral canina e humana.

Suppression of LPS-induced inflammatory responses in macrophages infected with Leishmania

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Kelly Ben L

2010-02-01

Full Text Available Abstract Background Chronic inflammation activated by macrophage innate pathogen recognition receptors such as TLR4 can lead to a range of inflammatory diseases, including atherosclerosis, Crohn's disease, arthritis and cancer. Unlike many microbes, the kinetoplastid protozoan pathogen Leishmania has been shown to avoid and even actively suppress host inflammatory cytokine responses, such as LPS-induced IL-12 production. The nature and scope of Leishmania-mediated inflammatory cytokine suppression, however, is not well characterized. Advancing our knowledge of such microbe-mediated cytokine suppression may provide new avenues for therapeutic intervention in inflammatory disease. Methods We explored the kinetics of a range of cytokine and chemokine responses in primary murine macrophages stimulated with LPS in the presence versus absence of two clinically distinct species of Leishmania using sensitive multiplex cytokine analyses. To confirm that these effects were parasite-specific, we compared the effects of Leishmania uptake on LPS-induced cytokine expression with uptake of inert latex beads. Results Whilst Leishmania uptake alone did not induce significant levels of any cytokine analysed in this study, Leishmania uptake in the presence of LPS caused parasite-specific suppression of certain LPS-induced pro-inflammatory cytokines, including IL-12, IL-17 and IL-6. Interestingly, L. amazonensis was generally more suppressive than L. major. We also found that other LPS-induced proinflammatory cytokines, such as IL-1α, TNF-α and the chemokines MIP-1α and MCP-1 and also the anti-inflammatory cytokine IL-10, were augmented during Leishmania uptake, in a parasite-specific manner. Conclusions During uptake by macrophages, Leishmania evades the activation of a broad range of cytokines and chemokines. Further, in the presence of a strong inflammatory stimulus, Leishmania suppresses certain proinflammatory cytokine responses in a parasite

46-kDa protein located in the flagellar pocket of Leishmania ...

Indian Academy of Sciences (India)

NII

Cloning and expression of endocytic Rab GTPases from Leishmania. Fractionation of early compartment from. Leishmania containing endocytic probes. Rab7:WT. Rab5: WT. Localization of Rab5 and Rab7 in Leishmania. Phase. LTR. Merge. Rab7-GFP. Rab5-GFP. LTR. Phase. Merge. Rab5-GFP. Rab7-GFP. Lysosomes.

Differentiation of Leishmania (Viannia) panamensis and Leishmania (V.) guyanensis using BccI for hsp70 PCR-RFLP.

Science.gov (United States)

Montalvo Alvarez, Ana Margarita; Nodarse, Jorge Fraga; Goodridge, Ivón Montano; Fidalgo, Lianet Monzote; Marin, Marcel; Van Der Auwera, Gert; Dujardin, Jean-Claude; Bernal, Iván Darío Velez; Muskus, Carlos

2010-05-01

Leishmania panamensis and Leishmania guyanensis are two species of the subgenus Viannia that are genetically very similar. Both parasites are usually associated with cutaneous leishmaniasis, but also have the potential to cause the mucocutaneous form of the disease. In addition, the study of foci and consequently the identification of vectors and probable reservoirs involved in transmission require a correct differentiation between both species, which is important at epidemiological level. We explored the possibility of identifying these species by using restriction fragment length polymorphisms (RFLP) in the gene coding for heat-shock protein 70 (hsp70). Previously, an hsp70 PCR-RFLP assay proved to be very effective in differentiating other Leishmania species when HaeIII is used as restriction enzyme. Based on hsp70 sequences analysis, BccI was found to generate species-specific fragments that can easily be recognized by agarose gel electrophoresis. Using the analysis of biopsies, scrapings, and parasite isolates previously grouped in a cluster comprising both L. panamensis and L. guyanensis, we showed that our approach allowed differentiation of both entities. This offers the possibility not only for identification of parasites in biological samples, but also to apply molecular epidemiology in certain countries of the New World, where several Leishmania species could coexist. Copyright 2009 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Clinical Efficacy Associated with Enhanced Antioxidant Enzyme Activities of Silver Nanoparticles Biosynthesized Using Moringa oleifera Leaf Extract, Against Cutaneous Leishmaniasis in a Murine Model of Leishmania major.

Science.gov (United States)

El-Khadragy, Manal; Alolayan, Ebtesam M; Metwally, Dina M; El-Din, Mohamed F Serag; Alobud, Sara S; Alsultan, Nour I; Alsaif, Sarah S; Awad, Manal A; Abdel Moneim, Ahmed E

2018-05-22

Leishmaniasis is one of the most significant vector-borne syndromes of individuals. This parasitic infection can be affected by many species of Leishmania, most of which are zoonotic. Natural products have made and are continuing to make important contributions to the search for new antileishmanial agents. The use of plants in the production assembly of silver nanoparticles has drawn attention because of its rapid, eco-friendly, non-pathogenic, economical protocol and provides a single step technique for the biosynthetic process. Hence, we aimed to biosynthesize silver nanoparticles (Ag-NPs) using Moringa oleifera leaf extract and investigated the antileishmanial activity of these nanoparticles in a murine model of Leishmania major infection. A total of 50 mice were used and divided into five groups-healthy control, infected, infected mice treated with pentostam, infected mice treated with Ag-NPs and infected mice pretreated with Ag-NPs. In the present study, the leaf extract of the plant species Moringa oleifera was found to be a good source for the synthesis of silver nanoparticles, their formation being confirmed by color change and stability in solution. In the present murine model of Leishmania major infection, we found that oral treatment with silver nanoparticles biosynthesized using Moringa oleifera extract resulted in a significant reduction in the average size of leishmaniasis cutaneous lesions compared with untreated mice. Furthermore, the clinical efficacy of Moringa oleifera extract was associated with enhanced antioxidant enzyme activities. In conclusion, treatment with silver nanoparticles biosynthesized using Moringa oleifera extract has higher and faster clinical efficacy than standard pentavalent antimonial treatment, probably by boosting the endogenous antioxidant activity.

The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis.

Science.gov (United States)

Ceccarelli, Marcello; Galluzzi, Luca; Diotallevi, Aurora; Andreoni, Francesca; Fowler, Hailie; Petersen, Christine; Vitale, Fabrizio; Magnani, Mauro

2017-05-16

Leishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as the target sequence. These assays had potential cross-species amplification, due to sequence similarity between Leishmania species. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high-resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographical area. DNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were amplified by a previously developed qPCR (qPCR-ML) and subjected to HRM analysis; selected PCR products were sequenced using an ABI PRISM 310 Genetic Analyzer. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed. The qPCR-ML followed by HRM analysis did not allow discrimination between L. (L.) amazonensis and L. (L.) infantum in 53.4% of cases. Hence, the novel SYBR green-based qPCR (qPCR-ama) has been tested. This assay achieved a detection limit of 0.1 pg of parasite DNA in samples spiked with host DNA and did not show cross amplification with Trypanosoma cruzi or host DNA. Although the qPCR-ama also amplified L. (L.) infantum strains, the C q values were dramatically increased compared to qPCR-ML. Therefore, the combined analysis of C q values from qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples

Sphingolipid base modifying enzymes in sunflower (Helianthus annuus): cloning and characterization of a C4-hydroxylase gene and a new paralogous Δ8-desaturase gene.

Science.gov (United States)

Moreno-Pérez, Antonio J; Martínez-Force, Enrique; Garcés, Rafael; Salas, Joaquín J

2011-05-15

Sphingolipids are components of plant cell membranes that participate in the regulation of important physiological processes. Unlike their animal counterparts, plant sphingolipids are characterized by high levels of base C4-hydroxylation. Moreover, desaturation at the Δ8 position predominates over the Δ4 desaturation typically found in animal sphingolipids. These modifications are due to the action of C4-hydroxylases and Δ8-long chain base desaturases, and they are important for complex sphingolipids finally becoming functional. The long chain bases of sunflower sphingolipids have high levels of hydroxylated and unsaturated moieties. Here, a C4-long chain base hydroxylase was functionally characterized in sunflower plant, an enzyme that could complement the sur2Δ mutation when heterologously expressed in this yeast mutant deficient in hydroxylation. This hydroxylase was ubiquitously expressed in sunflower, with the highest levels found in the developing cotyledons. In addition, we identified a new Δ8-long base chain desaturase gene that displays strong homology to a previously reported desaturase gene. This desaturase was also expressed in yeast and was able to change the long chain base composition of the transformed host. We studied the expression of this desaturase and compared it with that of the other isoform described in sunflower. The desaturase form studied in this paper displayed higher expression levels in developing seeds. Copyright © 2010 Elsevier GmbH. All rights reserved.

Genome-wide association study identifies novel loci associated with circulating phospho- and sphingolipid concentrations

DEFF Research Database (Denmark)

Demirkan, Ayşe; van Duijn, Cornelia M; Ugocsai, Peter

2012-01-01

, and metabolic consequences. A large number of phospholipid and sphingolipid species can be detected and measured in human plasma. We conducted a meta-analysis of five European family-based genome-wide association studies (N = 4034) on plasma levels of 24 sphingomyelins (SPM), 9 ceramides (CER), 57...

First molecular detection of Leishmania tarentolae-like DNA in Sergentomyia minuta in Spain.

Science.gov (United States)

Bravo-Barriga, Daniel; Parreira, Ricardo; Maia, Carla; Blanco-Ciudad, Juan; Afonso, Maria Odete; Frontera, Eva; Campino, Lenea; Pérez-Martín, Juan Enrique; Serrano Aguilera, Francisco Javier; Reina, David

2016-03-01

Phlebotomine sand flies (Diptera, Psychodidae) are vectors of multiple Leishmania species, among which Leishmania infantum stands out as a being frequently pathogenic to humans and dogs in Mediterranean countries. In this study, Sergentomyia minuta sand flies were collected using CDC miniature light traps in different 431 biotopes from Southwest Spain. A total of 114 females were tested for the presence of Leishmania DNA by targeting ITS-1 and cyt-B sequences by PCR. Leishmania DNA was detected in one S. minuta. Characterization of the obtained DNA sequences by phylogenetic analyses revealed close relatedness with Leishmania tarentolae Wenyon, 1921 as well as with both human and canine pathogenic strains of Asian origin (China), previously described as Leishmania sp. To our knowledge, this is the first report of phlebotomine sand flies naturally infected with L. tarentolae-like in Spain. The possible infection of sand flies with novel Leishmania species should be taken into consideration in epidemiological studies of vector species in areas where leishmaniosis is endemic.

From genomes to vaccines: Leishmania as a model.

Science.gov (United States)

Almeida, Renata; Norrish, Alan; Levick, Mark; Vetrie, David; Freeman, Tom; Vilo, Jaak; Ivens, Alasdair; Lange, Uta; Stober, Carmel; McCann, Sharon; Blackwell, Jenefer M

2002-01-01

The 35 Mb genome of Leishmania should be sequenced by late 2002. It contains approximately 8500 genes that will probably translate into more than 10 000 proteins. In the laboratory we have been piloting strategies to try to harness the power of the genome-proteome for rapid screening of new vaccine candidate. To this end, microarray analysis of 1094 unique genes identified using an EST analysis of 2091 cDNA clones from spliced leader libraries prepared from different developmental stages of Leishmania has been employed. The plan was to identify amastigote-expressed genes that could be used in high-throughput DNA-vaccine screens to identify potential new vaccine candidates. Despite the lack of transcriptional regulation that polycistronic transcription in Leishmania dictates, the data provide evidence for a high level of post-transcriptional regulation of RNA abundance during the developmental cycle of promastigotes in culture and in lesion-derived amastigotes of Leishmania major. This has provided 147 candidates from the 1094 unique genes that are specifically upregulated in amastigotes and are being used in vaccine studies. Using DNA vaccination, it was demonstrated that pooling strategies can work to identify protective vaccines, but it was found that some potentially protective antigens are masked by other disease-exacerbatory antigens in the pool. A total of 100 new vaccine candidates are currently being tested separately and in pools to extend this analysis, and to facilitate retrospective bioinformatic analysis to develop predictive algorithms for sequences that constitute potentially protective antigens. We are also working with other members of the Leishmania Genome Network to determine whether RNA expression determined by microarray analyses parallels expression at the protein level. We believe we are making good progress in developing strategies that will allow rapid translation of the sequence of Leishmania into potential interventions for disease

Asymptomatic dogs are highly competent to transmit Leishmania (Leishmania) infantum chagasi to the natural vector.

Science.gov (United States)

Laurenti, Márcia Dalastra; Rossi, Claudio Nazaretian; da Matta, Vânia Lúcia Ribeiro; Tomokane, Thaise Yumie; Corbett, Carlos Eduardo Pereira; Secundino, Nágila Francinete Costa; Pimenta, Paulo Filemon Paulocci; Marcondes, Mary

2013-09-23

We evaluated the ability of dogs naturally infected with Leishmania (Leishmania) infantum chagasi to transfer the parasite to the vector and the factors associated with transmission. Thirty-eight infected dogs were confirmed to be infected by direct observation of Leishmania in lymph node smears. Dogs were grouped according to external clinical signs and laboratory data into symptomatic (n=24) and asymptomatic (n=14) animals. All dogs were sedated and submitted to xenodiagnosis with F1-laboratory-reared Lutzomyia longipalpis. After blood digestion, sand flies were dissected and examined for the presence of promastigotes. Following canine euthanasia, fragments of skin, lymph nodes, and spleen were collected and processed using immunohistochemistry to evaluate tissue parasitism. Specific antibodies were detected using an enzyme-linked immunosorbent assay. Antibody levels were found to be higher in symptomatic dogs compared to asymptomatic dogs (p=0.0396). Both groups presented amastigotes in lymph nodes, while skin parasitism was observed in only 58.3% of symptomatic and in 35.7% of asymptomatic dogs. Parasites were visualized in the spleens of 66.7% and 71.4% of symptomatic and asymptomatic dogs, respectively. Parasite load varied from mild to intense, and was not significantly different between groups. All asymptomatic dogs except for one (93%) were competent to transmit Leishmania to the vector, including eight (61.5%) without skin parasitism. Sixteen symptomatic animals (67%) infected sand flies; six (37.5%) showed no amastigotes in the skin. Skin parasitism was not crucial for the ability to infect Lutzomyia longipalpis but the presence of Leishmania in lymph nodes was significantly related to a positive xenodiagnosis. Additionally, a higher proportion of infected vectors that fed on asymptomatic dogs was observed (p=0.0494). Clinical severity was inversely correlated with the infection rate of sand flies (p=0.027) and was directly correlated with antibody

Methods of staining and visualization of sphingolipid enriched and non-enriched plasma membrane regions of Arabidopsis thaliana with fluorescent dyes and lipid analogues

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Blachutzik Jörg O

2012-08-01

Full Text Available Abstract Background Sterols and Sphingolipids form lipid clusters in the plasma membranes of cell types throughout the animal and plant kingdoms. These lipid domains provide a medium for protein signaling complexes at the plasma membrane and are also observed to be principal regions of membrane contact at the inception of infection. We visualized different specific fluorescent lipophilic stains of the both sphingolipid enriched and non-sphingolipid enriched regions in the plasma membranes of live protoplasts of Arabidopsis thaliana. Results Lipid staining protocols for several fluorescent lipid analogues in plants are presented. The most emphasis was placed on successful protocols for the single and dual staining of sphingolipid enriched regions and exclusion of sphingolipid enriched regions on the plasma membrane of Arabidopsis thaliana protoplasts. A secondary focus was placed to ensure that these staining protocols presented still maintain cell viability. Furthermore, the protocols were successfully tested with the spectrally sensitive dye Laurdan. Conclusion Almost all existing staining procedures of the plasma membrane with fluorescent lipid analogues are specified for animal cells and tissues. In order to develop lipid staining protocols for plants, procedures were established with critical steps for the plasma membrane staining of Arabidopsis leaf tissue and protoplasts. The success of the plasma membrane staining protocols was additionally verified by measurements of lipid dynamics by the fluorescence recovery after photobleaching technique and by the observation of new phenomena such as time dependent lipid polarization events in living protoplasts, for which a putative physiological relevance is suggested.

Dihydroceramide Desaturase Inhibition by a Cyclopropanated Dihydroceramide Analog in Cultured Keratinocytes

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Susanne Brodesser

2011-01-01

Full Text Available Most mammalian sphingolipids contain a 4,5-(E-double bond. We report on the chemical synthesis of a dihydroceramide derivative that prevents the introduction of the double bond into sphingolipids. Minimal alteration of the parent structure by formally replacing the hydrogen atoms in the 5- and in the 6-position of the sphinganine backbone by a methylene group leads to an inhibitor of dihydroceramide desaturase in cultured cells. In the presence of 10–50 μM of compound (1, levels of biosynthetically formed dihydroceramide and—surprisingly—also of phytoceramide are elevated at the expense of ceramide. The cells respond to the lack of unsaturated sphingolipids by an elevation of mRNAs of enzymes required for sphingosine formation. At the same time, the analysis of proliferation and differentiation markers indicates that the sphingolipid double bond is required to keep the cells in a differentiated state.

Dihydroceramide desaturase inhibition by a cyclopropanated dihydroceramide analog in cultured keratinocytes.

Science.gov (United States)

Brodesser, Susanne; Kolter, Thomas

2011-01-01

Most mammalian sphingolipids contain a 4,5-(E)-double bond. We report on the chemical synthesis of a dihydroceramide derivative that prevents the introduction of the double bond into sphingolipids. Minimal alteration of the parent structure by formally replacing the hydrogen atoms in the 5- and in the 6-position of the sphinganine backbone by a methylene group leads to an inhibitor of dihydroceramide desaturase in cultured cells. In the presence of 10-50 μM of compound (1), levels of biosynthetically formed dihydroceramide and-surprisingly-also of phytoceramide are elevated at the expense of ceramide. The cells respond to the lack of unsaturated sphingolipids by an elevation of mRNAs of enzymes required for sphingosine formation. At the same time, the analysis of proliferation and differentiation markers indicates that the sphingolipid double bond is required to keep the cells in a differentiated state.

A comparison of molecular markers to detect Lutzomyia longipalpis naturally infected with Leishmania (Leishmania infantum

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Kárita Cláudia Freitas-Lidani

2014-07-01

Full Text Available The aim of the present study was to detect natural infection by Leishmania (Leishmania infantum in Lutzomyia longipalpis captured in Barcarena, state of Pará, Brazil, through the use of three primer sets. With this approach, it is unnecessary to previously dissect the sandfly specimens. DNA of 280 Lu. longipalpis female specimens were extracted from the whole insects. PCR primers for kinetoplast minicircle DNA (kDNA, the mini-exon gene and the small subunit ribosomal RNA (SSU-rRNA gene of Leishmania were used, generating fragments of 400 bp, 780 bp and 603 bp, respectively. Infection by the parasite was found with the kDNA primer in 8.6% of the cases, with the mini-exon gene primer in 7.1% of the cases and with the SSU-rRNA gene primer in 5.3% of the cases. These data show the importance of polymerase chain reaction as a tool for investigating the molecular epidemiology of visceral leishmaniasis by estimating the risk of disease transmission in endemic areas, with the kDNA primer representing the most reliable marker for the parasite.

Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs.

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Miura, Ryuichi; Kooriyama, Takanori; Yoneda, Misako; Takenaka, Akiko; Doki, Miho; Goto, Yasuyuki; Sanjoba, Chizu; Endo, Yasuyuki; Fujiyuki, Tomoko; Sugai, Akihiro; Tsukiyama-Kohara, Kyoko; Matsumoto, Yoshitsugu; Sato, Hiroki; Kai, Chieko

2015-01-01

Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV-LACK, rCDV-TSA, and rCDV-LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV-LACK showed markedly smaller nodules without ulceration. Although the rCDV-TSA- and rCDV-LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV-LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs.

A sphingolipid-dependent diffusion barrier confines ER stress to the yeast mother cell

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Clay, Lori; Caudron, Fabrice; Denoth-Lippuner, Annina; Boettcher, Barbara; Buvelot Frei, Stéphanie; Snapp, Erik Lee; Barral, Yves

2014-01-01

In many cell types, lateral diffusion barriers compartmentalize the plasma membrane and, at least in budding yeast, the endoplasmic reticulum (ER). However, the molecular nature of these barriers, their mode of action and their cellular functions are unclear. Here, we show that misfolded proteins of the ER remain confined into the mother compartment of budding yeast cells. Confinement required the formation of a lateral diffusion barrier in the form of a distinct domain of the ER-membrane at the bud neck, in a septin-, Bud1 GTPase- and sphingolipid-dependent manner. The sphingolipids, but not Bud1, also contributed to barrier formation in the outer membrane of the dividing nucleus. Barrier-dependent confinement of ER stress into the mother cell promoted aging. Together, our data clarify the physical nature of lateral diffusion barriers in the ER and establish the role of such barriers in the asymmetric segregation of proteotoxic misfolded proteins during cell division and aging. DOI: http://dx.doi.org/10.7554/eLife.01883.001 PMID:24843009

Biosynthetic origin of acetic acid using SNIF-NMR

International Nuclear Information System (INIS)

Boffo, Elisangela Fabiana; Ferreira, Antonio Gilberto

2006-01-01

The main purpose of this work is to describe the use of the technique Site-Specific Natural Isotopic Fractionation of hydrogen (SNIF-NMR), using 2 H and 1 H NMR spectroscopy, to investigate the biosynthetic origin of acetic acid in commercial samples of Brazilian vinegar. This method is based on the deuterium to hydrogen ratio at a specific position (methyl group) of acetic acid obtained by fermentation, through different biosynthetic mechanisms, which result in different isotopic ratios. We measured the isotopic ratio of vinegars obtained through C 3 , C 4 , and CAM biosynthetic mechanisms, blends of C 3 and C 4 (agrins) and synthetic acetic acid. (author)

Experimental Acquisition, Development, and Transmission of Leishmania tropica by Phlebotomus duboscqi

Science.gov (United States)

2013-01-01

ac ta t ropica Experimental acquisition, development, and transmission of Leishmania tropica by Phlebotomus duboscqi Hanafi A. Hanafi, El...August 2012 Accepted 2 September 2012 Available online 10 September 2012 Keywords: Phlebotomus duboscqi Leishmania tropica Transmission Vector...competency a b s t r a c t We report experimental infection and transmission of Leishmania tropica (Wright), by the blood-feeding sand

Real-time PCR for Leishmania species identification: Evaluation and comparison with classical techniques.

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de Morais, Rayana Carla Silva; da Costa Oliveira, Cintia Nascimento; de Albuquerque, Suênia da Cunha Gonçalves; Mendonça Trajano Silva, Lays Adrianne; Pessoa-E-Silva, Rômulo; Alves da Cruz, Heidi Lacerda; de Brito, Maria Edileuza Felinto; de Paiva Cavalcanti, Milena

2016-06-01

Cutaneous leishmaniasis (CL) is a parasitic disease caused by various Leishmania species. Several studies have shown that real time quantitative PCR (qPCR) can be used for Leishmania spp. identification by analyzing the melting temperature (Tm). Thus, the aim of this study was to evaluate the viability of qPCR for differentiating eight closely related Leishmania species that cause the same clinical form of the disease and to compare the results with classical techniques. qPCR assays for standardizing the Tm using reference strains were performed. After the CL diagnosis on blood samples of domestic animals, positive samples were analyzed by their Tm and qPCR products were purified and sequenced. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by Tm. A Restriction Fragment Length Polymorphism (RFLP) assay, a reference test, was also standardized, by using the reference strains. Through standardization of Tm for Leishmania spp., two Tm ranges were created for analysis: 1 (Tm = 78-79.99 °C) included Leishmania (V.) braziliensis, Leishmania (V.) panamensis, Leishmania (V.) lainsoni, Leishmania (V.) guyanensis and Leishmania (V.) shawi; and 2 (Tm = 80-82.2 °C) included Leishmania (V.) naiffi, Leishmania (L.) amazonensis and Leishmania (L.) mexicana. A total of 223 positive blood samples were analyzed, with 58 included in range 1 and 165 in range 2. L. (V.) braziliensis, L. (V.) panamensis and L. (V.) guyanensis were identified by sequencing, while L. (V.) braziliensis, L. (L.) mexicana and L. (V.) panamensis were identified by RFLP analysis. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by qPCR Tm analysis; five were classified in range 1 and five in range 2. A concordance of 80% was calculated between qPCR and the gold-standard (MLEE) with no significant difference between the methods (p = 0.6499); a similar result was observed for sequencing

Distinct Macrophage Fates after in vitro Infection with Different Species of Leishmania: Induction of Apoptosis by Leishmania (Leishmania) amazonensis, but Not by Leishmania (Viannia) guyanensis.

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DaMata, Jarina Pena; Mendes, Bárbara Pinheiro; Maciel-Lima, Kátia; Menezes, Cristiane Alves Silva; Dutra, Walderez Ornelas; Sousa, Lirlândia Pires; Horta, Maria Fátima

2015-01-01

Leishmania is an intracellular parasite in vertebrate hosts, including man. During infection, amastigotes replicate inside macrophages and are transmitted to healthy cells, leading to amplification of the infection. Although transfer of amastigotes from infected to healthy cells is a crucial step that may shape the outcome of the infection, it is not fully understood. Here we compare L. amazonensis and L. guyanensis infection in C57BL/6 and BALB/c mice and investigate the fate of macrophages when infected with these species of Leishmania in vitro. As previously shown, infection of mice results in distinct outcomes: L. amazonensis causes a chronic infection in both strains of mice (although milder in C57BL/6), whereas L. guyanensis does not cause them disease. In vitro, infection is persistent in L. amazonensis-infected macrophages whereas L. guyanensis growth is controlled by host cells from both strains of mice. We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy. None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells. L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice. Considering these two species of Leishmania and strains of mice, macrophage apoptosis, induced at the initial moments of infection, correlates with chronic infection, regardless of its severity. We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far. The ingestion of apoptotic infected macrophages by healthy macrophages could be a way of amastigote spreading, leading to the establishment of infection.

Distinct Macrophage Fates after in vitro Infection with Different Species of Leishmania: Induction of Apoptosis by Leishmania (Leishmania amazonensis, but Not by Leishmania (Viannia guyanensis.

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Jarina Pena DaMata

Full Text Available Leishmania is an intracellular parasite in vertebrate hosts, including man. During infection, amastigotes replicate inside macrophages and are transmitted to healthy cells, leading to amplification of the infection. Although transfer of amastigotes from infected to healthy cells is a crucial step that may shape the outcome of the infection, it is not fully understood. Here we compare L. amazonensis and L. guyanensis infection in C57BL/6 and BALB/c mice and investigate the fate of macrophages when infected with these species of Leishmania in vitro. As previously shown, infection of mice results in distinct outcomes: L. amazonensis causes a chronic infection in both strains of mice (although milder in C57BL/6, whereas L. guyanensis does not cause them disease. In vitro, infection is persistent in L. amazonensis-infected macrophages whereas L. guyanensis growth is controlled by host cells from both strains of mice. We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy. None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells. L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice. Considering these two species of Leishmania and strains of mice, macrophage apoptosis, induced at the initial moments of infection, correlates with chronic infection, regardless of its severity. We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far. The ingestion of apoptotic infected macrophages by healthy macrophages could be a way of amastigote spreading, leading to the establishment of infection.

First Cases of Cutaneous Leishmaniasis Caused by Leishmania (Viannia) naiffi Infection in Surinam

NARCIS (Netherlands)

van Thiel, Pieter-Paul A. M.; van Gool, Tom; Kager, Piet A.; Bart, Aldert

2010-01-01

Cutaneous leishmaniasis in Surinam is generally caused by infection by Leishmania guyanensis. We report three cases of infection with Leishmania (Viannia) naiffi, a Leishmania species not described from Surinam before. Treatment with pentamidine proved to be effective

Heterologous expression of pikromycin biosynthetic gene cluster using Streptomyces artificial chromosome system.

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Pyeon, Hye-Rim; Nah, Hee-Ju; Kang, Seung-Hoon; Choi, Si-Sun; Kim, Eung-Soo

2017-05-31

Heterologous expression of biosynthetic gene clusters of natural microbial products has become an essential strategy for titer improvement and pathway engineering of various potentially-valuable natural products. A Streptomyces artificial chromosomal conjugation vector, pSBAC, was previously successfully applied for precise cloning and tandem integration of a large polyketide tautomycetin (TMC) biosynthetic gene cluster (Nah et al. in Microb Cell Fact 14(1):1, 2015), implying that this strategy could be employed to develop a custom overexpression scheme of natural product pathway clusters present in actinomycetes. To validate the pSBAC system as a generally-applicable heterologous overexpression system for a large-sized polyketide biosynthetic gene cluster in Streptomyces, another model polyketide compound, the pikromycin biosynthetic gene cluster, was preciously cloned and heterologously expressed using the pSBAC system. A unique HindIII restriction site was precisely inserted at one of the border regions of the pikromycin biosynthetic gene cluster within the chromosome of Streptomyces venezuelae, followed by site-specific recombination of pSBAC into the flanking region of the pikromycin gene cluster. Unlike the previous cloning process, one HindIII site integration step was skipped through pSBAC modification. pPik001, a pSBAC containing the pikromycin biosynthetic gene cluster, was directly introduced into two heterologous hosts, Streptomyces lividans and Streptomyces coelicolor, resulting in the production of 10-deoxymethynolide, a major pikromycin derivative. When two entire pikromycin biosynthetic gene clusters were tandemly introduced into the S. lividans chromosome, overproduction of 10-deoxymethynolide and the presence of pikromycin, which was previously not detected, were both confirmed. Moreover, comparative qRT-PCR results confirmed that the transcription of pikromycin biosynthetic genes was significantly upregulated in S. lividans containing tandem

Molecular docking and molecular dynamics simulation study of inositol phosphorylceramide synthase – inhibitor complex in leishmaniasis: Insight into the structure based drug design [version 2; referees: 2 approved

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Vineetha Mandlik

2016-09-01

Full Text Available Inositol phosphorylceramide synthase (IPCS has emerged as an important, interesting and attractive target in the sphingolipid metabolism of Leishmania. IPCS catalyzes the conversion of ceramide to IPC which forms the most predominant sphingolipid in Leishmania. IPCS has no mammalian equivalent and also plays an important role in maintaining the infectivity and viability of the parasite. The present study explores the possibility of targeting IPCS; development of suitable inhibitors for the same would serve as a treatment strategy for the infectious disease leishmaniasis. Five coumarin derivatives were developed as inhibitors of IPCS protein. Molecular dynamics simulations of the complexes of IPCS with these inhibitors were performed which provided insights into the binding modes of the inhibitors. In vitro screening of the top three compounds has resulted in the identification of one of the compounds (compound 3 which shows little cytotoxic effects. This compound therefore represents a good starting point for further in vivo experimentation and could possibly serve as an important drug candidate for the treatment of leishmaniasis.

Ophiamides A-B, new potent urease inhibitory sphingolipids from Heliotropium ophioglossum.

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Firdous, Sadiqa; Ansari, Nida Hassan; Fatima, Itrat; Malik, Abdul; Afza, Nighat; Iqbal, Lubna; Lateef, Mehreen

2012-07-01

Ophiamides A (1) and B (2), two new sphingolipids have been isolated from the n-hexane subfraction of the MeOH extract of the whole plant of Heliotropium ophioglossum along with glycerol monopalmitate (3) and β-sitosterol 3-O-β-D: -glucoside (4) reported for the first time from this species. Their structures were elucidated by spectroscopic techniques including MS and 2D-NMR spectroscopy. Both the compounds 1 and 2 showed potent inhibitory activity against the enzyme urease.

Genetic Diversity in Natural Populations of New World Leishmania

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Cupolillo Elisa

1998-01-01

Full Text Available Our results have shown the wide diversity of parasites within New World Leishmania. Biochemical and molecular characterization of species within the genus has revealed that much of the population heterogeneity has a genetic basis. The source of genetic diversity among Leishmania appears to arise from predominantly asexual, clonal reproduction, although occasional bouts of sexual reproduction can not be ruled out. Genetic variation is extensive with some clones widely distributed and others seemingly unique and localized to a particular endemic focus. Epidemiological studies of leishmaniasis has been directed to the ecology and dynamics of transmission of Leishmania species/variants, particularly in localized areas. Future research using molecular techniques should aim to identify and follow Leishmania types in nature and correlate genetic typing with important clinical characteristics such as virulence, pathogenicity, drug resistance and antigenic variation. The epidemiological significance of such variation not only has important implications for the control of the leishmaniases, but would also help to elucidate the evolutionary biology of the causative agents.

Screening For Inhibitors Of Essential Leishmania Glucose Transporters

Science.gov (United States)

2011-07-01

parasite life cycle and, unlike he amastigote form that lives inside mammalian macrophages, s viable provided that an alternative energy source such as pro...glucose transporters havebeenvalidated asnewdrug targets for proto- zoan parasites including Plasmodium falciparum, Leishmania mexicana and Trypanosoma...such as Leishmania species, Trypanosoma rucei, and Plasmodium falciparum, the causative agents of leish- aniasis, African sleeping sickness, and malaria

Leishmania (Leishmania mexicana en el corregimiento de San Matías, municipio de Gómez Plata, Antioquia, Colombia

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Diana Sierra

2006-10-01

Full Text Available Introducción. La leishmaniaisis es una enfermedad encontrada en focos naturales de infección donde están presentes insectos vectores y mamíferos reservorios deLeishmania. Objetivo. Registrar por primera vez la presencia de Leishmania (Leishmania mexicana Biagi, 1953, en el corregimiento de San Matías, municipio de Gómez Plata, departamento de Antioquia. Materiales y métodos. La especie fue aislada de un paciente con leishmaniasis cutánea localizada e identificada por la técnica de Inmunofluorescencia utilizando anticuerpos monoclonales específicos de especie y electroforesis de enzimas . Resultados y conclusión. Su perfil isoenzimático similar al de las cepas de referencia L. (L. mexicana (MNCY/BZ/62/M379 y L. (L. mexicana (MHOM/BE/82/BEL21, permitió concluír que la especie aislada del paciente es L. (L. mexicana.

Molecular crosstalks in Leishmania-sandfly-host relationships

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Volf P.

2008-09-01

Full Text Available Sandflies (Diptera: Phlebotominae are vectors of Leishmania parasites, causative agents of important human and animal diseases with diverse manifestations. This review summarizes present knowledge about the vectorial part of Leishmania life cycle and parasite transmission to the vertebrate host. Particularly, it focuses on molecules that determine the establishment of parasite infection in sandfly midgut. It describes the concept of specific versus permissive sandfly vectors, explains the epidemiological consequences of broad susceptibility of permissive sandflies and demonstrates that genetic exchange may positively affect Leishmania fitness in the vector. Last but not least, the review describes recent knowledge about circulating antibodies produced by hosts in response to sandfly bites. Studies on specificity and kinetics of antibody response revealed that anti-saliva IgG could be used as a marker of host exposure to sandflies, i.e. as a useful tool for evaluation of vector control.

19q13.12 microdeletion syndrome fibroblasts display abnormal storage of cholesterol and sphingolipids in the endo-lysosomal system.

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Zhao, Kexin; van der Spoel, Aarnoud; Castiglioni, Claudia; Gale, Sarah; Fujiwara, Hideji; Ory, Daniel S; Ridgway, Neale D

2018-06-01

Microdeletions in 19q12q13.12 cause a rare and complex haploinsufficiency syndrome characterized by intellectual deficiency, developmental delays, and neurological movement disorders. Variability in the size and interval of the deletions makes it difficult to attribute the complex clinical phenotype of this syndrome to an underlying gene(s). As an alternate approach, we examined the biochemical and metabolic features of fibroblasts from an affected individual to derive clues as to the molecular basis for the syndrome. Immunofluorescence and electron microscopy of affected fibroblasts revealed an abnormal endo-lysosomal compartment that was characterized by rapid accumulation of lysosomotropic dyes, elevated LAMP1 and LAMP2 expression and vacuoles containing membrane whorls, common features of lysosomal lipid storage disorders. The late endosomes-lysosomes (LE/LY) of affected fibroblasts accumulated low-density lipoprotein cholesterol, and displayed reduced cholesterol esterification and increased de novo cholesterol synthesis, indicative of defective cholesterol transport to the endoplasmic reticulum. Affected fibroblasts also had increased ceramide and sphingolipid mass, altered glycosphingolipid species and accumulation of a fluorescent lactosylceramide probe in LE/LY. Autophagosomes also accumulated in affected fibroblasts because of decreased fusion with autolysosomes, a defect associated with other lysosomal storage diseases. Attempts to correct the cholesterol/sphingolipid storage defect in fibroblasts with cyclodextrin, sphingolipid synthesis inhibitors or by altering ion transport were unsuccessful. Our data show that 19q13.12 deletion fibroblasts have abnormal accumulation of cholesterol and sphingolipids in the endo-lysosomal system that compromises organelle function and could be an underlying cause of the clinical features of the syndrome. Copyright © 2018 Elsevier B.V. All rights reserved.

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

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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.

Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs.

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Ryuichi Miura

Full Text Available Canine distemper virus (CDV vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV-LACK, rCDV-TSA, and rCDV-LmSTI1, respectively. Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV-LACK showed markedly smaller nodules without ulceration. Although the rCDV-TSA- and rCDV-LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV-LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs.

Potential role for dog fleas in the cycle of Leishmania spp.

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Ferreira, Marilia Gabriele Prado Albuquerque; Fattori, Karina Reinaldo; Souza, Fausto; Lima, Valéria Marçal Felix

2009-10-28

Several species of Leishmania spp. cause diseases in humans that range from self-healing cutaneous lesions to fatal visceral leishmaniosis. It has been observed that besides being transmitted by sand flies, Leishmania spp. may also be transmitted by arthropods such as ticks and fleas. To investigate the possible role of dog fleas in the transmission of Leishmania spp., Ctenocefalides felis were removed from 22 dogs which were positive according to ELISA and rK-39 tests. A C. felis sample from each of the 22 dogs was used to infect a hamster. The 22 hamsters were euthanized 4 months after infection with the fleas and the blood was subjected to ELISA to detect antibody anti-Leishmania spp., and the spleen samples were submitted to PCR for detection of Leishmania spp. DNA. PCR and ELISA were both positive in 18.1% (4/22), with PCR alone being positive in 45% (10/22) and ELISA alone in only 9% (2/22). These results suggest the participation of dog fleas in the Leishmania spp. cycle. Confirmation that C. felis indeed transmit leishmaniosis to dogs requires new strategies against leishmaniosis to be enforced by public health authorities and which focus on better ways to keep dogs free of fleas.

Natural canine infection by Leishmania infantum and Leishmania amazonensis and their implications for disease control

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Letícia da Cruz Sanches

Full Text Available Abstract Leishmaniasis is a major public health problem worldwide. Because Leishmania can adapt to new hosts or vectors, knowledge concerning the current etiological agent in dogs is important in endemic areas. This study aimed to identify the Leishmania species detected in 103 samples of peripheral blood from dogs that were naturally infected with these protozoa. The diagnosis of leishmaniasis was determined through parasitological examination, the indirect enzyme-linked immunosorbent assay (ELISA and the polymerase chain reaction (PCR. The Leishmania species were identified by means of PCR-restriction fragment length polymorphism (PCR-RFLP. The samples were subjected to PCR using oligonucleotide primers that amplify the intergenic region ITS1 of the rRNA gene in order to identify the species. The amplified DNA was digested using the restriction enzyme HaeIII. A restriction profile identical to L. amazonensis was shown in 77/103 samples and the profile was similar to L. infantum in 17/103. However, a mixed profile was shown in 9/103 samples, which impeded species identification. In conclusion, the infection in these dogs was predominantly due to L. amazonensis, thus indicating that diagnosing of cases of canine leishmaniasis needs to be reexamined, since the causative agent identified is not restricted to L. infantum.

Sand fly captures with Disney traps in area of occurrence of Leishmania (Leishmania amazonensis in the state of Mato Grosso do Sul, mid-western Brazil Capturas de flebotomíneos com armadilhas de Disney em área de ocorrência de Leishmania (Leishmania amazonensis no estado de Mato Grosso do Sul, região Centro-Oeste do Brasil

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Maria Elizabeth Cavalheiros Dorval

2010-10-01

Full Text Available INTRODUCTION: The work was conducted to study phlebotomine fauna (Diptera: Psychodidae and aspects of American cutaneous leishmaniasis transmission in a forested area where Leishmania (Leishmania amazonensis occurs, situated in the municipality of Bela Vista, State of Mato Grosso do Sul, Brazil. METHODS: The captures were conducted with modified Disney traps, using hamster (Mesocricetus auratus as bait, from May 2004 to January 2006. RESULTS: Ten species of phlebotomine sandflies were captured: Brumptomyia avellari, Brumptomyia brumpti, Bichromomyia flaviscutellata, Evandromyia bourrouli, Evandromyia lenti, Lutzomyia longipalpis, Psathyromyia campograndensis, Psathyromyia punctigeniculata, Psathyromyia shannoni and Sciopemyia sordellii. The two predominant species were Ev bourrouli (57.3% and Bi flaviscutellata (41.4%, present at all sampling sites. Two of the 36 hamsters used as bait presented natural infection with Leishmania. The parasite was identified as Leishmania (Leishmania amazonensis. CONCLUSIONS: Analysis of the results revealed the efficiency of Disney traps for capturing Bichromomyia flaviscutellata and the simultaneous presence of both vector and the Leishmania species transmitted by the same can be considered a predictive factor of the occurrence of leishmaniasis outbreaks for the human population that occupies the location.INTRODUÇÃO: O estudo foi realizado com o objetivo de estudar a fauna de flebotomíneos (Diptera: Psychodidae e aspectos ligados à transmissão da leishmaniose tegumentar americana em uma área florestal com ocorrência de Leishmania (Leishmania amazonensis, situada no município de Bela Vista, Estado do Mato Grosso do Sul, Brasil. MÉTODOS: As capturas de flebotomíneos foram realizadas utilizando-se armadilhas tipo Disney modificadas, com isca roedor, Mesocricetus auratus, no período de maio de 2004 a janeiro de 2006. RESULTADOS: As coletas resultaram na identificação de 10 espécies de Phlebotominae

First occurrence of an autochthonous canine case of Leishmania (Leishmania infantum chagasi in the municipality of Campinas, State of São Paulo, Brazil

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Elisa San Martin Mouriz Savani

2011-08-01

Full Text Available An autochthonous case of visceral leishmaniasis is reported in a dog (Canis familiaris as an apparently natural infection in a non-endemic area. DNA obtained from spleen and liver samples produced the expected fragment in a Leishmania-specific rDNA-based nested-PCR assay. The PCR product, a 490 bp fragment, was sequenced and the nucleotide sequence was identical to that of Leishmania (Leishmania infantum chagasi. These results are surprising since no autochthonous human or canine cases of visceral leishmaniasis have ever been reported in this municipality. This case suggests that natural transmission of this disease is occurring in this area.

RIPK1 and PGAM5 Control Leishmania Replication through Distinct Mechanisms.

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Farias Luz, Nivea; Balaji, Sakthi; Okuda, Kendi; Barreto, Aline Silva; Bertin, John; Gough, Peter J; Gazzinelli, Ricardo; Almeida, Roque P; Bozza, Marcelo T; Borges, Valeria M; Chan, Francis Ka-Ming

2016-06-15

Leishmaniasis is an important parasitic disease found in the tropics and subtropics. Cutaneous and visceral leishmaniasis affect an estimated 1.5 million people worldwide. Despite its human health relevance, relatively little is known about the cell death pathways that control Leishmania replication in the host. Necroptosis is a recently identified form of cell death with potent antiviral effects. Receptor interacting protein kinase 1 (RIPK1) is a critical kinase that mediates necroptosis downstream of death receptors and TLRs. Heme, a product of hemoglobin catabolism during certain intracellular pathogen infections, is also a potent inducer of macrophage necroptosis. We found that human visceral leishmaniasis patients exhibit elevated serum levels of heme. Therefore, we examined the impact of heme and necroptosis on Leishmania replication. Indeed, heme potently inhibited Leishmania replication in bone marrow-derived macrophages. Moreover, we found that inhibition of RIPK1 kinase activity also enhanced parasite replication in the absence of heme. We further found that the mitochondrial phosphatase phosphoglycerate mutase family member 5 (PGAM5), a putative downstream effector of RIPK1, was also required for inhibition of Leishmania replication. In mouse infection, both PGAM5 and RIPK1 kinase activity are required for IL-1β expression in response to Leishmania However, PGAM5, but not RIPK1 kinase activity, was directly responsible for Leishmania-induced IL-1β secretion and NO production in bone marrow-derived macrophages. Collectively, these results revealed that RIPK1 and PGAM5 function independently to exert optimal control of Leishmania replication in the host. Copyright © 2016 by The American Association of Immunologists, Inc.

A Historical Overview of the Classification, Evolution, and Dispersion of Leishmania Parasites and Sandflies.

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Mohammad Akhoundi

2016-03-01

Full Text Available The aim of this study is to describe the major evolutionary historical events among Leishmania, sandflies, and the associated animal reservoirs in detail, in accordance with the geographical evolution of the Earth, which has not been previously discussed on a large scale.Leishmania and sandfly classification has always been a controversial matter, and the increasing number of species currently described further complicates this issue. Despite several hypotheses on the origin, evolution, and distribution of Leishmania and sandflies in the Old and New World, no consistent agreement exists regarding dissemination of the actors that play roles in leishmaniasis. For this purpose, we present here three centuries of research on sandflies and Leishmania descriptions, as well as a complete description of Leishmania and sandfly fossils and the emergence date of each Leishmania and sandfly group during different geographical periods, from 550 million years ago until now. We discuss critically the different approaches that were used for Leishmana and sandfly classification and their synonymies, proposing an updated classification for each species of Leishmania and sandfly. We update information on the current distribution and dispersion of different species of Leishmania (53, sandflies (more than 800 at genus or subgenus level, and animal reservoirs in each of the following geographical ecozones: Palearctic, Nearctic, Neotropic, Afrotropical, Oriental, Malagasy, and Australian. We propose an updated list of the potential and proven sandfly vectors for each Leishmania species in the Old and New World. Finally, we address a classical question about digenetic Leishmania evolution: which was the first host, a vertebrate or an invertebrate?We propose an updated view of events that have played important roles in the geographical dispersion of sandflies, in relation to both the Leishmania species they transmit and the animal reservoirs of the parasites.

A Historical Overview of the Classification, Evolution, and Dispersion of Leishmania Parasites and Sandflies

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Akhoundi, Mohammad; Kuhls, Katrin; Cannet, Arnaud; Votýpka, Jan; Marty, Pierre; Delaunay, Pascal; Sereno, Denis

2016-01-01

Background The aim of this study is to describe the major evolutionary historical events among Leishmania, sandflies, and the associated animal reservoirs in detail, in accordance with the geographical evolution of the Earth, which has not been previously discussed on a large scale. Methodology and Principal Findings Leishmania and sandfly classification has always been a controversial matter, and the increasing number of species currently described further complicates this issue. Despite several hypotheses on the origin, evolution, and distribution of Leishmania and sandflies in the Old and New World, no consistent agreement exists regarding dissemination of the actors that play roles in leishmaniasis. For this purpose, we present here three centuries of research on sandflies and Leishmania descriptions, as well as a complete description of Leishmania and sandfly fossils and the emergence date of each Leishmania and sandfly group during different geographical periods, from 550 million years ago until now. We discuss critically the different approaches that were used for Leishmana and sandfly classification and their synonymies, proposing an updated classification for each species of Leishmania and sandfly. We update information on the current distribution and dispersion of different species of Leishmania (53), sandflies (more than 800 at genus or subgenus level), and animal reservoirs in each of the following geographical ecozones: Palearctic, Nearctic, Neotropic, Afrotropical, Oriental, Malagasy, and Australian. We propose an updated list of the potential and proven sandfly vectors for each Leishmania species in the Old and New World. Finally, we address a classical question about digenetic Leishmania evolution: which was the first host, a vertebrate or an invertebrate? Conclusions and Significance We propose an updated view of events that have played important roles in the geographical dispersion of sandflies, in relation to both the Leishmania species they

A Historical Overview of the Classification, Evolution, and Dispersion of Leishmania Parasites and Sandflies.

Science.gov (United States)

Akhoundi, Mohammad; Kuhls, Katrin; Cannet, Arnaud; Votýpka, Jan; Marty, Pierre; Delaunay, Pascal; Sereno, Denis

2016-03-01

The aim of this study is to describe the major evolutionary historical events among Leishmania, sandflies, and the associated animal reservoirs in detail, in accordance with the geographical evolution of the Earth, which has not been previously discussed on a large scale. Leishmania and sandfly classification has always been a controversial matter, and the increasing number of species currently described further complicates this issue. Despite several hypotheses on the origin, evolution, and distribution of Leishmania and sandflies in the Old and New World, no consistent agreement exists regarding dissemination of the actors that play roles in leishmaniasis. For this purpose, we present here three centuries of research on sandflies and Leishmania descriptions, as well as a complete description of Leishmania and sandfly fossils and the emergence date of each Leishmania and sandfly group during different geographical periods, from 550 million years ago until now. We discuss critically the different approaches that were used for Leishmana and sandfly classification and their synonymies, proposing an updated classification for each species of Leishmania and sandfly. We update information on the current distribution and dispersion of different species of Leishmania (53), sandflies (more than 800 at genus or subgenus level), and animal reservoirs in each of the following geographical ecozones: Palearctic, Nearctic, Neotropic, Afrotropical, Oriental, Malagasy, and Australian. We propose an updated list of the potential and proven sandfly vectors for each Leishmania species in the Old and New World. Finally, we address a classical question about digenetic Leishmania evolution: which was the first host, a vertebrate or an invertebrate? We propose an updated view of events that have played important roles in the geographical dispersion of sandflies, in relation to both the Leishmania species they transmit and the animal reservoirs of the parasites.

Neutrophils reduce the parasite burden in Leishmania (Leishmania amazonensis-infected macrophages.

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Erico Vinícius de Souza Carmo

2010-11-01

Full Text Available Studies on the role of neutrophils in Leishmania infection were mainly performed with L. (L major, whereas less information is available for L. (L amazonensis. Previous results from our laboratory showed a large infiltrate of neutrophils in the site of infection in a mouse strain resistant to L. (L. amazonensis (C3H/HePas. In contrast, the susceptible strain (BALB/c displayed a predominance of macrophages harboring a high number of amastigotes and very few neutrophils. These findings led us to investigate the interaction of inflammatory neutrophils with L. (L. amazonensis-infected macrophages in vitro.Mouse peritoneal macrophages infected with L. (L. amazonensis were co-cultured with inflammatory neutrophils, and after four days, the infection was quantified microscopically. Data are representative of three experiments with similar results. The main findings were 1 intracellular parasites were efficiently destroyed in the co-cultures; 2 the leishmanicidal effect was similar when cells were obtained from mouse strains resistant (C3H/HePas or susceptible (BALB/c to L. (L. amazonensis; 3 parasite destruction did not require contact between infected macrophages and neutrophils; 4 tumor necrosis factor alpha (TNF-α, neutrophil elastase and platelet activating factor (PAF were involved with the leishmanicidal activity, and 5 destruction of the parasites did not depend on generation of oxygen or nitrogen radicals, indicating that parasite clearance did not involve the classical pathway of macrophage activation by TNF-α, as reported for other Leishmania species.The present results provide evidence that neutrophils in concert with macrophages play a previously unrecognized leishmanicidal effect on L. (L. amazonensis. We believe these findings may help to understand the mechanisms involved in innate immunity in cutaneous infection by this Leishmania species.

Molecular detection of Leishmania infection due to Leishmania major and Leishmania turanica in the vectors and reservoir host in Iran.

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Rassi, Yavar; Oshaghi, Mohammad Ali; Azani, Sadegh Mohammadi; Abaie, Mohammad Reza; Rafizadeh, Sina; Mohebai, Mehdi; Mohtarami, Fatemeh; Zeinali, Mohammad kazem

2011-02-01

An epidemiological study was carried out on the vectors and reservoirs of cutaneous leishmaniasis in rural areas of Damghan district, Semnan province, central Iran, during 2008-2009. Totally, 6110 sand flies were collected using sticky papers and were subjected to molecular methods for detection of Leishmania parasite. Phlebotomus papatasi Scopoli was the common species in outdoor and indoor resting places. Polymerase chain reaction technique showed that 24 out of 218 P. papatasi (11%) and 4 out of 62 Phlebotomus caucasicus Marzinovskyi (6.5%) were positive for parasites Leishmania major Yakimoff and Schokhor. Twenty-one rodent reservoir hosts captured using Sherman traps were identified as Rhombomys opimus Lichtenstein (95%) and Meriones libycus Lichtenstein (5%). Microscopic investigation on blood smear of the animals for amastigote parasites revealed 8 (40%) rodents infected with R. opimus. L. major infection in these animals was then confirmed by polymerase chain reaction against internal transcribed spacer ribosomal DNA (rDNA) loci of the parasite followed by restriction fragment length polymorphism. Further, sequence analysis of 297 bp of ITS1-rDNA loci revealed the presence of L. major and Leishmania turanica in P. papatasi, and L. major in R. opimus. This is the first molecular report of L. major infection in both vectors (P. papatasi and P. caucasicus) and reservoir host (R. opimus) in this region. The results indicated that P. papatas was the primary vector of the disease and circulating the parasite between human and reservoirs, and P. caucasicus could be considered as a secondary vector. Further, our study showed that R. opimus is the most important host reservoir for maintenance of the parasite source in the area.

immune response in human leishmania infections Respuesta inmune en infecciones humanas por Leishmania spp

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Sara María Robledo Restrepo

2000-03-01

Full Text Available This review summarizes relevant information about the immune response triggered during leishmaniosis, a disease of great importance from the epidemiological point of view, since it is endemic in Colombia and other countries. We emphasize on human leishmaniosis; nevertheless, some important findings in the murine model are also mentioned. This information allows to conclude that Leishmania infection is a complex and coordinated process, which includes adhesion and entrance of the parasite into the host cells and its survival inside them. Events that mediate the infection process may influence its result in terms of elimination of the parasite or development of the disease, through induction or not of an effective specific immune response which involves host cell activation and parasite destruction. La presente revisión tiene como objetivo resumir la información más relevante acerca de la respuesta inmune que se desencadena durante la leishmaniosis, una enfermedad de gran importancia desde el punto de vista epidemiológico dado que es endémica en Colombia y otros países. Aunque la respuesta inmune en la leishmaniosis es un tema que se ha estudiado ampliamente en las infecciones por especies de Leishmania del Viejo Mundo, particularmente Leishmania major y Leishmania donovani y en el modelo murino, la presente revisión hace énfasis en la leishmaniosis humana. Algunos hallazgos importantes en el modelo murino también se mencionan. La información contenida en la revisión, en su mayoría, proviene de publicaciones derivadas de investigaciones, las cuales se seleccionaron con base en la calidad del trabajo realizado y en los aportes de sus resultados en el avance del conocimiento sobre las infecciones en humanos. La síntesis de la información seleccionada nos permite concluir que la infección por Leishmania es un proceso complejo y coordinado que incluye la adherencia y entrada del parásito a la célula hospedera y su posterior

Innate Immunity to Leishmania Infection: Within Phagocytes

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Marcela Freitas Lopes

2014-01-01

Full Text Available Infection by Leishmania takes place in the context of inflammation and tissue repair. Besides tissue resident macrophages, inflammatory macrophages and neutrophils are recruited to the infection site and serve both as host cells and as effectors against infection. Recent studies suggest additional important roles for monocytes and dendritic cells. This paper addresses recent experimental findings regarding the regulation of Leishmania major infection by these major phagocyte populations. In addition, the role of IL-4 on dendritic cells and monocytes is discussed.

LeishCyc: a biochemical pathways database for Leishmania major

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Doyle Maria A

2009-06-01

Full Text Available Abstract Background Leishmania spp. are sandfly transmitted protozoan parasites that cause a spectrum of diseases in more than 12 million people worldwide. Much research is now focusing on how these parasites adapt to the distinct nutrient environments they encounter in the digestive tract of the sandfly vector and the phagolysosome compartment of mammalian macrophages. While data mining and annotation of the genomes of three Leishmania species has provided an initial inventory of predicted metabolic components and associated pathways, resources for integrating this information into metabolic networks and incorporating data from transcript, protein, and metabolite profiling studies is currently lacking. The development of a reliable, expertly curated, and widely available model of Leishmania metabolic networks is required to facilitate systems analysis, as well as discovery and prioritization of new drug targets for this important human pathogen. Description The LeishCyc database was initially built from the genome sequence of Leishmania major (v5.2, based on the annotation published by the Wellcome Trust Sanger Institute. LeishCyc was manually curated to remove errors, correct automated predictions, and add information from the literature. The ongoing curation is based on public sources, literature searches, and our own experimental and bioinformatics studies. In a number of instances we have improved on the original genome annotation, and, in some ambiguous cases, collected relevant information from the literature in order to help clarify gene or protein annotation in the future. All genes in LeishCyc are linked to the corresponding entry in GeneDB (Wellcome Trust Sanger Institute. Conclusion The LeishCyc database describes Leishmania major genes, gene products, metabolites, their relationships and biochemical organization into metabolic pathways. LeishCyc provides a systematic approach to organizing the evolving information about Leishmania

Leishmania mexicana Gp63 cDNA Using Gene Gun Induced Higher Immunity to L. mexicana Infection Compared to Soluble Leishmania Antigen in BALB/C

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Rezvan, H; Rees, R; Ali, SA

2011-01-01

Background Leishmaniasis is a worldwide disease prevalent in tropical and sub tropical countries. Many attempts have been made and different strategies have been approached to develop a potent vaccine against Leishmania. DNA immunisation is a method, which is shown to be effective in Leishmania vaccination. Leishmania Soluble Antigen (SLA) has also recently been used Leishmania vaccination. Methods The immunity generated by SLA and L. mexicana gp63 cDNA was compared in groups of 6 mice, which were statistically analysed by student t- test with the P-value of 0.05. SLA was administered by two different methods; intramuscular injection and injection of dendritic cells (DCs) loaded with SLA. L. mexicana gp63 cDNA was administered by the gene gun. Results Immunisation of BALB/c mice with L. mexicana gp63 resulted in high levels of Th1-type immune response and cytotoxic T lymphocytes (CTL) activity, which were accompanied with protection induced by the immunisation against L. mexicana infection. In contrast, administration of SLA, produced a mixed Th1/Th2-type immune responses as well as a high level of CTL activity but did not protect mice from the infection. Conclusion The results indicate higher protection by DNA immunisation using L. mexicana gp63 cDNA compared to SLA, which is accompanied by a high level of Th1 immune response. However, the CTL activity does not necessarily correlate with the protection induced by the vaccine. Also, gene gun immunisation is a potential approach in Leishmania vaccination. These findings would be helpful in opening new windows in Leishmania vaccine research. PMID:22347315

Leishmania serology in the diagnosis of cutaneous leishmaniasis

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Mashood, A.H.; Malik, N.; Abbasi, S.

2013-01-01

Background: The gold standard to diagnose cutaneous leishmaniasis is histopathology, but there has always been a need of a rapid, reliable, cheap and convenient laboratory investigation. Serological tests fulfill the above criteria. Objective: The objective of the study was to determine the sensitivity and specificity of enzyme linked immunosorbent assay (ELISA) in detection of leishmania antibodies, in comparison with the histopathology. Place and duration of study: The study was conducted in Military Hospital Rawalpindi from 1st November 2010 to 30th June 2011. Patients and methods: The study population included the patients who were clinically diagnosed with cutaneous leishmaniasis. All of them were biopsied and serum was sent for leishmania serology. Results: A total of 47 patients were included. They were all adult males. The histopathology was positive in 31/47 patients (65.95%), while the leishmania serology was positive in 36/47 cases (76.59%). The sensitiuites was 74.19%, specificity was 18.75%, positive predictive value has 63.88%, negative predicative value was 27% and accuracy was 55%. Conclusion: In the light of sensitivity analysis, it may be concluded that leishmania serology has moderate sensitivity and low specificity; hence it is not a reliable test for cutaneous leishmaniasis. (author)

Identification of geographically distributed sub-populations of Leishmania (Leishmania major by microsatellite analysis

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Schwenkenbecher Jan

2008-06-01

Full Text Available Abstract Background Leishmania (Leishmania major, one of the agents causing cutaneous leishmaniasis (CL in humans, is widely distributed in the Old World where different species of wild rodent and phlebotomine sand fly serve as animal reservoir hosts and vectors, respectively. Despite this, strains of L. (L. major isolated from many different sources over many years have proved to be relatively uniform. To investigate the population structure of the species highly polymorphic microsatellite markers were employed for greater discrimination among it's otherwise closely related strains, an approach applied successfully to other species of Leishmania. Results Multilocus Microsatellite Typing (MLMT based on 10 different microsatellite markers was applied to 106 strains of L. (L. major from different regions where it is endemic. On applying a Bayesian model-based approach, three main populations were identified, corresponding to three separate geographical regions: Central Asia (CA; the Middle East (ME; and Africa (AF. This was congruent with phylogenetic reconstructions based on genetic distances. Re-analysis separated each of the populations into two sub-populations. The two African sub-populations did not correlate well with strains' geographical origin. Strains falling into the sub-populations CA and ME did mostly group according to their place of isolation although some anomalies were seen, probably, owing to human migration. Conclusion The model- and distance-based analyses of the microsatellite data exposed three main populations of L. (L. major, Central Asia, the Middle East and Africa, each of which separated into two sub-populations. This probably correlates with the different species of rodent host.

Genome-wide mapping reveals single-origin chromosome replication in Leishmania, a eukaryotic microbe.

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Marques, Catarina A; Dickens, Nicholas J; Paape, Daniel; Campbell, Samantha J; McCulloch, Richard

2015-10-19

DNA replication initiates on defined genome sites, termed origins. Origin usage appears to follow common rules in the eukaryotic organisms examined to date: all chromosomes are replicated from multiple origins, which display variations in firing efficiency and are selected from a larger pool of potential origins. To ask if these features of DNA replication are true of all eukaryotes, we describe genome-wide origin mapping in the parasite Leishmania. Origin mapping in Leishmania suggests a striking divergence in origin usage relative to characterized eukaryotes, since each chromosome appears to be replicated from a single origin. By comparing two species of Leishmania, we find evidence that such origin singularity is maintained in the face of chromosome fusion or fission events during evolution. Mapping Leishmania origins suggests that all origins fire with equal efficiency, and that the genomic sites occupied by origins differ from related non-origins sites. Finally, we provide evidence that origin location in Leishmania displays striking conservation with Trypanosoma brucei, despite the latter parasite replicating its chromosomes from multiple, variable strength origins. The demonstration of chromosome replication for a single origin in Leishmania, a microbial eukaryote, has implications for the evolution of origin multiplicity and associated controls, and may explain the pervasive aneuploidy that characterizes Leishmania chromosome architecture.

Unraveling the genetic diversity and phylogeny of Leishmania RNA virus 1 strains of infected Leishmania isolates circulating in French Guiana.

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Tirera, Sourakhata; Ginouves, Marine; Donato, Damien; Caballero, Ignacio S; Bouchier, Christiane; Lavergne, Anne; Bourreau, Eliane; Mosnier, Emilie; Vantilcke, Vincent; Couppié, Pierre; Prevot, Ghislaine; Lacoste, Vincent

2017-07-01

Leishmania RNA virus type 1 (LRV1) is an endosymbiont of some Leishmania (Vianna) species in South America. Presence of LRV1 in parasites exacerbates disease severity in animal models and humans, related to a disproportioned innate immune response, and is correlated with drug treatment failures in humans. Although the virus was identified decades ago, its genomic diversity has been overlooked until now. We subjected LRV1 strains from 19 L. (V.) guyanensis and one L. (V.) braziliensis isolates obtained from cutaneous leishmaniasis samples identified throughout French Guiana with next-generation sequencing and de novo sequence assembly. We generated and analyzed 24 unique LRV1 sequences over their full-length coding regions. Multiple alignment of these new sequences revealed variability (0.5%-23.5%) across the entire sequence except for highly conserved motifs within the 5' untranslated region. Phylogenetic analyses showed that viral genomes of L. (V.) guyanensis grouped into five distinct clusters. They further showed a species-dependent clustering between viral genomes of L. (V.) guyanensis and L. (V.) braziliensis, confirming a long-term co-evolutionary history. Noteworthy, we identified cases of multiple LRV1 infections in three of the 20 Leishmania isolates. Here, we present the first-ever estimate of LRV1 genomic diversity that exists in Leishmania (V.) guyanensis parasites. Genetic characterization and phylogenetic analyses of these viruses has shed light on their evolutionary relationships. To our knowledge, this study is also the first to report cases of multiple LRV1 infections in some parasites. Finally, this work has made it possible to develop molecular tools for adequate identification and genotyping of LRV1 strains for diagnostic purposes. Given the suspected worsening role of LRV1 infection in the pathogenesis of human leishmaniasis, these data have a major impact from a clinical viewpoint and for the management of Leishmania-infected patients.

NKT cell activation by Leishmania mexicana LPG: Description of a novel pathway.

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Zamora-Chimal, Jaime; Fernández-Figueroa, Edith A; Ruiz-Remigio, Adriana; Wilkins-Rodríguez, Arturo A; Delgado-Domínguez, José; Salaiza-Suazo, Norma; Gutiérrez-Kobeh, Laila; Becker, Ingeborg

2017-02-01

NKT cells have been associated with protection against Leishmania donovani, yet their role in infections with Leishmania mexicana has not been addressed, nor has the activation pathway been defined after stimulation with Leishmania mexicana lipophosphoglycan (LPG). We analyzed the activation of NKT cells and their cytokine production in response to Leishmania mexicana LPG. Additionally we compared NKT-cell numbers and cytokine profile in lymph nodes of skin lesions induced by Leishmania mexicana in BALB/c and C57BL/6 mice. We show that LPG activates NKT cells primarily through the indirect pathway, initiating with TLR2 stimulation of dendritic cells (DC), thereby enhancing TLR2, MHC II, and CD86 expressions and IL-12p70 production. This leads to IFN-γ production by NKT cells. C57BL/6 mice showed enhanced DC activation, which correlated with augmented IFN-γ production by NKT cells. Additionally, infected C57BL/6 mice showed elevated percentages of NKT cells with higher IFN-γ and IL-4 production in lymph nodes. We conclude that the response of NKT cells towards Leishmania mexicana LPG initiates with the indirect activation, after binding of LPG to TLR2 in DC. This indirect activation pathway enables NKT cells to produce IFN-γ during the innate phase of Leishmania infection, the magnitude of which differs between mouse strains. Copyright © 2016 The Authors. Published by Elsevier GmbH.. All rights reserved.

Dihydrotestosterone enhances growth and infectivity of Leishmania Mexicana.

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Sánchez-García, L; Wilkins-Rodriguez, A; Salaiza-Suazo, N; Morales-Montor, J; Becker, I

2018-03-01

A strong sex-associated susceptibility towards Leishmania has been reported in males, yet little is known on the effect of hormones in Leishmania physiopathogenicity. Due to the enhanced susceptibility of males to Leishmania mexicana infections, we were interested in analysing the effect exerted by the main androgen produced in males (DHT) on L. mexicana promastigotes. Thus, the aim of this study was to assess the regulation exerted by dihydrotestosterone (DHT) on L. mexicana replication, infectivity, survival and development of tissue lesions. Experiments included growth curves of L. mexicana promastigotes incubated with different doses of DHT, their infection rate, intracellular survival and lesion development in BALB/c mice. Our data show that DHT significantly enhances parasite replication, infection rate and survival in bone marrow-derived macrophages (BMMФ). Promastigotes in the presence of DHT produced significantly larger lesions in BALB/c earlobes. These results suggest that DHT probably plays a critical role during L. mexicana infections, and the higher susceptibility of males possibly relates to benefits gained by the parasite from host-derived hormones. Our data shed new light on the physiopathology of Leishmania infections and are the first attempt to understand the direct interaction between Leishmania and androgens, particularly DHT. Understanding this trans-regulation process employed by parasites to exploit host molecules sheds new light on L. mexicana physiopathogenesis and opens a possible field for studies on drug development. © 2017 John Wiley & Sons Ltd.

Transport and sorting of sphingolipids in polarized cells : the involvement of the sub-apical compartment

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IJzendoorn, Sven Christian David van

1999-01-01

The work described in this thesis has provided a novel insight into the process of sphingolipid transport and sorting in polarized cells. We have used HepG2 cells as a model system to study polarized traffic in hepatic cells. Under specific culture conditions, HepG2 cells acquire a polarized

T-cell responses associated with resistance to Leishmania infection in individuals from endemic areas for Leishmania (Viannia braziliensis

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Rita C Bittar

2007-08-01

Full Text Available Subclinical or asymptomatic infection is documented in individuals living in endemic areas for leishmaniasis suggesting that the development of an appropriate immune response can control parasite replication and maintain tissue integrity. A low morbidity indicates that intrinsic factors could favor resistance to Leishmania infection. Herein, leishmanial T-cell responses induced in subjects with low susceptibility to leishmaniasis as asymptomatic subjects were compared to those observed in cured cutaneous leishmaniasis (CCL patients, who controlled the disease after antimonial therapy. All of them have shown maintenance of specific long-term immune responses characterized by expansion of higher proportions of CD4+ as compared to CD8+ Leishmania reactive T-lymphocytes. Asymptomatic subjects had lower indexes of in vitro Leishmania induced lymphoproliferative responses and interferon-gamma (IFN-gamma production in comparison to CCL patients. On the other hand, interleukin (IL-10 production was much higher in asymptomatics than in CCL, while no differences in IL-5 levels were found. In conclusion, long lived T-cell responses achieved by asymptomatic individuals differed from those who had developed symptomatic leishmaniasis in terms of intensity of lymphocyte activation (proliferation or IFN-gamma and regulatory mechanisms (IL-10. The absence of the disease in asymptomatics could be explained by their intrinsic ability to create a balance between immunoregulatory (IL-10 and effector cytokines (IFN-gamma, leading to parasite destruction without producing skin tissue damage. The establishment of profiles of cell-mediated immune responses associated with resistance against Leishmania infection is likely to make new inroads into understanding the long-lived immune protection against the disease.

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

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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.

Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania*

Science.gov (United States)

Bahl, Surbhi; Parashar, Smriti; Malhotra, Himanshu; Raje, Manoj; Mukhopadhyay, Amitabha

2015-01-01

Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania. PMID:26499792

Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania.

Science.gov (United States)

Bahl, Surbhi; Parashar, Smriti; Malhotra, Himanshu; Raje, Manoj; Mukhopadhyay, Amitabha

2015-12-11

Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Preparation of live attenuated leishmania parasites by using laser technology

Science.gov (United States)

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

Monocyte Chemotactic Protein 1 in Plasma from Soluble Leishmania Antigen-Stimulated Whole Blood as a Potential Biomarker of the Cellular Immune Response to Leishmania infantum

Directory of Open Access Journals (Sweden)

Ana V. Ibarra-Meneses

2017-09-01

Full Text Available New biomarkers are needed to identify asymptomatic Leishmania infection as well as immunity following vaccination or treatment. With the aim of finding a robust biomarker to assess an effective cellular immune response, monocyte chemotactic protein 1 (MCP-1 was examined in plasma from soluble Leishmania antigen (SLA-stimulated whole blood collected from subjects living in a Leishmania infantum-endemic area. MCP-1, expressed 110 times more strongly than IL-2, identified 87.5% of asymptomatic subjects and verified some asymptomatic subjects close to the cutoff. MCP-1 was also significantly elevated in all patients cured of visceral leishmaniasis (VL, unlike IL-2, indicating the specific memory response generated against Leishmania. These results show MCP-1 to be a robust candidate biomarker of immunity that could be used as a marker of cure and to both select and follow the population in vaccine phase I–III human clinical trials with developed rapid, easy-to-use field tools.

DSFL database: A hub of target proteins of Leishmania sp. to combat leishmaniasis

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Ameer Khusro

2017-07-01

Full Text Available Leishmaniasis is a vector-borne chronic infectious tropical dermal disease caused by the protozoa parasite of the genus Leishmania that causes high mortality globally. Among three different clinical forms of leishmaniasis, visceral leishmaniasis (VL or kala-azar is a systemic public health disease with high morbidity and mortality in developing countries, caused by Leishmania donovani, Leishmania infantum or Leishmania chagasi. Unfortunately, there is no vaccine available till date for the treatment of leishmaniasis. On the other hand, the therapeutics approved to treat this fatal disease is expensive, toxic, and associated with serious side effects. Furthermore, the emergence of drug-resistant Leishmania parasites in most endemic countries due to the incessant utilization of existing drugs is a major concern at present. Drug Search for Leishmaniasis (DSFL is a unique database that involves 50 crystallized target proteins of varied Leishmania sp. in order to develop new drugs in future by interacting several antiparasitic compounds or molecules with specific protein through computational tools. The structure of target protein from different Leishmania sp. is available in this database. In this review, we spotlighted not only the current global status of leishmaniasis in brief but also detailed information about target proteins of various Leishmania sp. available in DSFL. DSFL has created a new expectation for mankind in order to combat leishmaniasis by targeting parasitic proteins and commence a new era to get rid of drug resistance parasites. The database will substantiate to be a worthwhile project for further development of new, non-toxic, and cost-effective antileishmanial drugs as targeted therapies using in vitro/in vivo assays.

Molecular detection of Leishmania infantum and Leishmania tropica in rodent species from endemic cutaneous leishmaniasis areas in Morocco.

Science.gov (United States)

Echchakery, Mohamed; Chicharro, Carmen; Boussaa, Samia; Nieto, Javier; Carrillo, Eugenia; Sheila, Ortega; Moreno, Javier; Boumezzough, Ali

2017-10-02

Leishmaniasis remains a major public health problem in African nations, including Morocco, where little is known about the vertebrate reservoirs involved in the causal parasites' transmission cycles. The present study investigates the role of rodent species as potential reservoirs of Leishmania spp. in central Morocco, where both L. tropica and L. infantum have been reported. Rodents were caught from 22 sites in central Morocco, by using Sherman metal traps, and identified morphologically. For each specimen, genomic DNA was extracted from different tissues using the Speed Tools DNA extraction Kit. Then, samples were PCR-analyzed, targeting the SSU rRNA gene to detect Leishmania spp. DNA, followed by amplification of the internal transcribed spacer 1 (ITS1) and its sequencing to identify the species. A total of 197 rodents belonging to ten species were captured and identified: Rattus rattus (40.61%), Mus musculus (25.38%), Apodemus sylvaticus (8.63%), Mus spretus (7.11%), Meriones shawi (5.58%), Rattus norvegicus (4.57%), Meriones libycus (3.05%), Mastomys erythroleucus (2.03%), Gerbillus campestris (2.03%) and Lemniscomys barbarus (1.01%). Molecular analysis revealed the presence of Leishmania species in 18 specimens: six R. rattus (out of 80 captured; 7.5%), 11 M. musculus (out of 50 captured; 22%), and one R. norvegicus (out of 9 captured; 11.11%). To the best of our knowledge, L. infantum and L. tropica were identified in rodent species for the first time in Morocco. These findings suggest that rodent species may be involved in L. infantum and L. tropica transmission cycles in this country but that further studies are needed to confirm their role as reservoirs of Leishmania species in Morocco.

Serological and molecular survey of Leishmania parasites in apparently healthy dogs in the West Bank, Palestine

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Hamarsheh Omar

2012-08-01

Full Text Available Abstract Background Canine visceral leishmaniasis (CVL is caused by Leishmania infantum in all Mediterranean countries. The Leishmania parasite is transmitted by the bite of a corresponding sand fly vector and primarily maintained in nature by wild and domestic reservoirs, including dogs, foxes and jackals. Infected dogs are the primary reservoir host in endemic regions and are the most significant risk disposing humans to infection. The present study aimed at assessing the prevalence of infection with Leishmania and identification of Leishmania infantum in domestic dogs in the West Bank, Palestine. Methods The infection rate among domestic dogs collected from seven districts in the Palestinian West Bank was investigated by examination of parasites in culture from the buffy coat using serological and molecular methods; based on ELISA, internal transcribed spacer 1 (ITS1 and cysteine protease (CPB PCR. Results Out of 215 dogs examined for Leishmania, 36 (16.7% were positive in at least one method. Twenty three animals (11.5% were positive for Leishmania DNA, whereas, ELISA and culture revealed 16 (7.5%, and 4 (1.5% respectively. CPB-PCR on one of three culture-positive isolates revealed Leishmania infantum as the causative agent for Leishmania infection in dogs. Conclusions Our study showed that canine leishmania infection is prevalent with varying degrees in all the seven studied districts in Palestine despite the absence of human VL cases in 4 of these districts. The causative agent was confirmed to be Leishmania infantum.

Identification of Tunisian Leishmania spp. by PCR amplification of cysteine proteinase B (cpb) genes and phylogenetic analysis.

Science.gov (United States)

Chaouch, Melek; Fathallah-Mili, Akila; Driss, Mehdi; Lahmadi, Ramzi; Ayari, Chiraz; Guizani, Ikram; Ben Said, Moncef; Benabderrazak, Souha

2013-03-01

Discrimination of the Old World Leishmania parasites is important for diagnosis and epidemiological studies of leishmaniasis. We have developed PCR assays that allow the discrimination between Leishmania major, Leishmania tropica and Leishmania infantum Tunisian species. The identification was performed by a simple PCR targeting cysteine protease B (cpb) gene copies. These PCR can be a routine molecular biology tools for discrimination of Leishmania spp. from different geographical origins and different clinical forms. Our assays can be an informative source for cpb gene studying concerning drug, diagnostics and vaccine research. The PCR products of the cpb gene and the N-acetylglucosamine-1-phosphate transferase (nagt) Leishmania gene were sequenced and aligned. Phylogenetic trees of Leishmania based cpb and nagt sequences are close in topology and present the classic distribution of Leishmania in the Old World. The phylogenetic analysis has enabled the characterization and identification of different strains, using both multicopy (cpb) and single copy (nagt) genes. Indeed, the cpb phylogenetic analysis allowed us to identify the Tunisian Leishmania killicki species, and a group which gathers the least evolved isolates of the Leishmania donovani complex, that was originated from East Africa. This clustering confirms the African origin for the visceralizing species of the L. donovani complex. Copyright © 2012 Elsevier B.V. All rights reserved.

LR1: a candidate RNA virus of Leishmania.

OpenAIRE

Tarr, P I; Aline, R F; Smiley, B L; Scholler, J; Keithly, J; Stuart, K

1988-01-01

Although viruses are important biological agents and useful molecular tools, little is known about the viruses of parasites. We report here the discovery of a candidate for an RNA virus in a kinetoplastid parasite. This potential virus, which we term LR1, is present in the promastigote form of the human pathogen Leishmania braziliensis guyanensis CUMC1-1A but not in 11 other stocks of Leishmania that were examined nor in Trypanosoma brucei. The candidate viral RNA has a size of approximately ...

Characterization of Leishmania Soluble Exo-Antigen

National Research Council Canada - National Science Library

Cui, Liwang

2003-01-01

.... Vaccine development is the ultimate solution for this problem. Our previous research indicates that Leishmania parasites secrete, excrete, or shed antigens into the medium during in vitro culture...

Leishmania (L. mexicana infected bats in Mexico: novel potential reservoirs.

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Miriam Berzunza-Cruz

2015-01-01

Full Text Available Leishmania (Leishmania mexicana causes cutaneous leishmaniasis, an endemic zoonosis affecting a growing number of patients in the southeastern states of Mexico. Some foci are found in shade-grown cocoa and coffee plantations, or near perennial forests that provide rich breeding grounds for the sand fly vectors, but also harbor a variety of bat species that live off the abundant fruits provided by these shade-giving trees. The close proximity between sand flies and bats makes their interaction feasible, yet bats infected with Leishmania (L. mexicana have not been reported. Here we analyzed 420 bats from six states of Mexico that had reported patients with leishmaniasis. Tissues of bats, including skin, heart, liver and/or spleen were screened by PCR for Leishmania (L. mexicana DNA. We found that 41 bats (9.77%, belonging to 13 species, showed positive PCR results in various tissues. The infected tissues showed no evidence of macroscopic lesions. Of the infected bats, 12 species were frugivorous, insectivorous or nectarivorous, and only one species was sanguivorous (Desmodus rotundus, and most of them belonged to the family Phyllostomidae. The eco-region where most of the infected bats were caught is the Gulf Coastal Plain of Chiapas and Tabasco. Through experimental infections of two Tadarida brasiliensis bats in captivity, we show that this species can harbor viable, infective Leishmania (L. mexicana parasites that are capable of infecting BALB/c mice. We conclude that various species of bats belonging to the family Phyllostomidae are possible reservoir hosts for Leishmania (L. mexicana, if it can be shown that such bats are infective for the sand fly vector. Further studies are needed to determine how these bats become infected, how long the parasite remains viable inside these potential hosts and whether they are infective to sand flies to fully evaluate their impact on disease epidemiology.

Leishmania (L.) mexicana Infected Bats in Mexico: Novel Potential Reservoirs

Science.gov (United States)

Berzunza-Cruz, Miriam; Rodríguez-Moreno, Ángel; Gutiérrez-Granados, Gabriel; González-Salazar, Constantino; Stephens, Christopher R.; Hidalgo-Mihart, Mircea; Marina, Carlos F.; Rebollar-Téllez, Eduardo A.; Bailón-Martínez, Dulce; Balcells, Cristina Domingo; Ibarra-Cerdeña, Carlos N.; Sánchez-Cordero, Víctor; Becker, Ingeborg

2015-01-01

Leishmania (Leishmania) mexicana causes cutaneous leishmaniasis, an endemic zoonosis affecting a growing number of patients in the southeastern states of Mexico. Some foci are found in shade-grown cocoa and coffee plantations, or near perennial forests that provide rich breeding grounds for the sand fly vectors, but also harbor a variety of bat species that live off the abundant fruits provided by these shade-giving trees. The close proximity between sand flies and bats makes their interaction feasible, yet bats infected with Leishmania (L.) mexicana have not been reported. Here we analyzed 420 bats from six states of Mexico that had reported patients with leishmaniasis. Tissues of bats, including skin, heart, liver and/or spleen were screened by PCR for Leishmania (L.) mexicana DNA. We found that 41 bats (9.77%), belonging to 13 species, showed positive PCR results in various tissues. The infected tissues showed no evidence of macroscopic lesions. Of the infected bats, 12 species were frugivorous, insectivorous or nectarivorous, and only one species was sanguivorous (Desmodus rotundus), and most of them belonged to the family Phyllostomidae. The eco-region where most of the infected bats were caught is the Gulf Coastal Plain of Chiapas and Tabasco. Through experimental infections of two Tadarida brasiliensis bats in captivity, we show that this species can harbor viable, infective Leishmania (L.) mexicana parasites that are capable of infecting BALB/c mice. We conclude that various species of bats belonging to the family Phyllostomidae are possible reservoir hosts for Leishmania (L.) mexicana, if it can be shown that such bats are infective for the sand fly vector. Further studies are needed to determine how these bats become infected, how long the parasite remains viable inside these potential hosts and whether they are infective to sand flies to fully evaluate their impact on disease epidemiology. PMID:25629729

Disruption of sphingolipid biosynthesis in hepatocyte nodules: selective proliferative stimulus induced by fumonisin B{sub 1}

Energy Technology Data Exchange (ETDEWEB)

Westhuizen, Liana van der; Gelderblom, Wentzel C.A.; Shephard, Gordon S; Swanevelder, Sonja

2004-07-15

In order to investigate the role of sphingolipid disruption in the cancer promoting potential of fumonisin B{sub 1} (FB{sub 1}) in the development of hepatocyte nodules, male Fischer 344 rats were subjected to cancer initiation (FB{sub 1} containing diet or diethylnitrosamine (DEN) by i.p. injection) and promotion (2-acetylaminofluorene with partial hepatectomy, 2-AAF/PH) treatments followed by a secondary FB{sub 1} dietary regimen. Sphinganine (Sa) and sphingosine (So) levels were measured by high performance liquid chromatography in control, surrounding and nodular liver tissues of the rats. The disruption of sphingolipid biosynthesis by the secondary FB{sub 1} treatment in the control rats was significantly (P<0.05) enhanced by the 2-AAF/PH cancer promotion treatment. The nodular and surrounding Sa levels returned to baseline following FB{sub 1} initiation and 2-AAF/PH promotion. When comparing the groups subjected to the secondary FB{sub 1} treatment, the initiation effected by FB{sub 1} was less (P<0.01) sensitive to the accumulation of Sa in the nodular and surrounding tissues than DEN initiation and the 2-AAF/PH control treatment. In contrast, the So level of FB{sub 1} initiation was marginally increased in the nodules compared to the surrounding liver after 2-AAF/PH promotion and significantly (P<0.05) higher with the secondary FB{sub 1} treatment. Although, the FB{sub 1}-induced hepatocyte nodules were not resistant to the disruption of sphingolipid biosynthesis, the nodular So levels were increased and might provide a selective growth stimulus possibly induced by bio-active sphingoid intermediates such as sphingosine 1-phosphate (S1P)

Estudo histológico e parasitológico do trato gastrintestinal de cães infectados com Leishmania (Leishmania) chagasi

OpenAIRE

Aldair Junio Woyames Pinto

2011-01-01

São poucas as descrições das alterações patológicas e parasitológicas relacionadas ao envolvimento do trato gastrointestinal (TGI) na leishmaniose visceral canina e, sobretudo considerando-se o TGI de forma sistemática. Assim, neste trabalho objetivou-se um estudo sistemático, clínico, anatomopatológico e parasitológico do TGI de cães naturalmente infectados com Leishmania (Leishmania) chagasi provenientes da região metropolitana de Belo Horizonte, MG. Após confirmação sorológica (RIFI e ELIS...

Regulation of Flavonoid Biosynthetic Genes in Germinating Arabidopsis Seedlings.

Science.gov (United States)

Kubasek, WL; Shirley, BW; McKillop, A; Goodman, HM; Briggs, W; Ausubel, FM

1992-01-01

Many higher plants, including Arabidopsis, transiently display purple anthocyanin pigments just after seed germination. We observed that steady state levels of mRNAs encoded by four flavonoid biosynthetic genes, PAL1 (encoding phenylalanine ammonia-lyase 1), CHS (encoding chalcone synthase), CHI (encoding chalcone isomerase), and DFR (encoding dihydroflavonol reductase), were temporally regulated, peaking in 3-day-old seedlings grown in continuous white light. Except for the case of PAL1 mRNA, mRNA levels for these flavonoid genes were very low in seedlings grown in darkness. Light induction studies using seedlings grown in darkness showed that PAL1 mRNA began to accumulate before CHS and CHI mRNAs, which, in turn, began to accumulate before DFR mRNA. This order of induction is the same as the order of the biosynthetic steps in flavonoid biosynthesis. Our results suggest that the flavonoid biosynthetic pathway is coordinately regulated by a developmental timing mechanism during germination. Blue light and UVB light induction experiments using red light- and dark-grown seedlings showed that the flavonoid biosynthetic genes are induced most effectively by UVB light and that blue light induction is mediated by a specific blue light receptor. PMID:12297632

Anti-Leishmania and cytotoxic activities of perillaldehyde epoxide synthetic positional isomers.

Science.gov (United States)

Keesen, Tatjana Souza Lima; da Silva, Larisse Virgolino; da Câmara Rocha, Juliana; Andrade, Luciana Nalone; Lima, Tamires Cardoso; de Sousa, Damião Pergentino

2018-03-13

Leishmaniasis belongs to a complex of zoonotic disease caused by protozoa of the genus Leishmania and is considered a major public health problem. Several essential oil chemical components have inhibitory effect against protozoa, including Leishmania donovani. Thus, the aim of this study was to evaluate for the first time the anti-Leishmania activity of two p-menthane monoterpene isomers (EPER-1: perillaldehyde 1,2-epoxide and EPER-2: perillaldehyde 8,9-epoxide) against L. donovani promastigotes as well as evaluating cytotoxic effect on mononuclear peripheral blood cells. Results of anti-Leishmania assay revealed that EPER-2 (IC 50  = 3.8 μg.mL -1 ) was 16-fold more potent than its isomer EPER-1 (IC 50  = 64.6 μg.mL -1 ). In contrast to PBMC cells, EPER-2 was not cytotoxic (IC 50  > 400 μg.mL -1 ) when compared to positive control. These data suggest that the disposition of epoxide group into the p-menthane skeleton affects the anti-Leishmania activity, being that the presence of the exocyclic epoxide group considerably increased potency. Thus, it was possible to observe that the location of the epoxide group into the p-menthane skeleton resulted in different potencies.

Leishmania exosomes and other virulence factors: Impact on innate immune response and macrophage functions.

Science.gov (United States)

Atayde, Vanessa Diniz; Hassani, Kasra; da Silva Lira Filho, Alonso; Borges, Andrezza Raposo; Adhikari, Anupam; Martel, Caroline; Olivier, Martin

2016-11-01

Leishmania parasites are the causative agents of the leishmaniases, a collection of vector-borne diseases that range from simple cutaneous to fatal visceral forms. Employing potent immune modulation mechanisms, Leishmania is able to render the host macrophage inactive and persist inside its phagolysosome. In the last few years, the role of exosomes in Leishmania-host interactions has been increasingly investigated. For instance, it was reported that Leishmania exosome release is augmented following temperature shift, a condition mimicking parasite's entry into its mammalian host. Leishmania exosomes were found to strongly affect macrophage cell signaling and functions, similarly to whole parasites. Importantly, these vesicles were shown to be pro-inflammatory, capable to recruit neutrophils at their inoculation site exacerbating the pathology. In this review, we provide the most recent insights on the role of exosomes and other virulence factors, especially the surface protease GP63, in Leishmania-host interactions, deepening our knowledge on leishmaniasis and paving the way for the development of new therapeutics. Copyright © 2016 Elsevier Inc. All rights reserved.

The Dynamics of Lateral Gene Transfer in Genus Leishmania - A Route for Adaptation and Species Diversification

Science.gov (United States)

Vikeved, Elisabet; Backlund, Anders; Alsmark, Cecilia

2016-01-01

Background The genome of Leishmania major harbours a comparably high proportion of genes of prokaryote origin, acquired by lateral gene transfer (LGT). Some of these are present in closely related trypanosomatids, while some are detected in Leishmania only. We have evaluated the impact and destiny of LGT in genus Leishmania. Methodology/Principal Findings To study the dynamics and fate of LGTs we have performed phylogenetic, as well as nucleotide and amino acid composition analyses within orthologous groups of LGTs detected in Leishmania. A set of universal trypanosomatid LGTs was added as a reference group. Both groups of LGTs have, to some extent, ameliorated to resemble the recipient genomes. However, while virtually all of the universal trypanosomatid LGTs are distributed and conserved in the entire genus Leishmania, the LGTs uniquely present in genus Leishmania are more prone to gene loss and display faster rates of evolution. Furthermore, a PCR based approach has been employed to ascertain the presence of a set of twenty LGTs uniquely present in genus Leishmania, and three universal trypanosomatid LGTs, in ten additional strains of Leishmania. Evolutionary rates and predicted expression levels of these LGTs have also been estimated. Ten of the twenty LGTs are distributed and conserved in all species investigated, while the remainder have been subjected to modifications, or undergone pseudogenization, degradation or loss in one or more species. Conclusions/Significance LGTs unique to the genus Leishmania have been acquired after the divergence of Leishmania from the other trypanosomatids, and are evolving faster than their recipient genomes. This implies that LGT in genus Leishmania is a continuous and dynamic process contributing to species differentiation and speciation. This study also highlights the importance of carefully evaluating these dynamic genes, e.g. as LGTs have been suggested as potential drug targets. PMID:26730948

The Dynamics of Lateral Gene Transfer in Genus Leishmania - A Route for Adaptation and Species Diversification.

Science.gov (United States)

Vikeved, Elisabet; Backlund, Anders; Alsmark, Cecilia

2016-01-01

The genome of Leishmania major harbours a comparably high proportion of genes of prokaryote origin, acquired by lateral gene transfer (LGT). Some of these are present in closely related trypanosomatids, while some are detected in Leishmania only. We have evaluated the impact and destiny of LGT in genus Leishmania. To study the dynamics and fate of LGTs we have performed phylogenetic, as well as nucleotide and amino acid composition analyses within orthologous groups of LGTs detected in Leishmania. A set of universal trypanosomatid LGTs was added as a reference group. Both groups of LGTs have, to some extent, ameliorated to resemble the recipient genomes. However, while virtually all of the universal trypanosomatid LGTs are distributed and conserved in the entire genus Leishmania, the LGTs uniquely present in genus Leishmania are more prone to gene loss and display faster rates of evolution. Furthermore, a PCR based approach has been employed to ascertain the presence of a set of twenty LGTs uniquely present in genus Leishmania, and three universal trypanosomatid LGTs, in ten additional strains of Leishmania. Evolutionary rates and predicted expression levels of these LGTs have also been estimated. Ten of the twenty LGTs are distributed and conserved in all species investigated, while the remainder have been subjected to modifications, or undergone pseudogenization, degradation or loss in one or more species. LGTs unique to the genus Leishmania have been acquired after the divergence of Leishmania from the other trypanosomatids, and are evolving faster than their recipient genomes. This implies that LGT in genus Leishmania is a continuous and dynamic process contributing to species differentiation and speciation. This study also highlights the importance of carefully evaluating these dynamic genes, e.g. as LGTs have been suggested as potential drug targets.

The Dynamics of Lateral Gene Transfer in Genus Leishmania - A Route for Adaptation and Species Diversification.

Directory of Open Access Journals (Sweden)

Elisabet Vikeved

2016-01-01

Full Text Available The genome of Leishmania major harbours a comparably high proportion of genes of prokaryote origin, acquired by lateral gene transfer (LGT. Some of these are present in closely related trypanosomatids, while some are detected in Leishmania only. We have evaluated the impact and destiny of LGT in genus Leishmania.To study the dynamics and fate of LGTs we have performed phylogenetic, as well as nucleotide and amino acid composition analyses within orthologous groups of LGTs detected in Leishmania. A set of universal trypanosomatid LGTs was added as a reference group. Both groups of LGTs have, to some extent, ameliorated to resemble the recipient genomes. However, while virtually all of the universal trypanosomatid LGTs are distributed and conserved in the entire genus Leishmania, the LGTs uniquely present in genus Leishmania are more prone to gene loss and display faster rates of evolution. Furthermore, a PCR based approach has been employed to ascertain the presence of a set of twenty LGTs uniquely present in genus Leishmania, and three universal trypanosomatid LGTs, in ten additional strains of Leishmania. Evolutionary rates and predicted expression levels of these LGTs have also been estimated. Ten of the twenty LGTs are distributed and conserved in all species investigated, while the remainder have been subjected to modifications, or undergone pseudogenization, degradation or loss in one or more species.LGTs unique to the genus Leishmania have been acquired after the divergence of Leishmania from the other trypanosomatids, and are evolving faster than their recipient genomes. This implies that LGT in genus Leishmania is a continuous and dynamic process contributing to species differentiation and speciation. This study also highlights the importance of carefully evaluating these dynamic genes, e.g. as LGTs have been suggested as potential drug targets.

Neurochemical Metabolomics Reveals Disruption to Sphingolipid Metabolism Following Chronic Haloperidol Administration.

Science.gov (United States)

McClay, Joseph L; Vunck, Sarah A; Batman, Angela M; Crowley, James J; Vann, Robert E; Beardsley, Patrick M; van den Oord, Edwin J

2015-09-01

Haloperidol is an effective antipsychotic drug for treatment of schizophrenia, but prolonged use can lead to debilitating side effects. To better understand the effects of long-term administration, we measured global metabolic changes in mouse brain following 3 mg/kg/day haloperidol for 28 days. These conditions lead to movement-related side effects in mice akin to those observed in patients after prolonged use. Brain tissue was collected following microwave tissue fixation to arrest metabolism and extracted metabolites were assessed using both liquid and gas chromatography mass spectrometry (MS). Over 300 unique compounds were identified across MS platforms. Haloperidol was found to be present in all test samples and not in controls, indicating experimental validity. Twenty-one compounds differed significantly between test and control groups at the p < 0.05 level. Top compounds were robust to analytical method, also being identified via partial least squares discriminant analysis. Four compounds (sphinganine, N-acetylornithine, leucine and adenosine diphosphate) survived correction for multiple testing in a non-parametric analysis using false discovery rate threshold < 0.1. Pathway analysis of nominally significant compounds (p < 0.05) revealed significant findings for sphingolipid metabolism (p = 0.015) and protein biosynthesis (p = 0.024). Altered sphingolipid metabolism is suggestive of disruptions to myelin. This interpretation is supported by our observation of elevated N-acetyl-aspartyl-glutamate in the haloperidol-treated mice (p = 0.004), a marker previously associated with demyelination. This study further demonstrates the utility of murine neurochemical metabolomics as a method to advance understanding of CNS drug effects.

Peptone-yeast autolysate-fetal bovine serum 10, a simple, inexpensive liquid medium for cultivation of Leishmania spp.

OpenAIRE

Palomino, J C

1982-01-01

A simple liquid medium for the cultivation of Leishmania parasites is described. Leishmania brasiliensis and Leishmania peruviana cultured in this medium reached cell densities greater than 10(7) promastigotes per ml within 7 days. This medium compares very favorably with the more complex media used to cultivate Leishmania spp. and other hemoflagellates.

Biosynthetic Pathways of Ergot Alkaloids

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Nina Gerhards

2014-12-01

Full Text Available Ergot alkaloids are nitrogen-containing natural products belonging to indole alkaloids. The best known producers are fungi of the phylum Ascomycota, e.g., Claviceps, Epichloë, Penicillium and Aspergillus species. According to their structures, ergot alkaloids can be divided into three groups: clavines, lysergic acid amides and peptides (ergopeptines. All of them share the first biosynthetic steps, which lead to the formation of the tetracyclic ergoline ring system (except the simplest, tricyclic compound: chanoclavine. Different modifications on the ergoline ring by specific enzymes result in an abundance of bioactive natural products, which are used as pharmaceutical drugs or precursors thereof. From the 1950s through to recent years, most of the biosynthetic pathways have been elucidated. Gene clusters from several ergot alkaloid producers have been identified by genome mining and the functions of many of those genes have been demonstrated by knock-out experiments or biochemical investigations of the overproduced enzymes.

Heterologous Expression of the Oxytetracycline Biosynthetic Pathway in Myxococcus xanthus▿

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Stevens, D. Cole; Henry, Michael R.; Murphy, Kimberly A.; Boddy, Christopher N.

2010-01-01

New natural products for drug discovery may be accessed by heterologous expression of bacterial biosynthetic pathways in metagenomic DNA libraries. However, a “universal” host is needed for this experiment. Herein, we show that Myxococcus xanthus is a potential “universal” host for heterologous expression of polyketide biosynthetic gene clusters. PMID:20208031

Further support for a palaearctic origin of Leishmania

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Sara F Kerr

2000-08-01

Full Text Available The fossil record and systematics of murid rodents, reservoirs of zoonotic cutaneous leishmaniasis in the Palaearctic, Oriental, African, Nearctic and Neotropical, strongly support a Palaearctic origin of Leishmania. The fossil record and systematics of phlebotomine sand flies reinforce this idea. Interpretations of molecular data that place the origin of Leishmania in the Neotropical are inconsistent with the natural histories of reservoirs and vectors. The evolutionary pattern of New World rats (Sigmodontinae indicates that they may be the most important reservoirs of zoonotic cutaneous leishmaniasis throughout their range.

Eugenia uniflora L. Essential Oil as a Potential Anti-Leishmania Agent: Effects on Leishmania amazonensis and Possible Mechanisms of Action

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Amorim, Layane Valéria; de Oliveira, Jamylla Mirck Guerra; Dias, Clarice Noleto; Moraes, Denise Fernandes Coutinho; Andrade, Eloisa Helena de Aguiar; Maia, Jose Guilherme Soares; Carneiro, Sabrina Maria Portela; Carvalho, Fernando Aécio de Amorim

2013-01-01

Eugenia uniflora L. is a member of the Myrtaceae family and is commonly known as Brazilian cherry tree. In this study, we evaluated the chemical composition of Eugenia uniflora L. essential oil (EuEO) by using gas chromatography-mass spectrometry (GC-MS) and assessed its anti-Leishmania activity. We also explored the potential mechanisms of action and cytotoxicity of EuEO. Thirty-two compounds were identified, which constituted 92.65% of the total oil composition. The most abundant components were sesquiterpenes (91.92%), with curzerene (47.3%), γ-elemene (14.25%), and trans-β-elemenone (10.4%) being the major constituents. The bioactivity shown by EuEO against promastigotes (IC50, 3.04 μg·mL−1) and amastigotes (IC50, 1.92 μg·mL−1) suggested significant anti-Leishmania activity. In the cytotoxicity determination, EuEO was 20 times more toxic to amastigotes than to macrophages. Hemolytic activity was 63.22% at the highest concentration tested (400 μg·mL−1); however, there appeared to be no toxicity at 50 μg·mL−1. While the data show that EuEO activity is not mediated by nitric oxide production, they do suggest that macrophage activation may be involved in EuEO anti-Leishmania activity, as evidenced by increases in both the phagocytic capacity and the lysosomal activity. More studies are needed to determine in vivo activity as well as additional mechanisms of the anti-Leishmania activity. PMID:23533469

Eugenia uniflora L. Essential Oil as a Potential Anti-Leishmania Agent: Effects on Leishmania amazonensis and Possible Mechanisms of Action

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Klinger Antonio da Franca Rodrigues

2013-01-01

Full Text Available Eugenia uniflora L. is a member of the Myrtaceae family and is commonly known as Brazilian cherry tree. In this study, we evaluated the chemical composition of Eugenia uniflora L. essential oil (EuEO by using gas chromatography-mass spectrometry (GC-MS and assessed its anti-Leishmania activity. We also explored the potential mechanisms of action and cytotoxicity of EuEO. Thirty-two compounds were identified, which constituted 92.65% of the total oil composition. The most abundant components were sesquiterpenes (91.92%, with curzerene (47.3%, γ-elemene (14.25%, and trans-β-elemenone (10.4% being the major constituents. The bioactivity shown by EuEO against promastigotes (IC50, 3.04 μg·mL−1 and amastigotes (IC50, 1.92 μg·mL−1 suggested significant anti-Leishmania activity. In the cytotoxicity determination, EuEO was 20 times more toxic to amastigotes than to macrophages. Hemolytic activity was 63.22% at the highest concentration tested (400 μg·mL−1; however, there appeared to be no toxicity at 50 μg·mL−1. While the data show that EuEO activity is not mediated by nitric oxide production, they do suggest that macrophage activation may be involved in EuEO anti-Leishmania activity, as evidenced by increases in both the phagocytic capacity and the lysosomal activity. More studies are needed to determine in vivo activity as well as additional mechanisms of the anti-Leishmania activity.

Eugenia uniflora L. Essential Oil as a Potential Anti-Leishmania Agent: Effects on Leishmania amazonensis and Possible Mechanisms of Action.

Science.gov (United States)

Rodrigues, Klinger Antonio da Franca; Amorim, Layane Valéria; de Oliveira, Jamylla Mirck Guerra; Dias, Clarice Noleto; Moraes, Denise Fernandes Coutinho; Andrade, Eloisa Helena de Aguiar; Maia, Jose Guilherme Soares; Carneiro, Sabrina Maria Portela; Carvalho, Fernando Aécio de Amorim

2013-01-01

Eugenia uniflora L. is a member of the Myrtaceae family and is commonly known as Brazilian cherry tree. In this study, we evaluated the chemical composition of Eugenia uniflora L. essential oil (EuEO) by using gas chromatography-mass spectrometry (GC-MS) and assessed its anti-Leishmania activity. We also explored the potential mechanisms of action and cytotoxicity of EuEO. Thirty-two compounds were identified, which constituted 92.65% of the total oil composition. The most abundant components were sesquiterpenes (91.92%), with curzerene (47.3%), γ -elemene (14.25%), and trans- β -elemenone (10.4%) being the major constituents. The bioactivity shown by EuEO against promastigotes (IC50, 3.04  μ g·mL(-1)) and amastigotes (IC50, 1.92  μ g·mL(-1)) suggested significant anti-Leishmania activity. In the cytotoxicity determination, EuEO was 20 times more toxic to amastigotes than to macrophages. Hemolytic activity was 63.22% at the highest concentration tested (400  μ g·mL(-1)); however, there appeared to be no toxicity at 50  μ g·mL(-1). While the data show that EuEO activity is not mediated by nitric oxide production, they do suggest that macrophage activation may be involved in EuEO anti-Leishmania activity, as evidenced by increases in both the phagocytic capacity and the lysosomal activity. More studies are needed to determine in vivo activity as well as additional mechanisms of the anti-Leishmania activity.

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

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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

The preliminary research for biosynthetic engineering by radiation fusion technology

Energy Technology Data Exchange (ETDEWEB)

Roh, Chang Hyun; Jung, U Hee; Park, Hae Ran [KAERI, Daejeon (Korea, Republic of)

2012-01-15

The purpose of this project is to elucidate the solution to the production of bioactive substance using biotransformation process from core technology of biosynthetic engineering by radiation fusion technology. And, this strategy will provide core technology for development of drugs as new concept and category. Research scopes and contents of project include 1) The development of mutant for biosynthetic engineering by radiation fusion technology 2) The development of host for biosynthetic engineering by radiation fusion technology 3) The preliminary study for biosynthetic engineering of isoflavone by radiation fusion technology. The results are as follows. Isoflavone compounds(daidzein, hydroxylated isoflavone) were analyzed by GC-MS. The study of radiation doses and p-NCA high-throughput screening for mutant development were elucidated. And, it was carried out the study of radiation doses for host development. Furthermore, the study of redox partner and construction of recombinant strain for region-specific hydroxylation(P450, redox partner). In addition, the biological effect of 6,7,4'-trihydroxyisoflavone as an anti-obesity agent was elucidated in this study.

Species-Specific Antimonial Sensitivity in Leishmania Is Driven by Post-Transcriptional Regulation of AQP1

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Mandal, Goutam; Mandal, Srotoswati; Sharma, Mansi; Charret, Karen Santos; Papadopoulou, Barbara; Bhattacharjee, Hiranmoy; Mukhopadhyay, Rita

2015-01-01

Leishmania is a digenetic protozoan parasite causing leishmaniasis in humans. The different clinical forms of leishmaniasis are caused by more than twenty species of Leishmania that are transmitted by nearly thirty species of phlebotomine sand flies. Pentavalent antimonials (such as Pentostam or Glucantime) are the first line drugs for treating leishmaniasis. Recent studies suggest that pentavalent antimony (Sb(V)) acts as a pro-drug, which is converted to the more active trivalent form (Sb(III)). However, sensitivity to trivalent antimony varies among different Leishmania species. In general, Leishmania species causing cutaneous leishmaniasis (CL) are more sensitive to Sb(III) than the species responsible for visceral leishmaniasis (VL). Leishmania aquaglyceroporin (AQP1) facilitates the adventitious passage of antimonite down a concentration gradient. In this study, we show that Leishmania species causing CL accumulate more antimonite, and therefore exhibit higher sensitivity to antimonials, than the species responsible for VL. This species-specific differential sensitivity to antimonite is directly proportional to the expression levels of AQP1 mRNA. We show that the stability of AQP1 mRNA in different Leishmania species is regulated by their respective 3’-untranslated regions. The differential regulation of AQP1 mRNA explains the distinct antimonial sensitivity of each species. PMID:25714343

Differential Activation of Human Keratinocytes by Leishmania Species Causing Localized or Disseminated Disease.

Science.gov (United States)

Scorza, Breanna M; Wacker, Mark A; Messingham, Kelly; Kim, Peter; Klingelhutz, Aloysius; Fairley, Janet; Wilson, Mary E

2017-10-01

All Leishmania species parasites are introduced into mammalian skin through a sand fly bite, but different species cause distinct clinical outcomes. Mouse studies suggest that early responses are critical determinants of subsequent adaptive immunity in leishmaniasis, yet few studies address the role of keratinocytes, the most abundant cell in the epidermis. We hypothesized that Leishmania infection causes keratinocytes to produce immunomodulatory factors that influence the outcome of infection. Incubation of primary or immortalized human keratinocytes with Leishmania infantum or Leishmania major, which cause visceral or cutaneous leishmaniasis, respectively, elicited dramatically different responses. Keratinocytes incubated with L. infantum significantly increased expression of proinflammatory genes for IL-6, IL-8, tumor necrosis factor, and IL-1B, whereas keratinocytes exposed to several L. major isolates did not. Furthermore, keratinocyte-monocyte co-incubation studies across a 4 µM semipermeable membrane suggested that L. infantum-exposed keratinocytes release soluble factors that enhance monocyte control of intracellular L. infantum replication (P Leishmania species that may affect the course of disease. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Natural infection of Didelphis aurita (Mammalia: Marsupialia) with Leishmania infantum in Brazil.

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Carreira, João Carlos Araujo; da Silva, Alba Valéria Machado; de Pita Pereira, Daniela; Brazil, Reginaldo Peçanha

2012-06-07

The opossum Didelphis have been considered as natural hosts of Leishmania parasites in the New World, suggesting an important role in the epidemiology of Visceral Leishmaniasis (VL). Among six extant species that belong to the genus Didelphis, only two (D. marsupialis and D. albiventris), have been mentioned as natural hosts of Leishmania infantum in Brazil and Colombia. In the present paper, it is reported for the first time, the observation of intracellular parasites (amastigotes) in tissues of Didelphis aurita naturally infected with Leishmania infantum in Brazil. We also discuss some aspects associated to the relationship between L. infantum and the geographical distribution of some species of the genus Didelphis. The opossums studied were caught by wire traps (Tomahawk) in Barra de Guaratiba, a peri-urban area in Rio de Janeiro. The opossums were killed with an overdose of Thiopental sodium.At necropsy, macroscopic alterations were examined and samples from liver, spleen, lymph nodes, ear, abdominal skin, scent glands and bone marrow were collected for parasitological and molecular diagnoses. Forty-eight opossums were captured in an AVL endemic region, 30 being caught in a mangrove area and eighteen animals in a forest area near to some residential-yards. Among the thirty opossums trapped in the mangrove area, all of them were negative by both imprint and sera samples assayed on Dipstick Tests, that is a test based on a combination of protein-A colloidal gold conjugate and rk39 Leishmania antigen to detect anti-Leishmania antibody in serum or plasma. At the macroscopic examination one out of eighteen opossums, caught close to the forest, presented alterations compatible with spleen hypertrophy and three were positive by Dipstick Tests (16.6%) and presented amastigotes in the spleen and in one of them, the parasites were also observed in a submandibular lymph node. Leishmania infantum infections were confirmed through dot blot hybridization using a L. infantum

Progress towards a Leishmania vaccine.

Science.gov (United States)

Tabbara, Khaled S

2006-07-01

Leishmaniasis is a vector-born protozoan disease. Approximately 12 million individuals are affected worldwide with an estimated annual incidence of 1.5-2 million. Two clinical manifestations are recognized, cutaneous, and visceral, both of which are common in the Middle East. In both forms, infection is chronic, with potential deformities, persistence following cure, and lifelong risk of reactivation. Attempts to develop an effective human Leishmania vaccine have not yet succeeded. Leishmanization, a crude form of live vaccination historically originated in this part of the world. Experimental vaccination has been extensively studied in model animals in the past 2 decades. In this review, major human killed vaccine trials are surveyed, and modern trends in Leishmania vaccine development, including subunit vaccines, naked DNA vaccines, and transmission blocking vaccines are explored. Recent findings of a link between persistence of live parasites, and maintenance of long-term immunity suggest live vaccination with attenuated strains, as a future vaccination strategy.

Development of a dipstick assay for detection of Leishmania-specific canine antibodies

NARCIS (Netherlands)

Schallig, Henk D. F. H.; Cardoso, Luís; Hommers, Marieke; Kroon, Nel; Belling, Guus; Rodrigues, Manuela; Semião-Santos, Saul J.; Vetter, Hans

2004-01-01

A dipstick assay, based on Leishmania infantum antigen, for the rapid detection of Leishmania-specific antibodies in canine serum samples was developed and evaluated. After determination of optimal dipstick test conditions, test performance was compared with two existing serological tests, i.e., the

An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens

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Márcia Cristina Aquino Teixeira

2011-03-01

Full Text Available Domestic dogs are considered to be the main reservoirs of zoonotic visceral leishmaniasis. In this work, we evaluated a protocol to induce Leishmania infantum/Leishmania chagasi-specific cellular and humoral immune responses in dogs, which consisted of two injections of Leishmania promastigote lysate followed by a subcutaneous inoculation of viable promastigotes. The primary objective was to establish a canine experimental model to provide positive controls for testing immune responses to Leishmania in laboratory conditions. After inoculation of viable promastigotes, specific proliferative responses of peripheral blood mononuclear cells (PBMCs to either Leishmania lysate or recombinant proteins, the in vitro production of interferon-γ by antigen-stimulated PBMCs and a significant increase in circulating levels of anti-Leishmania antibodies were observed. The immunized dogs also displayed positive delayed-type hypersensitivity reactions to Leishmania crude antigens and to purified recombinant proteins. An important finding that supports the suitability of the dogs as positive controls is that they remained healthy for the entire observation period, i.e., more than seven years after infection. Following the Leishmania antigen lysate injections, the infection of dogs by the subcutaneous route appears to induce a sustained cellular immune response, leading to an asymptomatic infection. This provides a useful model for both the selection of immunogenic Leishmania antigens and for immunobiological studies on their possible immunoprotective activities.

Structurally optimized analogs of the retrograde trafficking inhibitor Retro-2cycl limit Leishmania infections.

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Craig, Evan; Huyghues-Despointes, Charles-Eugene; Yu, Chun; Handy, Emma L; Sello, Jason K; Kima, Peter E

2017-05-01

In infected mammalian cells, Leishmania parasites reside within specialized compartments called parasitophorous vacuoles (LPVs). We have previously shown that Retro-2, a member of a novel class of small retrograde pathway inhibitors caused reduced LPV sizes and lower parasite numbers during experimental L. mexicana sp. infections. The purpose of this study was to determine if structural analogs of Retro-2cycl reported to have superior potency in the inhibition of retrograde pathway-dependent phenomena (i.e., polyomavirus cellular infection by polyomavrius and Shiga toxin trafficking in cells) are also more effective than the parent compound at controlling Leishmania infections. In addition to their effects on LPV development, we show that two optimized analogs of Retro-2cycl, DHQZ 36 and DHQZ 36.1 limit Leishmania amazonensis infection in macrophages at EC50 of 13.63+/-2.58μM and10.57+/-2.66μM, respectively, which is significantly lower than 40.15μM the EC50 of Retro-2cycl. In addition, these analogs caused a reversal in Leishmania induced suppression of IL-6 release by infected cells after LPS activation. Moreover, we show that in contrast to Retro-2cycl that is Leishmania static, the analogs can kill Leishmania parasites in axenic cultures, which is a desirable attribute for any drug to treat Leishmania infections. Together, these studies validate and extend the published structure-activity relationship analyses of Retro-2cycl.

The flagellar protein FLAG1/SMP1 is a candidate for Leishmania-sand fly interaction.

Science.gov (United States)

Di-Blasi, Tatiana; Lobo, Amanda R; Nascimento, Luanda M; Córdova-Rojas, Jose L; Pestana, Karen; Marín-Villa, Marcel; Tempone, Antonio J; Telleria, Erich L; Ramalho-Ortigão, Marcelo; McMahon-Pratt, Diane; Traub-Csekö, Yara M

2015-03-01

Leishmaniasis is a serious problem that affects mostly poor countries. Various species of Leishmania are the agents of the disease, which take different clinical manifestations. The parasite is transmitted by sandflies, predominantly from the Phlebotomus genus in the Old World and Lutzomyia in the New World. During development in the gut, Leishmania must survive various challenges, which include avoiding being expelled with blood remnants after digestion. It is believed that attachment to the gut epithelium is a necessary step for vector infection, and molecules from parasites and sand flies have been implicated in this attachment. In previous work, monoclonal antibodies were produced against Leishmania. Among these an antibody was obtained against Leishmania braziliensis flagella, which blocked the attachment of Leishmania panamensis flagella to Phlebotomus papatasi guts. The protein recognized by this antibody was identified and named FLAG1, and the complete FLAG1 gene sequence was obtained. This protein was later independently identified as a small, myristoylated protein and called SMP1, so from now on it will be denominated FLAG1/SMP1. The FLAG1/SMP1 gene is expressed in all developmental stages of the parasite, but has higher expression in promastigotes. The anti-FLAG1/SMP1 antibody recognized the flagellum of all Leishmania species tested and generated the expected band by western blots. This antibody was used in attachment and infection blocking experiments. Using the New World vector Lutzomyia longipalpis and Leishmania infantum chagasi, no inhibition of attachment ex vivo or infection in vivo was seen. On the other hand, when the Old World vectors P. papatasi and Leishmania major were used, a significant decrease of both attachment and infection were seen in the presence of the antibody. We propose that FLAG1/SMP1 is involved in the attachment/infection of Leishmania in the strict vector P. papatasi and not the permissive vector L. longipalpis.

Severity of tegumentary leishmaniasis is not exclusively associated with Leishmania RNA virus 1 infection in Brazil

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Luiza de Oliveira Ramos Pereira

2013-08-01

Full Text Available Leishmania RNA virus (LRV has been shown to be a symbiotic component of Leishmania parasites in South America. Nested retro-transcription polymerase chain reaction was employed to investigate LRV1 presence in leishmaniasis lesions from Brazil. In endemic areas of Rio de Janeiro (RJ, no LRV1 infection was observed even with mucosal involvement. LRV1 was only detected in Leishmania (V. guyanensis cutaneous lesions from the northern region, which were obtained from patients presenting with disease reactivation after clinical cure of their primary lesions. Our results indicated that the severity of leishmaniasis in some areas of RJ, where Leishmania (V. brazi-liensis is the primary etiological agent, was not associated with Leishmania LRV1 infection.

Evaluation of four molecular methods to detect Leishmania infection in dogs.

Science.gov (United States)

Albuquerque, Andreia; Campino, Lenea; Cardoso, Luís; Cortes, Sofia

2017-03-13

Canine leishmaniasis, a zoonotic disease caused by Leishmania infantum vectored by phlebotomine sand flies, is considered a relevant veterinary and public health problem in various countries, namely in the Mediterranean basin and Brazil, where dogs are considered the main reservoir hosts. Not only diseased dogs but also those subclinically infected play a relevant role in the transmission of L. infantum to vectors; therefore, early diagnosis is essential, under both a clinical and an epidemiological perspective. Molecular tools can be a more accurate and sensitive approach for diagnosis, with a wide range of protocols currently in use. The aim of the present report was to compare four PCR based protocols for the diagnosis of canine Leishmania infection in a cohort of dogs from the Douro region, Portugal. A total of 229 bone marrow samples were collected from dogs living in the Douro region, an endemic region for leishmaniasis. Four PCR protocols were evaluated for Leishmania DNA detection in canine samples, three single (ITS1-PCR, MC-PCR and Uni21/Lmj4-PCR) and one nested (nested SSU rRNA-PCR). Two of the protocols were based on nuclear targets and the other two on kinetoplastid targets. The higher overall percentage of infected dogs was detected with the nested SSU rRNA-PCR (37.6%), which also was able to detect Leishmania DNA in a higher number of samples from apparently healthy dogs (25.3%). The ITS1-PCR presented the lowest level of Leishmania detection. Nested SSU rRNA-PCR is an appropriate method to detect Leishmania infection in dogs. Accurate and early diagnosis in clinically suspect as well as apparently healthy dogs is essential, in order to treat and protect animals and public health and contribute to the control and awareness of the disease.

Leishmania isoenzyme polymorphisms in Ecuador: Relationships with geographic distribution and clinical presentation

Science.gov (United States)

Calvopina, Manuel; Armijos, Rodrigo X; Marco, Jorge D; Uezato, Hiroshi; Kato, Hirotomo; Gomez, Eduardo A; Korenaga, Masataka; Barroso, Paola A; Mimori, Tatsuyuki; Cooper, Philip J; Nonaka, Shigeo; Hashiguchi, Yoshihisa

2006-01-01

Background Determinants of the clinical presentation of the leishmaniases are poorly understood but Leishmania species and strain differences are important. To examine the relationship between clinical presentation, species and isoenzyme polymorphisms, 56 Leishmania isolates from distinct presentations of American tegumentary leishmaniasis (ATL) from Ecuador were analyzed. Methods Isolates were characterized by multilocus enzyme electrophoresis for polymorphisms of 11 isoenzymes. Patients were infected in four different ecologic regions: highland and lowland jungle of the Pacific coast, Amazonian lowlands and Andean highlands. Results Six Leishmania species constituting 21 zymodemes were identified: L. (Viannia) panamensis (21 isolates, 7 zymodemes), L. (V.) guyanensis (7 isolates, 4 zymodemes), L. (V.) braziliensis (5 isolates, 3 zymodemes), L. (Leishmania) mexicana (11 isolates, 4 zymodemes), L. (L.) amazonensis (10 isolates, 2 zymodemes) and L. (L.) major (2 isolates, 1 zymodeme). L. panamensis was the species most frequently identified in the Pacific region and was associated with several clinical variants of cutaneous disease (CL); eight cases of leishmaniasis recidiva cutis (LRC) found in the Pacific highlands were associated with 3 zymodemes of this species. Mucocutaneous leishmaniasis found only in the Amazonian focus was associated with 3 zymodemes of L. braziliensis. The papular variant of CL, Uta, found in the Andean highlands was related predominantly with a single zymodeme of L. mexicana. Conclusion Our data show a high degree of phenotypic variation within species, and some evidence for associations between specific variants of ATL (i.e. Uta and LRC) and specific Leishmania zymodemes. This study further defines the geographic distribution of Leishmania species and clinical variants of ATL in Ecuador. PMID:16968553

Vector Competence of Lutzomyia cruzi Naturally Demonstrated for Leishmania infantum and Suspected for Leishmania amazonensis.

Science.gov (United States)

de Oliveira, Everton Falcão; Oshiro, Elisa Teruya; Fernandes, Wagner Souza; Ferreira, Alda Maria Teixeira; de Oliveira, Alessandra Gutierrez; Galati, Eunice Aparecida Bianchi

2017-01-11

Corumbá city is one of the oldest visceral leishmaniasis-endemic foci in the state of Mato Grosso do Sul, Brazil, where the transmission of Leishmania infantum has been attributed to Lutzomyia cruzi Aiming at investigating the parameters of the vectorial capacity of Lu. cruzi for L. infantum, a project was undertaken in this city. Among these parameters, vector competence was investigated and the results obtained are reported herein. Of the 12 hamsters exposed to feed wild-caught female sandflies, two developed infection with L. infantum and surprisingly, one with Leishmania amazonensis In addition, hamsters with L. infantum infection were bitten only by females of Lu. cruzi, whereas the hamster infected with L. amazonensis was bitten by 124 Lu. cruzi females and one of Evandromyia corumbaensis Although there is a strong suspicion regarding the competence of Lu. cruzi in transmitting L. amazonensis naturally, it was not demonstrated. © The American Society of Tropical Medicine and Hygiene.

Identificación de una nueva proteína en Leishmania (Viannia peruviana

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Maxy De los Santos

1998-01-01

Full Text Available El análisis de la secuencia nucleotídica y aminoacídica de un clon de la biblioteca de expresión en fago λgt11 de Leishmania (Viannia peruviana, estableció identidad parcial con los genes de las proteínas acídicas ribosomales P2 de Leishmania (Leishmania infantum. Este hallazgo unido a ciertos dominios geonómicos conservados, sugeridos de la comparación de 14 secuencias de otras proteínas P1 eucarióticas, confirman que la secuencia del inserto de clon codifica la proteína acídica ribosomal P1 de L. (V. peruviana denominada LpP1. Este es el primer reporte sobre este tipo de proteína en el género Leishmania.

Mitochondrial associated ubiquitin fold modifier-1 mediated protein conjugation in Leishmania donovani.

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Sreenivas Gannavaram

2011-01-01

Full Text Available In this report, we demonstrate the existence of the ubiquitin fold modifier-1 (Ufm1 and its conjugation pathway in trypanosomatid parasite Leishmania donovani. LdUfm1 is activated by E1-like enzyme LdUba5. LdUfc1 (E2 specifically interacted with LdUfm1 and LdUba5 to conjugate LdUfm1 to proteinaceous targets. Mass spectrometry analysis revealed that LdUfm1 is conjugated to Leishmania protein targets that are associated with mitochondria. Immunofluorescence experiments showed that Leishmania Ufm1, Uba5 and Ufc1 are associated with the mitochondria. The demonstration that all the components of this system as well as the substrates are associated with mitochondrion suggests it may have physiological roles not yet described in any other organism. Overexpression of a non-conjugatable form of LdUfm1 and an active site mutant of LdUba5 resulted in reduced survival of Leishmania in the macrophage. Since mitochondrial activities are developmentally regulated in the life cycle of trypanosomatids, Ufm1 mediated modifications of mitochondrial proteins may be important in such regulation. Thus, Ufm1 conjugation pathway in Leishmania could be explored as a potential drug target in the control of Leishmaniasis.

Detection of Leishmania spp in silvatic mammals and isolation of Leishmania (Viannia braziliensis from Rattus rattus in an endemic area for leishmaniasis in Minas Gerais State, Brazil.

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Agnes Antônia Sampaio Pereira

Full Text Available Knowledge of potential reservoirs of Leishmania spp. in an anthropic environment is important so that surveillance and control measures can be implemented. The aim of this study was to investigate the infection by Leishmania in small mammals in an area located in Minas Gerais, Brazil, that undergoes changes in its natural environment and presents autochthonous human cases of cutaneous leishmaniasis (CL and visceral leishmaniasis (VL. For the capture of the animals, Sherman and Tomahawk traps were used and distributed in the peridomicile of houses with reports of autochthonous cases of CL or VL. Six catches were carried out on two consecutive nights with intervals of two months during one year and samples of spleen, liver, tail skin, ear skin and bone marrow of the animals were obtained. Parasitological and molecular methods were used to detect the infection. Identification of the Leishmania species was performed by PCR RFLPhsp70. Twenty five animals of four species were captured: ten Rattus rattus, nine Didelphis albiventris, five Cerradomys subflavus and one Marmosops incanus. In the PCR-hsp70, five animals were positive (20%. The Leishmania species identified in PCR-RFLPhsp70 were: Leishmania braziliensis in D. albiventris (2, C. subflavus (1 and R. rattus (1 and Leishmania infantum in R. rattus (1. The highest positivity rate for L. braziliensis was obtained in the liver samples. The spleen was the only tissue positive for L. infantum. It was isolated in culture medium L. braziliensis from two samples (liver and spleen of R. rattus. This is the first record of isolation of L. braziliensis from R. rattus in the southeastern region of Brazil. These results are relevant to the knowledge of the epidemiology of leishmaniasis in the region, mainly in the investigation of the presence of hosts and possible reservoirs of the parasite.

Activity evaluation from different native or irradiated with 60 Co gamma rays snake venoms and their inhibitory effect on Leishmania (Leishmania) amazonensis

International Nuclear Information System (INIS)

Lourenco, Cecilia de Oliveira

2000-01-01

Cutaneous leishmaniasis is a disease, caused by Leishmania parasites, that occurs frequently in tropical and sub-tropical regions of the world. Skin lesions that could results in disfiguring aspect characterize it. The treatment is based on few drugs as antimony salts or pentamidine that are toxic with increasing resistance by the parasite. Alternative forms of disease treatment are in constant search, including natural components as snake venoms. Previous studies demonstrate that some components of snake venoms have an inhibitory effect against those parasites, including Leishmania species. Although snake venoms presented high toxicity, several methods have been described to detoxify most or some of their toxic components, with favorable results by the use of gamma irradiation. In this report we tested several native and irradiated snake venoms for inhibitory effect against Leishmania (Leishmania) amazonensis parasite and LLCMK 2 mammalian cells, with enzymatic tests and electrophoresis. There are significant activity in Acanthophis antarcticus, Agkistrodon bilineatus, Bothrops moojeni, Bothrops jararaca, Hoplocephalus stephensi, Naja melanoleuca, Naja mossambica, Pseudechis australis, Pseudechis colletti, Pseudechis guttatus and Pseudechis porphyriacus, venom being inactive Pseudonaja textilis, Notechis ater niger, Notechis scutatus. Oxyuranus microlepidotus and Oxyuranus scutellatus venoms. After 2 KGy of 60 Co irradiation most venom loses significantly their activity. Venoms with antileishmanial activity presented L-amino acid oxidase (L-AO) activity and showed common protein with a molecular weight about 60kDa in SDS-PAGE. These results indicate that L-AO activity in those venoms are probably related with antileishmanial effect. (author)

Detection of Leishmania donovani and L. tropica in ethiopian wild rodents

Czech Academy of Sciences Publication Activity Database

Kassahun, A.; Sádlová, J.; Dvořák, V.; Košťálová, T.; Rohoušová, I.; Frynta, D.; Aghová, Tatiana; Yasur-Landau, D.; Lemma, W.; Hailu, A.; Baneth, G.; Warburg, A.; Volf, P.; Votýpka, J.

2015-01-01

Roč. 145, May 2015 (2015), s. 39-44 ISSN 0001-706X R&D Projects: GA ČR GAP506/10/0983 EU Projects: European Commission(XE) 261504 - EDENEXT Institutional support: RVO:68081766 Keywords : Leishmania donovani * Leishmania tropica * Phlebotomine sand fly * Rodents * kDNA * ITS1 Subject RIV: EG - Zoology Impact factor: 2.380, year: 2015

Impact of Leishmania metalloprotease GP63 on macrophage signaling

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Isnard, Amandine; Shio, Marina T.; Olivier, Martin

2012-01-01

The intramacrophage protozoan parasites of Leishmania genus have developed sophisticated ways to subvert the innate immune response permitting their infection and propagation within the macrophages of the mammalian host. Several Leishmania virulence factors have been identified and found to be of importance for the development of leishmaniasis. However, recent findings are now further reinforcing the critical role played by the zinc-metalloprotease GP63 as a virulence factor that greatly influence host cell signaling mechanisms and related functions. GP63 has been found to be involved not only in the cleavage and degradation of various kinases and transcription factors, but also to be the major molecule modulating host negative regulatory mechanisms involving for instance protein tyrosine phosphatases (PTPs). Those latter being well recognized for their pivotal role in the regulation of a great number of signaling pathways. In this review article, we are providing a complete overview about the role of Leishmania GP63 in the mechanisms underlying the subversion of macrophage signaling and functions. PMID:22919663

Ocorrência de Leishmania spp. em felinos do município de Araçatuba, SP Occurrence de Leishmania spp. in domestic cats from Araçatuba, SP

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Katia Denise Saraiva Bresciani

2010-06-01

Full Text Available Este trabalho teve como objetivo comparar a ocorrência de Leishmania spp. em gatos por dois métodos (citológico e sorológico, bem como associar a ocorrência deste protozoário com as variáveis sexo, idade e raça. Amostras séricas de 283 felinos domésticos foram testadas pela Reação de Imunofluorescência Indireta (RIFI, e o exame parasitológico direto de linfonodos também foi realizado para a verificação da positividade para Leishmania spp. Ocorrência de 0,7% (2/283 foi observada nos felinos examinados, por meio de imprint de linfonodos e nenhum animal apresentou títulos de anticorpos para Leishmania spp. As duas fêmeas positivas eram sem raça definida, sendo uma jovem e outra adulta. Por meio dos resultados obtidos, não foi constatada diferença estatisticamente significante em relação às variáveis sexo, raça e idade nos gatos desta pesquisa (p > 0,05. Ocorrência de Leishmania spp. nos gatos deste estudo foi baixa. Devido a esta baixa incidência sugere-se que estes não assumem importância epidemiológica na área do estudo.This study had the purpose to compare the occurrence of Leishmania spp. in felines through two methods (cytological and serological, as well as to associate the occurrence of this protozoan with the sex, age and breed variables. Serum samples from 283 domestic felines were processed by means of Indirect Immunofluorescence Reaction (IIR, and the direct parasitological test for linfonodes was also carried out in order to verify positivity for Leishmania spp. Occurrence of 0.7% (2/283 was observed in the tested felines by means of linfonode imprinting and no animal showed title of antibodies for Leishmania spp. The two positive females were mongrel, a young female and an adult female feline. From the obtained results, no statistically significant difference was observed as regards the sex, breed and age variables in this research (p > 0.05. Occurrence of Leishmania spp. in the cats of this study was

Rattus norvegicus (Rodentia: Muridae Infected by Leishmania (Leishmania infantum (syn. Le. chagasi in Brazil

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Fabiana de Oliveira Lara-Silva

2014-01-01

Full Text Available In the present study we surveyed the fauna of phlebotomine sand flies and small mammals in peridomestic areas from a Brazilian municipality where the American cutaneous leishmaniasis (ACL is endemic. A total of 608 female phlebotomine sand flies were captured during nine months in 2009 and 2010. Seven different species were represented with 60% of them being Lutzomyia intermedia and Lu. whitmani, both incriminated vectors of ACL. Lu. longipalpis, a proven vector of visceral leishmaniasis (VL was also captured at high proportion (12.8%. Genomic DNA analysis of 136 species-specific pools of female sand flies followed by molecular genotyping showed the presence of Leishmania infantum DNA in two pools of Lu. longipalpis. The same Leishmania species was found in one blood sample from Rattus norvegicus among 119 blood and tissue samples analysed. This is the first report of Le. infantum in R. norvegicus in the Americas and suggests a possible role for this rodent species in the zoonotic cycle of VL. Our study coincided with the reemergence of VL in Governador Valadares.

Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

International Nuclear Information System (INIS)

Neto, J.L. Siqueira; Lira, C.B.B.; Giardini, M.A.; Khater, L.; Perez, A.M.; Peroni, L.A.; Reis, J.R.R. dos; Freitas-Junior, L.H.; Ramos, C.H.I.; Cano, M.I.N.

2007-01-01

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres

Exosome Secretion by the Parasitic Protozoan Leishmania within the Sand Fly Midgut

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Vanessa Diniz Atayde

2015-11-01

Full Text Available Despite several studies describing the secretion of exosomes by Leishmania in vitro, observation of their formation and release in vivo has remained a major challenge. Herein, we show that Leishmania constitutively secretes exosomes within the lumen of the sand fly midgut through a mechanism homologous to the mammalian pathway. Through egestion experiments, we demonstrate that Leishmania exosomes are part of the sand fly inoculum and are co-egested with the parasite during the insect’s bite, possibly influencing the host infectious process. Indeed, co-inoculation of mice footpads with L. major plus midgut-isolated or in-vitro-isolated L. major exosomes resulted in a significant increase in footpad swelling. Notably, co-injections produced exacerbated lesions through overinduction of inflammatory cytokines, in particular IL-17a. Our data indicate that Leishmania exosomes are an integral part of the parasite’s infectious life cycle, and we propose to add these vesicles to the repertoire of virulence factors associated with vector-transmitted infections.

Methodology optimizing SAGE library tag-to-gene mapping: application to Leishmania

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Smandi Sondos

2012-01-01

Full Text Available Abstract Background Leishmaniasis are widespread parasitic-diseases with an urgent need for more active and less toxic drugs and for effective vaccines. Understanding the biology of the parasite especially in the context of host parasite interaction is a crucial step towards such improvements in therapy and control. Several experimental approaches including SAGE (Serial analysis of gene expression have been developed in order to investigate the parasite transcriptome organisation and plasticity. Usual SAGE tag-to-gene mapping techniques are inadequate because almost all tags are normally located in the 3'-UTR outside the CDS, whereas most information available for Leishmania transcripts is restricted to the CDS predictions. The aim of this work is to optimize a SAGE libraries tag-to-gene mapping technique and to show how this development improves the understanding of Leishmania transcriptome. Findings The in silico method implemented herein was based on mapping the tags to Leishmania genome using BLAST then mapping the tags to their gene using a data-driven probability distribution. This optimized tag-to-gene mappings improved the knowledge of Leishmania genome structure and transcription. It allowed analyzing the expression of a maximal number of Leishmania genes, the delimitation of the 3' UTR of 478 genes and the identification of biological processes that are differentially modulated during the promastigote to amastigote differentiation. Conclusion The developed method optimizes the assignment of SAGE tags in trypanosomatidae genomes as well as in any genome having polycistronic transcription and small intergenic regions.

Evolutionary comparison of prenylation pathway in kinetoplastid Leishmania and its sister Leptomonas.

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Chauhan, Indira Singh; Kaur, Jaspreet; Krishna, Shagun; Ghosh, Arpita; Singh, Prashant; Siddiqi, Mohammad Imran; Singh, Neeloo

2015-11-21

Leptomonas is monogenetic kinetoplastid parasite of insects and is primitive in comparison to Leishmania. Comparative studies of these two kinetoplastid may share light on the evolutionary transition to dixenous parasitism in Leishmania. In order to adapt and survive within two hosts, Leishmania species must have acquired virulence factors in addition to mechanisms that mediate susceptibility/resistance to infection in the pathology associated with disease. Rab proteins are key mediators of vesicle transport and contribute greatly to the evolution of complexity of membrane transport system. In this study we used our whole genome sequence data of these two divergent kinetoplastids to analyze the orthologues/paralogues of Rab proteins. During change of lifestyle from monogenetic (Leptomonas) to digenetic (Leishmania), we found that the prenyl machinery remained unchanged. Geranylgeranyl transferase-I (GGTase-I) was absent in both Leishmania and its sister Leptomonas. Farnesyltransferase (FTase) and geranylgeranyl transferase-II (GGTase-II) were identified for protein prenylation. We predict that activity of the missing alpha-subunit (α-subunit) of GGTase-II in Leptomonas was probably contributed by the α-subunit of FTase, while beta-subunit (β-subunit) of GGTase-II was conserved and indicated functional conservation in the evolution of these two kinetoplastids. Therefore the β-subunit emerges as an excellent target for compounds inhibiting parasite activity in clinical cases of co-infections. We also confirmed that during the evolution to digenetic life style in Leishmania, the parasite acquired capabilities to evade drug action and maintain parasite virulence in the host with the incorporation of short-chain dehydrogenase/reductase (SDR/MDR) superfamily in Rab genes. Our study based on whole genome sequences is the first to build comparative evolutionary analysis and identification of prenylation proteins in Leishmania and its sister Leptomonas. The information

Affinity labeling of the folate-methotrexate transporter from Leishmania donovani

International Nuclear Information System (INIS)

Beck, J.T.; Ullman, B.

1989-01-01

An affinity labeling technique has been developed to identify the folate-methotrexate transporter of Leishmania donovani promastigotes using activated derivatives of the ligands. These activated derivatives were synthesized by incubating folate and methotrexate with a 10-fold excess of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) for 10 min at ambient temperature in dimethyl sulfoxide. When intact wild-type (DI700) Leishmania donovani or preparations of their membranes were incubated with a 0.4 μM concentration of either activated [ 3 H]folate or activated [ 3 H]methotrexate, the radiolabeled ligands were covalently incorporated into a polypeptide with a molecular weight of approximately 46,000, as demonstrated by SDS-polyacrylamide gel electrophoresis. No affinity labeling of a 46,000-dalton protein was observed when equimolar concentrations of activated radiolabeled ligands were incubated with intact cells or membranes prepared from a methotrexate-resistant mutant clone of Leishmania donovani, MTXA5, that is genetically defective in folate-methotrexate transport capability. Time course studies indicated that maximal labeling of the 46,000-dalton protein occurred within 5-10 min of incubation of intact cells with activated ligand. These studies provide biochemical evidence that the folate-methotrexate transporter of Leishmania donovani can be identified in crude extracts by an affinity labeling technique and serve as a prerequisite to further analysis of the transport protein by providing a vehicle for subsequent purification of this membrane carrier. Moreover, these investigations suggest that the affinity labeling technique using EDC-activated ligands may be exploitable to analyze other cell surface binding proteins in Leishmania donovani, as well as in other organisms

Genomic confirmation of hybridisation and recent inbreeding in a vector-isolated Leishmania population.

Science.gov (United States)

Rogers, Matthew B; Downing, Tim; Smith, Barbara A; Imamura, Hideo; Sanders, Mandy; Svobodova, Milena; Volf, Petr; Berriman, Matthew; Cotton, James A; Smith, Deborah F

2014-01-01

Although asexual reproduction via clonal propagation has been proposed as the principal reproductive mechanism across parasitic protozoa of the Leishmania genus, sexual recombination has long been suspected, based on hybrid marker profiles detected in field isolates from different geographical locations. The recent experimental demonstration of a sexual cycle in Leishmania within sand flies has confirmed the occurrence of hybridisation, but knowledge of the parasite life cycle in the wild still remains limited. Here, we use whole genome sequencing to investigate the frequency of sexual reproduction in Leishmania, by sequencing the genomes of 11 Leishmania infantum isolates from sand flies and 1 patient isolate in a focus of cutaneous leishmaniasis in the Çukurova province of southeast Turkey. This is the first genome-wide examination of a vector-isolated population of Leishmania parasites. A genome-wide pattern of patchy heterozygosity and SNP density was observed both within individual strains and across the whole group. Comparisons with other Leishmania donovani complex genome sequences suggest that these isolates are derived from a single cross of two diverse strains with subsequent recombination within the population. This interpretation is supported by a statistical model of the genomic variability for each strain compared to the L. infantum reference genome strain as well as genome-wide scans for recombination within the population. Further analysis of these heterozygous blocks indicates that the two parents were phylogenetically distinct. Patterns of linkage disequilibrium indicate that this population reproduced primarily clonally following the original hybridisation event, but that some recombination also occurred. This observation allowed us to estimate the relative rates of sexual and asexual reproduction within this population, to our knowledge the first quantitative estimate of these events during the Leishmania life cycle.

Leishmania carbon metabolism in the macrophage phagolysosome- feast or famine?

Science.gov (United States)

McConville, Malcolm J; Saunders, Eleanor C; Kloehn, Joachim; Dagley, Michael J

2015-01-01

A number of medically important microbial pathogens target and proliferate within macrophages and other phagocytic cells in their mammalian hosts. While the majority of these pathogens replicate within the host cell cytosol or non-hydrolytic vacuolar compartments, a few, including protists belonging to the genus Leishmania, proliferate long-term within mature lysosome compartments.  How these parasites achieve this feat remains poorly defined. In this review, we highlight recent studies that suggest that Leishmania virulence is intimately linked to programmed changes in the growth rate and carbon metabolism of the obligate intra-macrophage stages. We propose that activation of a slow growth and a stringent metabolic response confers resistance to multiple stresses (oxidative, temperature, pH), as well as both nutrient limitation and nutrient excess within this niche. These studies highlight the importance of metabolic processes as key virulence determinants in Leishmania.

Serological and molecular survey of Leishmania infection in dogs from Luanda, Angola

NARCIS (Netherlands)

Vilhena, Hugo; Granada, Sara; Oliveira, Ana Cristina; Schallig, Henk D. F. H.; Nachum-Biala, Yaarit; Cardoso, Luís; Baneth, Gad

2014-01-01

Canine leishmaniosis (CanL) due to Leishmania infantum is a global zoonosis endemic in more than 70 countries in Europe, North Africa, Asia and America; however, data on this infection is scarce from southern Africa. The aim of this study was to survey dogs in Luanda, Angola, for Leishmania

Evolutionary systems biology of amino acid biosynthetic cost in yeast.

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Michael D Barton

2010-08-01

Full Text Available Every protein has a biosynthetic cost to the cell based on the synthesis of its constituent amino acids. In order to optimise growth and reproduction, natural selection is expected, where possible, to favour the use of proteins whose constituents are cheaper to produce, as reduced biosynthetic cost may confer a fitness advantage to the organism. Quantifying the cost of amino acid biosynthesis presents challenges, since energetic requirements may change across different cellular and environmental conditions. We developed a systems biology approach to estimate the cost of amino acid synthesis based on genome-scale metabolic models and investigated the effects of the cost of amino acid synthesis on Saccharomyces cerevisiae gene expression and protein evolution. First, we used our two new and six previously reported measures of amino acid cost in conjunction with codon usage bias, tRNA gene number and atomic composition to identify which of these factors best predict transcript and protein levels. Second, we compared amino acid cost with rates of amino acid substitution across four species in the genus Saccharomyces. Regardless of which cost measure is used, amino acid biosynthetic cost is weakly associated with transcript and protein levels. In contrast, we find that biosynthetic cost and amino acid substitution rates show a negative correlation, but for only a subset of cost measures. In the economy of the yeast cell, we find that the cost of amino acid synthesis plays a limited role in shaping transcript and protein expression levels compared to that of translational optimisation. Biosynthetic cost does, however, appear to affect rates of amino acid evolution in Saccharomyces, suggesting that expensive amino acids may only be used when they have specific structural or functional roles in protein sequences. However, as there appears to be no single currency to compute the cost of amino acid synthesis across all cellular and environmental

Molecular and serological detection of Leishmania spp. in captive wild animals from Ilha Solteira, SP, Brazil Detecção sorológica e molecular de Leishmania spp. em animais selvagens do zoológico de Ilha Solteira, SP, Brasil

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Márcia Mariza Gomes Jusi

2011-09-01

Full Text Available Leishmaniasis is a zoonotic disease that affects 12 million people worldwide. Several mammalian species can serve as a reservoir for this disease. Dogs are the main reservoir for visceral leishmaniasis in urban areas, which has become a serious public health concern in Brazil. The aim of this study was to evaluate the presence of Leishmania spp. in captive wild animals from Ilha Solteira, São Paulo, Brazil. Blood and various tissues samples were collected from animals of five different species: Speothos venaticus, Chrysocyon brachyurus, Cerdocyon thous, Pseudalopex vetulus, and Procyon cancrivorus. Antibodies against Leishmania spp. were detected in three wild canids by indirect fluorescent antibody test (IFAT and enzyme-linked immunosorbent assay (ELISA. PCR analyses of blood and bone marrow from all animals were negative, but Leishmania DNA was found in the tissues and skin of seropositive animals. Positive PCR samples were also positive for Leishmania donovani complex. Analysis of sequenced PCR products showed similarities with different regions of Leishmania (Leishmania infantum and Leishmania (Leishmania chagasi kinetoplastids. Measures to control visceral leishmaniasis in wild animals kept in Brazilian zoos should be established, as no disease control programs are currently available.Leishmaniose é uma doença zoonótica que afeta cerca de 12 milhões de pessoas no mundo todo. Várias espécies mamíferas podem servir de reservatório para a doença. Os cães são considerados os principais reservatórios para a leishmaniose visceral em áreas urbanas, o que tem se tornado um sério problema de saúde pública no Brasil. O objetivo deste trabalho foi avaliar a presença de Leishmania spp. em animais selvagens mantidos no zoológico de Ilha Solteira, São Paulo, Brasil. Foram coletados amostras de sangue e tecidos de cinco espécies diferentes: Speothos venaticus, Chrysocyon brachyurus, Cerdocyon thous, Pseudalopex vetulus, e Procyon

Leishmania isoenzyme polymorphisms in Ecuador: Relationships with geographic distribution and clinical presentation

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Mimori Tatsuyuki

2006-09-01

Full Text Available Abstract Background Determinants of the clinical presentation of the leishmaniases are poorly understood but Leishmania species and strain differences are important. To examine the relationship between clinical presentation, species and isoenzyme polymorphisms, 56 Leishmania isolates from distinct presentations of American tegumentary leishmaniasis (ATL from Ecuador were analyzed. Methods Isolates were characterized by multilocus enzyme electrophoresis for polymorphisms of 11 isoenzymes. Patients were infected in four different ecologic regions: highland and lowland jungle of the Pacific coast, Amazonian lowlands and Andean highlands. Results Six Leishmania species constituting 21 zymodemes were identified: L. (Viannia panamensis (21 isolates, 7 zymodemes, L. (V. guyanensis (7 isolates, 4 zymodemes, L. (V. braziliensis (5 isolates, 3 zymodemes, L. (Leishmania mexicana (11 isolates, 4 zymodemes, L. (L. amazonensis (10 isolates, 2 zymodemes and L. (L. major (2 isolates, 1 zymodeme. L. panamensis was the species most frequently identified in the Pacific region and was associated with several clinical variants of cutaneous disease (CL; eight cases of leishmaniasis recidiva cutis (LRC found in the Pacific highlands were associated with 3 zymodemes of this species. Mucocutaneous leishmaniasis found only in the Amazonian focus was associated with 3 zymodemes of L. braziliensis. The papular variant of CL, Uta, found in the Andean highlands was related predominantly with a single zymodeme of L. mexicana. Conclusion Our data show a high degree of phenotypic variation within species, and some evidence for associations between specific variants of ATL (i.e. Uta and LRC and specific Leishmania zymodemes. This study further defines the geographic distribution of Leishmania species and clinical variants of ATL in Ecuador.

Activity evaluation from different native or irradiated with {sup 60} Co gamma rays snake venoms and their inhibitory effect on Leishmania (Leishmania) amazonensis; Avaliacao da atividade de diferentes venenos de serpentes, nativos ou irradiados, com radiacao gama de {sup 60} Co, quanto ao poder inibitorio do crescimento de Leishmania (Leishmania) amazonensis

Energy Technology Data Exchange (ETDEWEB)

Lourenco, Cecilia de Oliveira

2000-07-01

Cutaneous leishmaniasis is a disease, caused by Leishmania parasites, that occurs frequently in tropical and sub-tropical regions of the world. Skin lesions that could results in disfiguring aspect characterize it. The treatment is based on few drugs as antimony salts or pentamidine that are toxic with increasing resistance by the parasite. Alternative forms of disease treatment are in constant search, including natural components as snake venoms. Previous studies demonstrate that some components of snake venoms have an inhibitory effect against those parasites, including Leishmania species. Although snake venoms presented high toxicity, several methods have been described to detoxify most or some of their toxic components, with favorable results by the use of gamma irradiation. In this report we tested several native and irradiated snake venoms for inhibitory effect against Leishmania (Leishmania) amazonensis parasite and LLCMK{sub 2} mammalian cells, with enzymatic tests and electrophoresis. There are significant activity in Acanthophis antarcticus, Agkistrodon bilineatus, Bothrops moojeni, Bothrops jararaca, Hoplocephalus stephensi, Naja melanoleuca, Naja mossambica, Pseudechis australis, Pseudechis colletti, Pseudechis guttatus and Pseudechis porphyriacus, venom being inactive Pseudonaja textilis, Notechis ater niger, Notechis scutatus. Oxyuranus microlepidotus and Oxyuranus scutellatus venoms. After 2 KGy of {sup 60}Co irradiation most venom loses significantly their activity. Venoms with antileishmanial activity presented L-amino acid oxidase (L-AO) activity and showed common protein with a molecular weight about 60kDa in SDS-PAGE. These results indicate that L-AO activity in those venoms are probably related with antileishmanial effect. (author)

A diagnostic assay based on variable intergenic region distinguishes between Leishmania donovani and Leishmania infantum

Czech Academy of Sciences Publication Activity Database

Chocholová, Eva; Jirků, Milan; Lukeš, Julius

2008-01-01

Roč. 55, č. 1 (2008), s. 75-78 ISSN 0015-5683 R&D Projects: GA MŠk LC07032; GA MŠk 2B06129 Institutional research plan: CEZ:AV0Z60220518 Keywords : Leishmania * assay * diagnosis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.307, year: 2008

Detection of Leishmania spp. in Bats from an Area of Brazil Endemic for Visceral Leishmaniasis.

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de Rezende, M B; Herrera, H M; Carvalho, C M E; Carvalho Anjos, E A; Ramos, C A N; de Araújo, F R; Torres, J M; de Oliveira, C E

2017-12-01

The multihost parasites Leishmania spp. infect a broad range of wild mammalian species including bats. Several species of bats have adapted to a variety of food resources and shelters in urban areas. This study aimed to detect Leishmania spp. DNA in bats present in forest fragments located in metropolitan areas endemic for leishmaniasis in Campo Grande, Mato Grosso do Sul (MS), Brazil. Blood samples were obtained from 80 individuals, including eight species of Phyllostomidae and one species of Vespertilionidae. Thirty of the 80 bats were positive for Leishmania spp. using conventional PCR, all belonging to the family Phyllostomidae. Eighteen samples tested by real-time PCR (qPCR) using specific primers for the kDNA of Leishmania infantum were positive. To the best of our knowledge, this is the first report detecting Leishmania spp. in Platyrrhinus incarum in addition to being the first reported detection of L. infantum in the bat species Phyllostomus discolor, Platyrrhinus lineatus, Artibeus planirostris and Artibeus lituratus. Our results show that bats can host Leishmania spp. in areas endemic for leishmaniasis, which must be taken into account in disease control operations by public health authorities. © 2017 Blackwell Verlag GmbH.

Leishmania development in sand flies: parasite-vector interactions overview.

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Dostálová, Anna; Volf, Petr

2012-12-03

Leishmaniases are vector-borne parasitic diseases with 0.9 - 1.4 million new human cases each year worldwide. In the vectorial part of the life-cycle, Leishmania development is confined to the digestive tract. During the first few days after blood feeding, natural barriers to Leishmania development include secreted proteolytic enzymes, the peritrophic matrix surrounding the ingested blood meal and sand fly immune reactions. As the blood digestion proceeds, parasites need to bind to the midgut epithelium to avoid being excreted with the blood remnant. This binding is strictly stage-dependent as it is a property of nectomonad and leptomonad forms only. While the attachment in specific vectors (P. papatasi, P. duboscqi and P. sergenti) involves lipophosphoglycan (LPG), this Leishmania molecule is not required for parasite attachment in other sand fly species experimentally permissive for various Leishmania. During late-stage infections, large numbers of parasites accumulate in the anterior midgut and produce filamentous proteophosphoglycan creating a gel-like plug physically obstructing the gut. The parasites attached to the stomodeal valve cause damage to the chitin lining and epithelial cells of the valve, interfering with its function and facilitating reflux of parasites from the midgut. Transformation to metacyclic stages highly infective for the vertebrate host is the other prerequisite for effective transmission. Here, we review the current state of knowledge of molecular interactions occurring in all these distinct phases of parasite colonization of the sand fly gut, highlighting recent discoveries in the field.

Natural infection of Didelphis aurita (Mammalia: Marsupialia with Leishmania infantum in Brazil

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Carreira João Carlos

2012-06-01

Full Text Available Abstract Background The opossum Didelphis have been considered as natural hosts of Leishmania parasites in the New World, suggesting an important role in the epidemiology of Visceral Leishmaniasis (VL. Among six extant species that belong to the genus Didelphis, only two (D. marsupialis and D. albiventris, have been mentioned as natural hosts of Leishmania infantum in Brazil and Colombia. In the present paper, it is reported for the first time, the observation of intracellular parasites (amastigotes in tissues of Didelphis aurita naturally infected with Leishmania infantum in Brazil. We also discuss some aspects associated to the relationship between L. infantum and the geographical distribution of some species of the genus Didelphis. Methods The opossums studied were caught by wire traps (Tomahawk in Barra de Guaratiba, a peri-urban area in Rio de Janeiro. The opossums were killed with an overdose of Thiopental sodium.At necropsy, macroscopic alterations were examined and samples from liver, spleen, lymph nodes, ear, abdominal skin, scent glands and bone marrow were collected for parasitological and molecular diagnoses. Results Forty-eight opossums were captured in an AVL endemic region, 30 being caught in a mangrove area and eighteen animals in a forest area near to some residential-yards. Among the thirty opossums trapped in the mangrove area, all of them were negative by both imprint and sera samples assayed on Dipstick Tests, that is a test based on a combination of protein-A colloidal gold conjugate and rk39 Leishmania antigen to detect anti-Leishmania antibody in serum or plasma. At the macroscopic examination one out of eighteen opossums, caught close to the forest, presented alterations compatible with spleen hypertrophy and three were positive by Dipstick Tests (16.6% and presented amastigotes in the spleen and in one of them, the parasites were also observed in a submandibular lymph node. Leishmania infantum infections were confirmed

PKC/ROS-Mediated NLRP3 Inflammasome Activation Is Attenuated by Leishmania Zinc-Metalloprotease during Infection

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Jung, Jee Yong; Chang, Kwang-Poo; Olivier, Martin

2015-01-01

Parasites of the Leishmania genus infect and survive within macrophages by inhibiting several microbicidal molecules, such as nitric oxide and pro-inflammatory cytokines. In this context, various species of Leishmania have been reported to inhibit or reduce the production of IL-1β both in vitro and in vivo. However, the mechanism whereby Leishmania parasites are able to affect IL-1β production and secretion by macrophages is still not fully understood. Dependent on the stimulus at hand, the maturation of IL-1β is facilitated by different inflammasome complexes. The NLRP3 inflammasome has been shown to be of pivotal importance in the detection of danger molecules such as inorganic crystals like asbestos, silica and malarial hemozoin, (HZ) as well as infectious agents. In the present work, we investigated whether Leishmania parasites modulate NLRP3 inflammasome activation. Using PMA-differentiated THP-1 cells, we demonstrate that Leishmania infection effectively inhibits macrophage IL-1β production upon stimulation. In this context, the expression and activity of the metalloprotease GP63 - a critical virulence factor expressed by all infectious Leishmania species - is a prerequisite for a Leishmania-mediated reduction of IL-1β secretion. Accordingly, L. mexicana, purified GP63 and GP63-containing exosomes, caused the inhibition of macrophage IL-1β production. Leishmania-dependent suppression of IL-1β secretion is accompanied by an inhibition of reactive oxygen species (ROS) production that has previously been shown to be associated with NLRP3 inflammasome activation. The observed loss of ROS production was due to an impaired PKC-mediated protein phosphorylation. Furthermore, ROS-independent inflammasome activation was inhibited, possibly due to an observed GP63-dependent cleavage of inflammasome and inflammasome-related proteins. Collectively for the first time, we herein provide evidence that the protozoan parasite Leishmania, through its surface

Using Proteomics to Understand How Leishmania Parasites Survive inside the Host and Establish Infection.

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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.

Molecular Characterization of Leishmania Species Isolated from Cutaneous Leishmaniasis in Yemen

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Mahdy, Mohammed A. K.; Al-Mekhlafi, Hesham M.; Al-Mekhlafi, Abdulsalam M.; Lim, Yvonne A. L.; Bin Shuaib, Naemah O. M.; Azazy, Ahmed A.; Mahmud, Rohela

2010-01-01

Background Cutaneous leishmaniasis (CL) is a neglected tropical disease endemic in the tropics and subtropics with a global yearly incidence of 1.5 million. Although CL is the most common form of leishmaniasis, which is responsible for 60% of DALYs lost due to tropical-cluster diseases prevalent in Yemen, available information is very limited. Methodology/Principal Findings This study was conducted to determine the molecular characterization of Leishmania species isolated from human cutaneous lesions in Yemen. Dermal scrapes were collected and examined for Leishmania amastigotes using the Giemsa staining technique. Amplification of the ribosomal internal transcribed spacer 1(ITS-1) gene was carried out using nested PCR and subsequent sequencing. The sequences from Leishmania isolates were subjected to phylogenetic analysis using the neighbor-joining and maximum parsimony methods. The trees identified Leishmania tropica from 16 isolates which were represented by two sequence types. Conclusions/Significance The predominance of the anthroponotic species (i.e. L. tropica) indicates the probability of anthroponotic transmission of cutaneous leishmaniasis in Yemen. These findings will help public health authorities to build an effective control strategy taking into consideration person–to-person transmission as the main dynamic of transmission of CL. PMID:20862227

Leishmaniasis in Turkey: Visceral and cutaneous leishmaniasis caused by Leishmania donovani in Turkey

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Özbilgin, Ahmet; Harman, Mehmet; Karakuş, Mehmet; Bart, Aldert; Töz, Seray; Kurt, Özgür; Çavuş, İbrahim; Polat, Erdal; Gündüz, Cumhur; van Gool, Tom; Özbel, Yusuf

2017-01-01

In Turkey, the main causative agents are Leishmania tropica (L. tropica) and Leishmania infantum (L. infantum) for cutaneous leishmaniasis (CL) and L. infantum for visceral leishmaniasis (VL). In this study, we investigated leishmaniasis cases caused by L. donovani and established animal models for

Curcumin overcomes the inhibitory effect of nitric oxide on Leishmania.

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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.

The current status of phlebotomine sandflies (Diptera: Psychodidae) in Tunisia and their role on Leishmania transmission: A review

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Ahmed Tabbabi; Sajida Sboui; Jabeur Daaboub

2017-01-01

In Tunisia, the epidemiological situation of leishmaniasis is characterized by the coexistence in a rather circumscribed territory (165000 km2, including the Sahara) of 4 forms of leishmaniasis caused by 3 species: Leishmania infantum, Leishmania major and Leishmania tropica (L. tropica) (synonymous Leishmania killicki). One of the factors determining the clinical, epidemiological and immunological diversity of leishmanioses is certainly the existence of a vector-parasite specificity of of...

Current relevance of fungal and trypanosomatid glycolipids and sphingolipids: studies defining structures conspicuously absent in mammals

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Helio K. Takahashi

2009-09-01

Full Text Available Recently, glycosphingolipids have been attracting attention due to their role on biological systems as second messengers or modulators of signal transduction, affecting several events, which range from apoptosis to regulation of the cell cycle. In pathogenic fungi, glycolipids are expressed in two classes: neutral monohexosylceramides (glucosyl-or galactosylceramide and acidic glycosylinositol phosphorylceramides (the latter class carries longer glycan chains. It is worth to mention that monohexosylceramides exhibit significant structural differences in their lipid moieties compared to their mammalian counterparts, whereas the glycosylinositol phosphorylceramides exhibit remarkable structural differences in their carbohydrate moieties in comparison to mammal glycosphingolipids counterpart. We observed that glycosylinositol phosphorylceramides are capable of promoting immune response in infected humans. In addition, inhibiting fungal glycosphingolipid biosynthetic pathways leads to an inhibition of colony formation, spore germination, cell cycle, dimorphism and hyphal growth. Other pathogens, such as trypanosomatids, also present unique glycolipids, which may have an important role for the parasite development and/or disease establishment. Regarding host-pathogen interaction, cell membrane rafts, which are enriched in sphingolipids and sterols, participate in parasite/fungal infection. In this review, it is discussed the different biological roles of (glyco (sphingolipids of pathogenic/opportunistic fungi and trypanosomatids.Recentemente, glicoesfingolipídeos têm atraído atenção devido ao seu papel na biologia celular como segundo-mensageiro ou moduladores da transdução de sinal, afetando vários eventos, desde apoptose até a regulação do ciclo celular. Em fungos patogênicos, existem duas classes de glicolipídeos: monohexosil ceramidas neutras (glucosil-ou galactosilceramida e glicosilinositol fosforilceramidas (os quais apresentam

Comparison of Tetrazolium Salt Assays for Evaluation of Drug Activity against Leishmania spp.

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Ginouves, Marine; Carme, Bernard; Couppie, Pierre

2014-01-01

In French Guiana, leishmaniasis is an essentially cutaneous infection. It constitutes a major public health problem, with a real incidence of 0.2 to 0.3%. Leishmania guyanensis is the causal species most frequently encountered in French Guiana. The treatment of leishmaniasis is essentially drug based, but the therapeutic compounds available have major side effects (e.g., liver damage and diabetes) and must be administered parenterally or are costly. The efficacy of some of these agents has declined due to the emergence of resistance in certain strains of Leishmania. There is currently no vaccine against leishmaniasis, and it is therefore both necessary and urgent to identify new compounds effective against Leishmania. The search for new drugs requires effective tests for evaluations of the leishmanicidal activity of a particular molecule or extract. Microculture tetrazolium assays (MTAs) are colorimetric tests based on the use of tetrazolium salts. We compared the efficacies of three tetrazolium salts—3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8)—for quantification of the promastigotes of various species of Leishmania. We found that the capacity of Leishmania to metabolize a tetrazolium salt depended on the salt used and the species of Leishmania. WST-8 was the tetrazolium salt best metabolized by L. guyanensis and gave the best sensitivity. PMID:24719447

Arginase activity in pathogenic and non-pathogenic species of Leishmania parasites.

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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

AVALIAÇÃO DA TERAPIA FOTODINÂMICA COM AZUL DE METILENO EM Leishmania major e Leishmania braziliensis: ESTUDO in vitro

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Danielle El Atra Coelho

2017-03-01

Full Text Available A Leishmaníase é uma doença crônica causada pelo protozoário do gênero Leishmania, cujo tratamento é agressivo. A Terapia Fotodinâmica (TFD é uma alternativa promissora que combina luz, fotossensibilizador (FS e oxigênio molecular, para causar a morte celular. O objetivo desse trabalho foi avaliar, in vitro, a ação da TFD com Azul de metileno (AM em promastigotas de Leishmania, por teste de MTT, curva de crescimento e morfologia do parasito. O teste de MTT demonstrou alteração de ambas as espécies após interação com o AM no escuro e após TFD. A análise das curvas demonstrou que a TFD influenciou o crescimento das espécies. A análise morfológica revelou que o AM no escuro não causou alterações expressivas como a TFD, sendo a cepa de L. braziliensis mais afetada que a cepa de L. major. Pode-se concluir que a TFD com AM foi promissora contra promastigotas de Leishmania, pois foi capaz de diminuir o crescimento e alterar a morfologia dos parasitos em cultura.

Surveillance for antibodies to Leishmania spp. in dogs from Sri Lanka and India

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The global distribution of leishmaniasis is rapidly expanding into new geographic regions. Dogs are the primary reservoir hosts for human visceral leishmaniasis (VL) caused by infection with Leishmania infantum. Natural infections with other Leishmania species can occur in dogs, but their role as re...

Application of the microscopic method in cutaneous leishmania diagnosis

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Mohammed Wael Daboul

2011-10-01

Full Text Available Introduction: Cutaneous leishmania is spreading fast. This study aims at developing the microscopic method to achieve a full detection of all positive cases of leishmania.Methods: 50 human cases have been studied by applying microscopic smears stained with Wright stain. Microscopic photos were taken for the presumed unfamiliar figures.Results: Mononuclear cells with tails are present at a rate of (98%. They are associated with Leishman Donovan (LD bodies in 50% of the cases. The polygonal figures and the spherical forms are present at the same rate (60% and are associated with LD bodies in 24% of the cases. The small promastigote like forms are seen at a rate of (76% and are associated with LD bodies in 26% of the cases. The giant promastigotes like forms are present in (80% of the cases and are associated with LD bodies in 28% of the cases. Candle flame forms are present in (40% of the cases and are associated with the LD bodies in 21% of the cases.Discussion: It is applicable to use those discovered figures in diagnosing cutaneous leishmania.

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

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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.

Natural infection of phlebotomines (Diptera: Psychodidae) by Leishmania (Leishmania) amazonensis in an area of ecotourism in Central-Western Brazil.

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Brilhante, Andreia Fernandes; Nunes, Vânia Lúcia Brandão; Kohatsu, Kleber Augusto; Galati, Eunice Aparecida Bianchi; Rocca, Maria Elizabeth Ghizzi; Ishikawa, Edna Aoba Yassui

2015-01-01

Bonito municipality, known as an area of ecoturism, in Mato Grosso do Sul state, Brazil, is also a focus of visceral and cutaneous leishmaniases, with cases registered in both human and canine populations. This study sought to investigate natural infection by flagellate forms of Leishmania in phlebotomines of the urban area of Bonito. Sand flies were collected fortnightly from October 2005 to July 2006 with modified automatic light traps installed in peridomiciles and animal shelters in the center and on the outskirts of the city. The females were dissected and their guts observed under an optical microscope. A total of 1977 specimens were captured, Lutzomyia longipalpis (88.4 %) and Bichromomyia flaviscutelata (3.0 %) being the most frequent species. Bi. flaviscutellata was found infected by flagellates that were identified as Leishmania (Leishmania) amazonensis by indirect immunofluorescence reaction, employing monoclonal antibodies and the biotin-avidin system. This is the first report of natural infection by L. amazonensis in Bi. flaviscutellata in a Brazilian urban area. As Bi. flaviscutellata is only slightly attracted by humans, the transmission of L. amazonensis in the study area may have a zoonotic character; however, the sympatric occurrence of this parasite and Lu. longipalpis should be taken into consideration by the local health authorities since this sand fly has already been found with L. amazonensis DNA in a focus of canine visceral leishmaniasis in Bonito municipality.

Lipid Biosynthesis as an Antifungal Target

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Jiao Pan

2018-04-01

Full Text Available Lipids, commonly including phospholipids, sphingolipids, fatty acids, sterols, and triacylglycerols (TAGs, are important biomolecules for the viability of all cells. Phospholipids, sphingolipids, and sterols are important constituents of biological membranes. Many lipids play important roles in the regulation of cell metabolism by acting as signaling molecules. Neutral lipids, including TAGs and sterol esters (STEs, are important storage lipids in cells. In view of the importance of lipid molecules, this review briefly summarizes the metabolic pathways for sterols, phospholipids, sphingolipids, fatty acids, and neutral lipids in fungi and illustrates the differences between fungal and human (or other mammalian cells, especially in relation to lipid biosynthetic pathways. These differences might provide valuable clues for us to find target proteins for novel antifungal drugs. In addition, the development of lipidomics technology in recent years has supplied us with a shortcut for finding new antifungal drug targets; this ability is important for guiding our research on pathogenic fungi.

Leishmania-specific surface antigens show sub-genus sequence variation and immune recognition.

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Daniel P Depledge

2010-09-01

Full Text Available A family of hydrophilic acylated surface (HASP proteins, containing extensive and variant amino acid repeats, is expressed at the plasma membrane in infective extracellular (metacyclic and intracellular (amastigote stages of Old World Leishmania species. While HASPs are antigenic in the host and can induce protective immune responses, the biological functions of these Leishmania-specific proteins remain unresolved. Previous genome analysis has suggested that parasites of the sub-genus Leishmania (Viannia have lost HASP genes from their genomes.We have used molecular and cellular methods to analyse HASP expression in New World Leishmania mexicana complex species and show that, unlike in L. major, these proteins are expressed predominantly following differentiation into amastigotes within macrophages. Further genome analysis has revealed that the L. (Viannia species, L. (V. braziliensis, does express HASP-like proteins of low amino acid similarity but with similar biochemical characteristics, from genes present on a region of chromosome 23 that is syntenic with the HASP/SHERP locus in Old World Leishmania species and the L. (L. mexicana complex. A related gene is also present in Leptomonas seymouri and this may represent the ancestral copy of these Leishmania-genus specific sequences. The L. braziliensis HASP-like proteins (named the orthologous (o HASPs are predominantly expressed on the plasma membrane in amastigotes and are recognised by immune sera taken from 4 out of 6 leishmaniasis patients tested in an endemic region of Brazil. Analysis of the repetitive domains of the oHASPs has shown considerable genetic variation in parasite isolates taken from the same patients, suggesting that antigenic change may play a role in immune recognition of this protein family.These findings confirm that antigenic hydrophilic acylated proteins are expressed from genes in the same chromosomal region in species across the genus Leishmania. These proteins are

Effect of Kelussia odoratissima Mozaff essential oil on promastigot form of Leishmania major (in vitro)

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Pirali Kheirabadi Khodadad; Saei Dehkordi Siavash; Kheibari Parviz

2015-01-01

Introduction: Leishmaniasis is a zoonotic disease caused by a protozoan of the genus Leishmania. In this study, the effects of Kelussia odoratissima Mozaff essential oil on the promastigot form of Leishmania major were studied. Methods: In this study, the effects of Kelussia odoratissima Mozaff essential oil on the promastigot form of Leishmania major were assessed by calculating the average number of surviving promastigots after exposure to different concentrations of essential oil, relativ...

Sand fly-Leishmania interactions: long relationships are not necessarily easy

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Ramalho-Ortigao, Marcelo; Saraiva, Elvira M.; Traub-Csekö, Yara M.

2010-01-01

Sand fly and Leishmania are one of the best studied vector-parasite models. Much is known about the development of these parasites within the sand fly, and how transmission to a suitable vertebrate host takes place. Various molecules secreted by the vector assist the establishment of the infection in a vertebrate, and changes to the vector are promoted by the parasites in order to facilitate or enhance transmission. Despite a generally accepted view that sand flies and Leishmania are also one...

Skin-resident memory CD4+ T cells enhance protection against Leishmania major infection.

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Glennie, Nelson D; Yeramilli, Venkata A; Beiting, Daniel P; Volk, Susan W; Weaver, Casey T; Scott, Phillip

2015-08-24

Leishmaniasis causes a significant disease burden worldwide. Although Leishmania-infected patients become refractory to reinfection after disease resolution, effective immune protection has not yet been achieved by human vaccines. Although circulating Leishmania-specific T cells are known to play a critical role in immunity, the role of memory T cells present in peripheral tissues has not been explored. Here, we identify a population of skin-resident Leishmania-specific memory CD4+ T cells. These cells produce IFN-γ and remain resident in the skin when transplanted by skin graft onto naive mice. They function to recruit circulating T cells to the skin in a CXCR3-dependent manner, resulting in better control of the parasites. Our findings are the first to demonstrate that CD4+ TRM cells form in response to a parasitic infection, and indicate that optimal protective immunity to Leishmania, and thus the success of a vaccine, may depend on generating both circulating and skin-resident memory T cells. © 2015 Glennie et al.

The development of Leishmania turanica in sand flies and competition with L. major.

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Chajbullinova, Alsu; Votypka, Jan; Sadlova, Jovana; Kvapilova, Katerina; Seblova, Veronika; Kreisinger, Jakub; Jirku, Milan; Sanjoba, Chizu; Gantuya, Sambuu; Matsumoto, Yoshitsugu; Volf, Petr

2012-10-02

In Central Asian foci of zoonotic cutaneous leishmaniases, mixed infections of Leishmania turanica and L. major have been found in a reservoir host (the great gerbil, Rhombomys opimus) as well as in the sand fly vector Phlebotomus papatasi, but hybrids between these two Leishmania species have never been reported. In addition, the role of sand fly species other than P. papatasi in L. turanica circulation is not clear. In this work we compared the development of L. turanica in three sand fly species belonging to different subgenera. In addition, we studied experimental co-infections of sand flies by both Leishmania species using GFP transfected L. turanica (MRHO/MN/08/BZ18(GFP+)) and RFP transfected L. major (WHOM/IR/-/173-DsRED(RFP+)). The possibility of Leishmania genetic exchange during the vectorial part of the life cycle was studied using flow cytometry combined with immunofluorescent microscopy. Late-stage infections of L. turanica with frequent colonization of the stomodeal valve were observed in the specific vector P. (Phlebotomus) papatasi and in the permissive vector P. (Adlerius) arabicus. On the other hand, in P. sergenti (the specific vector of L. tropica), L. turanica promatigotes were present only until the defecation of bloodmeal remnants. In their natural vector P. papatasi, L. turanica and L. major developed similarly, and the spatiotemporal dynamics of localization in the sand fly gut was the same for both leishmania species. Fluorescence microscopy in combination with FACS analyses did not detect any L. major / L. turanica hybrids in the experimental co-infection of P. papatasi and P. duboscqi. Our data provide new insight into the development of different leishmania parasite species during a mixed infection in the sand fly gut. Despite the fact that both Leishmania species developed well in P. papatasi and P. duboscqi and did not outcompete each other, no genetic exchange was found. However, the ability of L. turanica to establish late

Total Leishmania antigens with Poly(I:C) induce Th1 protective response.

Science.gov (United States)

Sanchez, M V; Eliçabe, R J; Di Genaro, M S; Germanó, M J; Gea, S; García Bustos, M F; Salomón, M C; Scodeller, E A; Cargnelutti, D E

2017-11-01

Our proposal was to develop a vaccine based on total Leishmania antigens (TLA) adjuvanted with polyinosinic-polycytidylic acid [Poly(I:C)] able to induce a Th1 response which can provide protection against Leishmania infection. Mice were vaccinated with two doses of TLA-Poly(I:C) administered by subcutaneous route at 3-week interval. Humoral and cellular immune responses induced by the immunization were measured. The protective efficacy of the vaccine was evaluated by challenging mice with infective promastigotes of Leishmania (Leishmania) amazonensis into the footpad. Mice vaccinated with TLA-Poly(I:C) showed a high anti-Leishmania IgG titre, as well as increased IgG1 and IgG2a subclass titres compared with mice vaccinated with the TLA alone. The high IgG2a indicated a Th1 bias response induced by the TLA-Poly(I:C) immunization. Accordingly, the cellular immune response elicited by the formulation was characterized by an increased production of IFN-γ and no significant production of IL-4. The TLA-Poly(I:C) immunization elicited good protection, which was associated with decreased footpad swelling, a lower parasite load and a reduced histopathological alteration in the footpad. Our findings demonstrate a promising vaccine against cutaneous leishmaniasis that is relatively economic and easy to develop and which should be taken into account for preventing leishmaniasis in developing countries. © 2017 John Wiley & Sons Ltd.

Leishmania chagasi/infantum : further investigations on Leishmania tropisms in atypical cutaneous and visceral leishmaniasis foci in Central America

NARCIS (Netherlands)

Campos Ponce, M.; Ponce, C.; Ponce, E; Maingon, R.D.

2005-01-01

In Central America, apparently genetically identical Leishmania chagasi/infantum parasites cause cutaneous (CL) and visceral leishmaniasis (VL), the latter being more frequent in young children. The present study investigated if there were pathology-related differences in virulence between Honduran

Leishmania chagasi/infantum: further investigations on Leishmania tropisms in atypical cutaneous and visceral leishmaniasis foci in Central America.

NARCIS (Netherlands)

Campos Ponce, M.; Ponce, C.; Ponce, E.; Maingon, R.D.

2005-01-01

In Central America, apparently genetically identical Leishmania chagasi/infantum parasites cause cutaneous (CL) and visceral leishmaniasis (VL), the latter being more frequent in young children. The present study investigated if there were pathology-related differences in virulence between Honduran

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

Science.gov (United States)

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.

HIV aspartyl peptidase inhibitors interfere with cellular proliferation, ultrastructure and macrophage infection of Leishmania amazonensis.

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Lívia O Santos

Full Text Available BACKGROUND: Leishmania is the etiologic agent of leishmanisais, a protozoan disease whose pathogenic events are not well understood. Current therapy is suboptimal due to toxicity of the available therapeutic agents and the emergence of drug resistance. Compounding these problems is the increase in the number of cases of Leishmania-HIV coinfection, due to the overlap between the AIDS epidemic and leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: In the present report, we have investigated the effect of HIV aspartyl peptidase inhibitors (PIs on the Leishmania amazonensis proliferation, ultrastructure, interaction with macrophage cells and expression of classical peptidases which are directly involved in the Leishmania pathogenesis. All the HIV PIs impaired parasite growth in a dose-dependent fashion, especially nelfinavir and lopinavir. HIV PIs treatment caused profound changes in the leishmania ultrastructure as shown by transmission electron microscopy, including cytoplasm shrinking, increase in the number of lipid inclusions and some cells presenting the nucleus closely wrapped by endoplasmic reticulum resembling an autophagic process, as well as chromatin condensation which is suggestive of apoptotic death. The hydrolysis of HIV peptidase substrate by L. amazonensis extract was inhibited by pepstatin and HIV PIs, suggesting that an aspartyl peptidase may be the intracellular target of the inhibitors. The treatment with HIV PIs of either the promastigote forms preceding the interaction with macrophage cells or the amastigote forms inside macrophages drastically reduced the association indexes. Despite all these beneficial effects, the HIV PIs induced an increase in the expression of cysteine peptidase b (cpb and the metallopeptidase gp63, two well-known virulence factors expressed by Leishmania spp. CONCLUSIONS/SIGNIFICANCE: In the face of leishmaniasis/HIV overlap, it is critical to further comprehend the sophisticated interplays among Leishmania

Impact of Leishmania mexicana infection on dendritic cell signaling and functions.

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Irazú Contreras

2014-09-01

Full Text Available Leishmania parasites have the ability to modify macrophage signaling pathways in order to survive and multiply within its mammalian host. They are also known to invade other cells including neutrophils, fibroblasts and dendritic cells (DCs. DCs have an important role in immunity as the link between innate and adaptive immunity, necessary for the development of an effective response; however, the impact of Leishmania mexicana infection on DCs has been poorly studied. Herein, we report that Leishmania infection rapidly induced DC protein tyrosine phosphatases activity, leading to MAP kinases inactivation. In line with this, L. mexicana was found to decrease the nuclear translocation of transcription factors such as AP-1 and NF-κB. Concomitantly, L. mexicana-infected DCs showed reduced expression of several surface antigen-presenting and co-stimulatory molecules upon LPS stimulation. Leishmania-induced interference on DC maturation was further reflected by their reduced capacity to present OVA antigen to OVA-specific T cells, as shown by abrogation of IL-2 production by the T cells. Collectively, our data revealed that DC infection by L. mexicana appears to affect the cellular and immunological mechanisms necessary for the development of an effective and protective immune response, therefore favouring the survival and propagation of the parasite within its host.

Riesgo de transmisión de Leishmania (Kinetoplastida: Trypanosomatidae en Mérida Venezuela

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Elsa Nieves

2014-09-01

Full Text Available La leishmaniasis es una enfermedad causada por la infección de un parásito protozoario del género Leishmania, transmitido por la picada de insectos hematófagos conocidos como flebotominos. El estudio tiene como objetivo determinar la presencia de flebotominos en los Distritos Sanitarios del estado Mérida y diseñar un mapa de riesgo de transmisión entomológico. Se utilizaron cuatro métodos de captura de flebotominos, los ejemplares se identificaron y se les determinó la infección natural por Leishmania. Se estimó la riqueza de especies, y se realizó un proceso analítico Jerárquico. Los resultados muestran la presencia de diversas especies de flebotominos en los Distritos Sanitarios del estado Mérida, siendo las especies de mayor frecuencia L. youngi, L. gomezi, L. ovallesi y L. walkeri. Se detectó 2,1% de infección natural con Leishmania, la cual se encontró en las 4 especies más frecuentes. Se presenta un mapa de riesgo de transmisión entomológico para el estado Mérida. El conocimiento de la situación actual de los vectores de Leishmania en el estado Mérida y el riesgo de transmisión son relevantes a la hora de considerar la prevención y posible surgimiento de nuevos brotes de leishmaniasis. Abstract (english The leishmaniasis is a disease caused by infection with a protozoan parasite of the genus Leishmania, transmitted by the bite of blood-sucking insects known as sandflies. The study aims to determine the presence of sandflies in Merida state health districts and design a map of entomological risk of transmission. Four methods capture sandflies were used, the specimens were identified and natural Leishmania infection was determined. The richness species was estimated and analityc Hierarchie procesess was performed. The results show the presence of various species of sandflies in Merida state health districts, L. youngi, L. gomezi, L. ovallesi and L. walkeri were most abundant species. The 2.1% of natural infection

Extracellular Expression in Aspergillus niger of an Antibody Fused to Leishmania sp. Antigens.

Science.gov (United States)

Magaña-Ortíz, Denis; Fernández, Francisco; Loske, Achim M; Gómez-Lim, Miguel A

2018-01-01

Nucleoside hydrolase and sterol 24-c-methyltransferase, two antigenic proteins of Leishmania sp., were expressed in Aspergillus niger. Genetic transformation of conidia was achieved using underwater shock waves. scFv antibody addressed to DEC205, a receptor of dendritic cells, was fused to two proteins of Leishmania sp. Receptor 205 has a relevant role in the immune system in mammals; it can modulate T cell response to different antigens. Extracellular expression strategy of recombinant antibody was achieved using a fragment of native glucoamylase A (514 aa) as a carrier. Fermentations in shake flasks showed that the recombinant protein (104 kDa) was expressed and secreted only when maltose was used as carbon source; on the contrary, the expression was highly repressed in presence of xylose. Noteworthy, recombinant protein was secreted without glucoamylase-carrier and accumulation at intracellular level was not observed. The results presented here demonstrate the high value of Aspergillus niger as biotechnological platform for recombinant antibodies against Leishmania sp. at low cost. To the best of our knowledge, this is the first report about the recombinant expression of antigenic proteins of Leishmania sp. in filamentous fungi. The protein obtained can be used to explore novel strategies to induce immunity against Leishmania sp. or it can be employed in diagnostic kits to detect this neglected disease.

Comparative effects of fumonisins on sphingolipid metabolism and toxicity in ducks and turkeys.

Science.gov (United States)

Benlasher, Emad; Geng, Xiuyu; Nguyen, Ngoc Thanh Xuan; Tardieu, Didier; Bailly, Jean-Denis; Auvergne, Alain; Guerre, Philippe

2012-03-01

Fumonisins (FBs) are mycotoxins that are found worldwide in maize and maize products. Their main toxic effects have been well characterized in poultry, but differences between species have been demonstrated. Ducks appeared very sensitive to toxicity, whereas turkeys are more resistant. At the same time, alterations of sphingolipid metabolism, with an increase of the concentration of the free sphinganine (Sa) in serum and liver, have been demonstrated in the two species, but the link between the toxicity of FBs and Sa accumulation remains difficult to interpret. The aim of the present work was to compare the effects of FBs (10 mg FB1 + FB2/kg body weight) on sphingolipid metabolism in ducks and turkeys. Growth, feed consumption, and serum biochemistry were also investigated to evaluate toxicity. The main results showed that FBs increased Sa concentrations in liver and serum in ducks and turkeys, but these accumulations were not directly correlated with toxicity. Sa accumulation was higher in the livers of turkeys than in ducks, whereas Sa levels were higher in the sera of ducks than in turkeys. Hepatic toxicity was more pronounced in ducks than in turkeys and accompanied a decrease of body weight and an increase of serum biochemistry in ducks but not in turkeys. So, although FBs increase Sa concentration in the livers of both species, this effect is not directly proportional to toxicity. The mechanisms of FB toxicity and/or the mechanisms of protection of ducks and turkeys to the Sa accumulation within the liver remain to be established.

Antileishmanial activity of licochalcone A in mice infected with Leishmania major and in hamsters infected with Leishmania donovani

DEFF Research Database (Denmark)

Chen, M; Christensen, S B; Theander, T G

1994-01-01

This study was designed to examine the antileishmanial activity of the oxygenated chalcone licochalcone A in mice and hamsters infected with Leishmania parasites. Intraperitoneal administration of licochalcone A at doses of 2.5 and 5 mg/kg of body weight per day completely prevented lesion...... development in BALB/c mice infected with Leishmania major. Treatment of hamsters infected with L. donovani with intraperitoneal administration of licochalcone A at a dose of 20 mg/kg of body weight per day for 6 consecutive days resulted in a > 96% reduction of parasite load in the liver and the spleen...... consecutive days resulted in > 65 and 85% reductions of L. donovani parasite loads in the liver and the spleen, respectively, compared with those of untreated control hamsters. These data clearly demonstrate that licochalcone A is a promising lead for the development of a new drug against leishmaniases....

Characterization of monomeric DNA-binding protein Histone H1 in Leishmania braziliensis.

Science.gov (United States)

Carmelo, Emma; González, Gloria; Cruz, Teresa; Osuna, Antonio; Hernández, Mariano; Valladares, Basilio

2011-08-01

Histone H1 in Leishmania presents relevant differences compared to higher eukaryote counterparts, such as the lack of a DNA-binding central globular domain. Despite that, it is apparently fully functional since its differential expression levels have been related to changes in chromatin condensation and infectivity, among other features. The localization and the aggregation state of L. braziliensis H1 has been determined by immunolocalization, mass spectrometry, cross-linking and electrophoretic mobility shift assays. Analysis of H1 sequences from the Leishmania Genome Database revealed that our protein is included in a very divergent group of histones H1 that is present only in L. braziliensis. An antibody raised against recombinant L. braziliensis H1 recognized specifically that protein by immunoblot in L. braziliensis extracts, but not in other Leishmania species, a consequence of the sequence divergences observed among Leishmania species. Mass spectrometry analysis and in vitro DNA-binding experiments have also proven that L. braziliensis H1 is monomeric in solution, but oligomerizes upon binding to DNA. Finally, despite the lack of a globular domain, L. braziliensis H1 is able to form complexes with DNA in vitro, with higher affinity for supercoiled compared to linear DNA.

Serological and molecular survey of Leishmania infection in dogs from Luanda, Angola.

Science.gov (United States)

Vilhena, Hugo; Granada, Sara; Oliveira, Ana Cristina; Schallig, Henk D F H; Nachum-Biala, Yaarit; Cardoso, Luís; Baneth, Gad

2014-03-24

Canine leishmaniosis (CanL) due to Leishmania infantum is a global zoonosis endemic in more than 70 countries in Europe, North Africa, Asia and America; however, data on this infection is scarce from southern Africa. The aim of this study was to survey dogs in Luanda, Angola, for Leishmania infection. One hundred-and-three dogs presented to a veterinary medical centre in Luanda were serologically and molecularly assessed for Leishmania with the direct agglutination test (DAT) and polymerase chain reaction (PCR). Two dogs were seropositive, with DAT titres of 800 and ≥6400; the latter was also found to be PCR-positive and confirmed to be infected with L. infantum by DNA sequence analysis. No other dog was found to be PCR-positive. The first dog had been imported from Portugal, but the latter had never left Angola (neither had its parents), strongly suggesting an autochthonous infection. Although other cases of CanL have previously been described in the country, this is the first reported study of canine Leishmania infection at the population level, as well as the first report on the molecular characterization of L. infantum in dogs from Angola.

Natural Product Biosynthetic Diversity and Comparative Genomics of the Cyanobacteria.

Science.gov (United States)

Dittmann, Elke; Gugger, Muriel; Sivonen, Kaarina; Fewer, David P

2015-10-01

Cyanobacteria are an ancient lineage of slow-growing photosynthetic bacteria and a prolific source of natural products with intricate chemical structures and potent biological activities. The bulk of these natural products are known from just a handful of genera. Recent efforts have elucidated the mechanisms underpinning the biosynthesis of a diverse array of natural products from cyanobacteria. Many of the biosynthetic mechanisms are unique to cyanobacteria or rarely described from other organisms. Advances in genome sequence technology have precipitated a deluge of genome sequences for cyanobacteria. This makes it possible to link known natural products to biosynthetic gene clusters but also accelerates the discovery of new natural products through genome mining. These studies demonstrate that cyanobacteria encode a huge variety of cryptic gene clusters for the production of natural products, and the known chemical diversity is likely to be just a fraction of the true biosynthetic capabilities of this fascinating and ancient group of organisms. Copyright © 2015. Published by Elsevier Ltd.

Synthesis and processing of sphingolipid activator protein-2 (SAP-2) in cultured human fibroblasts

International Nuclear Information System (INIS)

Fujibayashi, S.; Wenger, D.A.

1986-01-01

Sphingolipid activator proteins (SAP) are relatively small molecular weight proteins that stimulate the enzymatic hydrolysis of sphingolipids in the presence of specific lysosomal hydrolases. SAP-2 has previously been demonstrated to activate the hydrolysis of glucosylceramide, galactosylceramide, and, possibly, sphingomyelin. Using monospecific rabbit antibodies against human spleen SAP-2, the synthesis and processing of SAP-2 were studied in cultured human fibroblasts. When [ 35 S]methionine was presented in the medium to control human cells for 4 h, five major areas of radiolabeling were found. These had apparent molecular weights of 73,000, 68,000, 50,000, 12,000, and 9000. Further studies indicated that the major extracellular product in normal cells given NH4Cl along with the [ 35 S]methionine and in medium from cultures from patients with I cell disease had an apparent molecular weight of 73,000. The Mr = 68,000 and 73,000 species can be converted to a species with an apparent molecular weight of 50,000 by the action of endoglycosidase F. After labeling cells for 1 h followed by a 1-h chase, the Mr = 12,000 and 9000 species appear. Treatment of the immunoprecipitated mixture with endoglycosidase F resulted in conversion of these species to one band with an apparent molecular weight of 7600. These studies indicate that this relatively low molecular weight protein is rapidly synthesized from a relatively large molecular weight highly glycosylated precursor

Key roles of Arf small G proteins and biosynthetic trafficking for animal development.

Science.gov (United States)

Rodrigues, Francisco F; Harris, Tony J C

2017-04-14

Although biosynthetic trafficking can function constitutively, it also functions specifically for certain developmental processes. These processes require either a large increase to biosynthesis or the biosynthesis and targeted trafficking of specific players. We review the conserved molecular mechanisms that direct biosynthetic trafficking, and discuss how their genetic disruption affects animal development. Specifically, we consider Arf small G proteins, such as Arf1 and Sar1, and their coat effectors, COPI and COPII, and how these proteins promote biosynthetic trafficking for cleavage of the Drosophila embryo, the growth of neuronal dendrites and synapses, extracellular matrix secretion for bone development, lumen development in epithelial tubes, notochord and neural tube development, and ciliogenesis. Specific need for the biosynthetic trafficking system is also evident from conserved CrebA/Creb3-like transcription factors increasing the expression of secretory machinery during several of these developmental processes. Moreover, dysfunctional trafficking leads to a range of developmental syndromes.

An agent-based model for Leishmania major infection

Science.gov (United States)

Dancik, Garrett M.; Jones, Douglas E.; Dorman, Karin S.

Leishmania are protozoan parasites transmitted by bites of infected sandflies. Over 20 species of Leishmania, endemic in 88 countries, are capable of causing human disease. Disease is either cutaneous, where skin ulcers occur on exposed surfaces of the body, or visceral, with near certain mortality if untreated. C3HeB/FeJ mice are resistant to L. major, but develop chronic cutaneous lesions when infected with another species L. amazonensis. The well-characterized mechanism of resistance to L. major depends on a CD4+ Thl immune response, macrophage activation, and elimination of the parasite [Sacks 2002]. The factors that account for host susceptibility to L. Amazonensis, however, are not completely understood, despite being generally attributed to a weakened Th1 response [Vanloubbeck 2004].

Detection of Leishmania parasites in the blood of patients with isolated cutaneous leishmaniasis.

Science.gov (United States)

Nakkash-Chmaisse, Hania; Makki, Raja; Nahhas, Georges; Knio, Khouzama; Nuwayri-Salti, Nuha

2011-07-01

The consequences of the spread of Leishmania parasites to the blood from lesions in patients with cutaneous leishmaniasis are numerous. To assess the magnitude of this invasion we conducted the present study on patients referred to the American University of Beirut Medical Center for cutaneous leishmaniasis. Patients referred for the management of cutaneous leishmaniasis were included in the study. Skin and blood cultures for Leishmania were taken from these patients. One hundred sixty-two patients were proven to have cutaneous leishmaniasis by pathology; 52% were males and 44% females (gender information was missing for 4%). Patient age ranged from 5 months to 70 years. None of the patients had received treatment for Leishmania. We obtained parasite isolates from 85 patients (52.5%), proven by cultures from skin and blood/blood components. Interestingly, the parasite was isolated in the blood and blood components of 50 patients (30.9%). Isoenzyme analysis confirmed the fact that the organisms in blood and skin were the same; from the 28 isolates that were positive in both skin and blood, eight isolates were Leishmania major and two were Leishmania tropica. The remaining isolates, whether positive in both blood and skin or in either of these tissues, skin or blood and its products, were Leishmania infantum sensu lato. In the current study, the detection rate of parasites in the blood of patients with cutaneous leishmaniasis was high. This illustrates the invasive characteristic of the parasite that has escaped the skin. Testing should be considered in areas other than Lebanon, especially around the Mediterranean basin. Whether these findings support the administration of systemic treatment for cutaneous leishmaniasis or not needs to be confirmed in larger prospective studies. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

Dual Transcriptome Profiling of Leishmania-Infected Human Macrophages Reveals Distinct Reprogramming Signatures.

Science.gov (United States)

Fernandes, Maria Cecilia; Dillon, Laura A L; Belew, Ashton Trey; Bravo, Hector Corrada; Mosser, David M; El-Sayed, Najib M

2016-05-10

Macrophages are mononuclear phagocytes that constitute a first line of defense against pathogens. While lethal to many microbes, they are the primary host cells of Leishmania spp. parasites, the obligate intracellular pathogens that cause leishmaniasis. We conducted transcriptomic profiling of two Leishmania species and the human macrophage over the course of intracellular infection by using high-throughput RNA sequencing to characterize the global gene expression changes and reprogramming events that underlie the interactions between the pathogen and its host. A systematic exclusion of the generic effects of large-particle phagocytosis revealed a vigorous, parasite-specific response of the human macrophage early in the infection that was greatly tempered at later time points. An analogous temporal expression pattern was observed with the parasite, suggesting that much of the reprogramming that occurs as parasites transform into intracellular forms generally stabilizes shortly after entry. Following that, the parasite establishes an intracellular niche within macrophages, with minimal communication between the parasite and the host cell later during the infection. No significant difference was observed between parasite species transcriptomes or in the transcriptional response of macrophages infected with each species. Our comparative analysis of gene expression changes that occur as mouse and human macrophages are infected by Leishmania spp. points toward a general signature of the Leishmania-macrophage infectome. Little is known about the transcriptional changes that occur within mammalian cells harboring intracellular pathogens. This study characterizes the gene expression signatures of Leishmania spp. parasites and the coordinated response of infected human macrophages as the pathogen enters and persists within them. After accounting for the generic effects of large-particle phagocytosis, we observed a parasite-specific response of the human macrophages early in

Occurrence of bioactive sphingolipids in meat and fish products

DEFF Research Database (Denmark)

Hellgren, Lars

2001-01-01

/neutral glycolipids varied from 1 to 2.9, while in poultry this ratio varied between 5.2 to 19.2 and in red meat it varied from 1.6 to 8.3. The fatty acid composition of sphingomyelin in fish was dominated by C24:1 (Delta (9)) or C22:1 (Delta (9)), while C16:0 and C18:0 were the dominating sphingomyelin species....... Therefore we investigated the contents of sphingomyelin and neutral glycosphingolipids in commonly consumed meat and fish products. Sphingomyelin and glycosphingolipids were found in all foodstuffs studied. The total amount varied between 118 +/- 17 nmol/g (cod) to 589 +/- 39 nmol/g (chicken leg). Generally......, lower amounts of sphingolipids were determined in fish meat than in red meat and poultry, while poultry was the richest source of this class of lipids. However, fish meat contained a relatively high content of neutral glycolipids compared with other types of meat. Thus, in fish the ratio sphingomyelin...

Trypanosome lytic factor, an antimicrobial high-density lipoprotein, ameliorates Leishmania infection.

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Marie Samanovic

2009-01-01

Full Text Available Innate immunity is the first line of defense against invading microorganisms. Trypanosome Lytic Factor (TLF is a minor sub-fraction of human high-density lipoprotein that provides innate immunity by completely protecting humans from infection by most species of African trypanosomes, which belong to the Kinetoplastida order. Herein, we demonstrate the broader protective effects of human TLF, which inhibits intracellular infection by Leishmania, a kinetoplastid that replicates in phagolysosomes of macrophages. We show that TLF accumulates within the parasitophorous vacuole of macrophages in vitro and reduces the number of Leishmania metacyclic promastigotes, but not amastigotes. We do not detect any activation of the macrophages by TLF in the presence or absence of Leishmania, and therefore propose that TLF directly damages the parasite in the acidic parasitophorous vacuole. To investigate the physiological relevance of this observation, we have reconstituted lytic activity in vivo by generating mice that express the two main protein components of TLFs: human apolipoprotein L-I and haptoglobin-related protein. Both proteins are expressed in mice at levels equivalent to those found in humans and circulate within high-density lipoproteins. We find that TLF mice can ameliorate an infection with Leishmania by significantly reducing the pathogen burden. In contrast, TLF mice were not protected against infection by the kinetoplastid Trypanosoma cruzi, which infects many cell types and transiently passes through a phagolysosome. We conclude that TLF not only determines species specificity for African trypanosomes, but can also ameliorate an infection with Leishmania, while having no effect on T. cruzi. We propose that TLFs are a component of the innate immune system that can limit infections by their ability to selectively damage pathogens in phagolysosomes within the reticuloendothelial system.

Infección de fibroblastos de piel de animales con distinto grado de susceptibilidad a Leishmania infantum y Leishmania mexicana (Kinetoplastida: Trypanosomatidae

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Miguel Angel Minero

2004-03-01

Full Text Available En este estudio se presenta un modelo in vitro de cultivo de fibroblastos de piel de hámster, ratón y rata hecho con el propósito de determinar diferencias en cuanto a la susceptibilidad a la infección por dos especies del género Leishmania (Kinetoplastida: Trypanosomatidae. Se realizó además un estudio ultraestructural por microscopía electrónica de transmisión con el fin de establecer si las formas intracelulares observadas correspondían a multiplicación interna o a fagocitosis múltiple. Se estudió la multiplicación de los parásitos en los fibroblastos de las tres especies de roedores infectados tanto por Leishmania infantum como por L. mexicana (cepa OCR y las diferencias entre las tres fueron estadísticamente significativas (pInfection and multiplication of Leishmania infantum and L. mexicana inside of skin fibroblasts from hamsters, mice and rats was achieved. This process was demonstrated either by counting parasites inside the stained cells or by electronic microscopy studies. In addition multiplication rate differences in the cells from these rodent species were determined, for L. infantum as well as for L. mexicana. Parasite development in hamsters and mice fibroblasts was evident but there was not multiplication in rat cells showing that apparently they are refractory to Leishmania infection. These results suggest that the parasite affinity for each animal, as well as any intracellular environment resistance, could involve genetic factors in the parasite multiplication. On the other hand, presence of amastigote multiplication inside of parasitophorus vacuole, showed by electronic microscopy images, probes a true parasite transformation. Therefore it is suggested that fibroblasts could work as host cells for parasite survival and permanency in the infected animals

Leishmania infection modulates beta-1 integrin activation and alters the kinetics of monocyte spreading over fibronectin

Science.gov (United States)

Figueira, Cláudio Pereira; Carvalhal, Djalma Gomes Ferrão; Almeida, Rafaela Andrade; Hermida, Micely d’ El-Rei; Touchard, Dominique; Robert, Phillipe; Pierres, Anne; Bongrand, Pierre; dos-Santos, Washington LC

2015-01-01

Contact with Leishmania leads to a decreases in mononuclear phagocyte adherence to connective tissue. In this work, we studied the early stages of bond formation between VLA4 and fibronectin, measured the kinetics of membrane alignment and the monocyte cytoplasm spreading area over a fibronectin-coated surface, and studied the expression of high affinity integrin epitope in uninfected and Leishmania-infected human monocytes. Our results show that the initial VLA4-mediated interaction of Leishmania-infected monocyte with a fibronectin-coated surface is preserved, however, the later stage, leukocyte spreading over the substrate is abrogated in Leishmania-infected cells. The median of spreading area was 72 [55–89] μm2 for uninfected and 41 [34–51] μm2 for Leishmania-infected monocyte. This cytoplasm spread was inhibited using an anti-VLA4 blocking antibody. After the initial contact with the fibronectrin-coated surface, uninfected monocyte quickly spread the cytoplasm at a 15 μm2 s−1 ratio whilst Leishmania-infected monocytes only made small contacts at a 5.5 μm2 s−1 ratio. The expression of high affinity epitope by VLA4 (from 39 ± 21% to 14 ± 3%); and LFA1 (from 37 ± 32% to 18 ± 16%) molecules was reduced in Leishmania-infected monocytes. These changes in phagocyte function may be important for parasite dissemination and distribution of lesions in leishmaniasis. PMID:26249106

Disseminated Leishmaniasis Caused by Leishmania Tropica in a Puppy from Karaj, Central Iran

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M Mohebali

2011-06-01

Full Text Available A 5-month old puppy with muco-cutaneous lesions in the chin, around lips and eyes was exam­ined physically and microscopically for leishmaniasis. Muco-cutaneous lesions containing a large num­ber of amastigotes of Leishmania spp. were observed. Amastigotes were also detected in liver and spleen of the puppy. The animal was positive with Dipstick rK39 kit and high level of anti-Leishmania antibodies was detected by direct agglutination test (DAT. DNA, Using PCR-RFLP technique extracted from cultured Leishmania promastigotes and L. tropica was identified. This is the first report of concurrent mucosal and visceral involvement of L. tropica in a puppy from Iran.

Cyclic nucleotide specific phosphodiesterases of Leishmania major

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Linder Markus

2006-03-01

Full Text Available Abstract Background Leishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major. Results This paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range

Kinetics of growth of Leishmania (Leishmania chagasi cycle in McCoy cell culture Cinéticas de crescimento do ciclo da Leishmania (Leishmania chagasi em cultura de células McCoy

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Yeda L. Nogueira

2006-12-01

Full Text Available The kinetics of growth of Leishmania performed in vitro after internalization of the promastigote form in the cell and the occurrence of the transformation of the parasite into the amastigote form have been described by several authors. They used explants of macrophages in hamster spleen cell culture or in a human macrophage lineage cell, the U937. Using microscopy, the description of morphologic inter-relationship and the analysis of the production of specific molecules, it has been possible to define some of the peculiarities of the biology of the parasite. The present study shows the growth cycle of Leishmania chagasi during the observation of kinetic analysis undertaken with a McCoy cell lineage that lasted for a period of 144 hours. During the process, the morphologic transformation was revealed by indirect immunofluorescence (IF and the molecules liberated in the extra cellular medium were observed by SDS-PAGE at 24-hour intervals during the whole 144-hour period. It was observed that in the first 72 hours the promastigote form of L. chagasi adhered to the cell membranes and assumed a rounded (amastigote-like form. At 96 hours the infected cells showed morphologic alterations; at 120 hours the cells had liberated soluble fluorescent antigens into the extra cellular medium. At 144 hours, new elongated forms of the parasites, similar to promastigotes, were observed. In the SDS-PAGE, specific molecular weight proteins were observed at each point of the kinetic analysis showing that the McCoy cell imitates the macrophage and may be considered a useful model for the study of the infection of the Leishmania/cell binomial.Cinéticas de crescimento de Leishmania realizadas in vitro após a internalização da forma promastigota na célula e a ocorrência da transformação do parasito na forma amastigota foram descritas por vários autores, seja com a utilização de explantes de macrófagos em células de baço de hamster ou atualmente da c

Canine cutaneous leishmaniasis caused by neotropical Leishmania infantum despite of systemic disease: A case report.

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Cavalcanti, Amanda; Lobo, Rogério; Cupolillo, Elisa; Bustamante, Fábio; Porrozzi, Renato

2012-12-01

Visceral leishmaniasis is an anthropozoonosis caused by a protozoan Leishmania infantum (syn. Leishmania chagasi). Here, we report a typical case of canine cutaneous leishmaniasis due to L. infantum infection without any other systemic symptom in one dog in the city of Rio de Janeiro, Brazil. A mongrel female dog was admitted in a veterinary clinic with reports of chronic wounds in the body. Physical examination revealed erosive lesions in the limbs, nasal ulcers, presence of ectoparasites and seborrheic dermatitis. Blood samples and fragments of healthy and injured skin were collected. The complete hemogram revealed aregenerative normocytic normochromic anemia and erythrocyte rouleaux, and biochemical analysis revealed normal renal and hepatic functions. Cytology of the muzzle and skin lesions suggested pyogranulomatous inflammatory process. The histopathology of a skin fragment was performed and revealed suspicion of protozoa accompanied by necrotizing dermatitis. The diagnosis of leishmaniasis was accomplished by positive serology, isolation of Leishmania from the skin lesion, and also by molecular test (PCR targeting the conserved region of Leishmania kDNA). Culture was positive for damaged skin samples. PCR targeting a fragment of Leishmania hsp70 gene was performed employing DNA extracted from damaged skin. RFLP of the amplified hsp70 fragment identified the parasite as L. infantum, instead of Leishmania braziliensis, the main agent of cutaneous leishmaniasis in Rio de Janeiro. Characterization of isolated promastigotes by five different enzymatic systems confirmed the species identification of the etiological agent. Serology was positive by ELISA and rapid test. This case warns to the suspicion of viscerotropic Leishmania in cases of chronic skin lesions and brings the discussion of the mechanisms involved in the parasite tissue tropism. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Could Phlebotomus mascittii play a role as a natural vector for Leishmania infantum? New data.

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Obwaller, Adelheid G; Karakus, Mehmet; Poeppl, Wolfgang; Töz, Seray; Özbel, Yusuf; Aspöck, Horst; Walochnik, Julia

2016-08-19

The occurrence of phlebotomine sand flies in Central Europe was questioned until they were recorded for the first time in Germany in 1999, and ten years later also in Austria. The aim of this study was to investigate sand flies collected in Austria for their carrier status of Leishmania spp. From 2012 to 2013 field studies were conducted in eastern Austria. Altogether, 22 individuals of sand flies were found, all morphologically identified as Phlebotomus (Transphlebotomus) mascittii Grassi, 1908. Twelve non-engorged female specimens with no visible remnants of a blood meal in their bodies were individually investigated for Leishmania spp. by ITS-1 real-time PCR. One out of these was positive for Leishmania, identified as Leishmania infantum by DNA sequencing. This finding suggests that L. infantum is not excreted by P. mascittii and possibly can establish an infection within P. mascittii. Interestingly, an asymptomatic dog living on the farm where this sand fly had been caught was also Leishmania-positive. This study provides new data on the suspected vector capacity of P. mascittii, being the northernmost sand fly species in Europe and in most central European regions the only sand fly species found. Proven vector capacity of P. mascittii for Leishmania spp. would be of significant medico-veterinary importance, not only with respect to expanding sand fly populations in Central Europe related to global warming, but also in the light of globalization and increasing movements of humans.

Leishmania and its quest for iron: An update and overview.

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Zaidi, Amir; Singh, Krishn Pratap; Ali, Vahab

2017-01-01

Parasites of genus Leishmania are the causative agents of complex neglected diseases called leishmaniasis and continue to be a significant health concern globally. Iron is a vital nutritional requirement for virtually all organisms, including pathogenic trypanosomatid parasites, and plays a crucial role in many facets of cellular metabolism as a cofactor of several enzymes. Iron acquisition is essential for the survival of parasites. Yet parasites are also vulnerable to the toxicity of iron and reactive oxygen species. The aim of this review is to provide an update on the current knowledge about iron acquisition and usage by Leishmania species. We have also discussed about host strategy to modulate iron availability and the strategies deployed by Leishmania parasites to overcome iron withholding defences and thus favour parasite growth within host macrophages. Since iron plays central roles in the host's response and parasite metabolism, a comprehensive understanding of the iron metabolism is beneficial to identify potential viable therapeutic opportunities against leishmaniasis. Copyright © 2016 Elsevier B.V. All rights reserved.

Deception and Manipulation: The Arms of Leishmania, a Successful Parasite

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Cecílio, Pedro; Pérez-Cabezas, Begoña; Santarém, Nuno; Maciel, Joana; Rodrigues, Vasco; Cordeiro da Silva, Anabela

2014-01-01

Leishmania spp. are intracellular parasitic protozoa responsible for a group of neglected tropical diseases, endemic in 98 countries around the world, called leishmaniasis. These parasites have a complex digenetic life cycle requiring a susceptible vertebrate host and a permissive insect vector, which allow their transmission. The clinical manifestations associated with leishmaniasis depend on complex interactions between the parasite and the host immune system. Consequently, leishmaniasis can be manifested as a self-healing cutaneous affliction or a visceral pathology, being the last one fatal in 85–90% of untreated cases. As a result of a long host–parasite co-evolutionary process, Leishmania spp. developed different immunomodulatory strategies that are essential for the establishment of infection. Only through deception and manipulation of the immune system, Leishmania spp. can complete its life cycle and survive. The understanding of the mechanisms associated with immune evasion and disease progression is essential for the development of novel therapies and vaccine approaches. Here, we revise how the parasite manipulates cell death and immune responses to survive and thrive in the shadow of the immune system. PMID:25368612

Deception and Manipulation: the arms of Leishmania, a successful parasite

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Pedro eCecílio

2014-10-01

Full Text Available Leishmania spp. are intracellular parasitic protozoa responsible for a group of neglected tropical diseases, endemic in 98 countries around the world, called leishmaniasis. These parasites have a complex digenetic life cycle requiring a susceptible vertebrate host and a permissive insect vector, which allow their transmission. The clinical manifestations associated with leishmaniasis depend on complex interactions between the parasite and the host immune system. Consequently, leishmaniasis can be manifested as a self-healing cutaneous affliction or a visceral pathology, being the last one fatal in 85-90% of untreated cases. As a result of a long host-parasite co-evolutionary process, Leishmania spp. developed different immunomodulatory strategies that are essential for the establishment of infection. Only through deception and manipulation of the immune system, Leishmania spp. can complete its life cycle and survive. The understanding of the mechanisms associated with immune evasion and disease progression is essential for the development of novel therapies and vaccine approaches. Here, we revise how the parasite manipulates cell death and immune responses to survive and thrive in the shadow of the immune system.

Molecular Identification of Leishmania spp. in Sand Flies (Diptera: Psychodidae: Phlebotominae) in the Lençóis Maranhenses National Park, Brazil.

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Pereira-Filho, Adalberto Alves; Fonteles, Raquel Silva; Bandeira, Maria da Conceição Abreu; Moraes, Jorge Luiz Pinto; Rebêlo, José Manuel Macário; Melo, Maria Norma

2018-02-20

Sand flies are very common in the region of Lençóis Maranhenses National Park, an important tourist attraction in Brazil. However, the role of some species and their relative importance locally in Leishmania Ross 1903 transmission is unclear. The objective of this study was to identify Leishmania infection in phlebotomine sand flies collected around the Lençóis Maranhenses National Park, an important conservation area and popular international/national tourist destination with a high incidence of leishmaniasis. Sand flies were collected in peridomiciliary areas on the tourist route from September 2012 to August 2013. The captured females were subjected to molecular analyses for the detection of Leishmania DNA. Sand flies were infected with four Leishmania species: Leishmania (Viannia) braziliensis (Vianna, 1911) was found in Lutzomyia whitmani (Antunes and Coutinho, 1939) (2.1%) and Lutzomyia longipalpis (Lutz and Neiva, 1912) (1.7%); Leishmania (Leishmania) infantum (Nicole, 1908) infected Lutzomyia wellcomei (Fraiha, Shaw, and Lainson, 1971) (20%), Lutzomyia sordellii (Shannon and Del Ponte, 1927) (4.3%), Lu. longipalpis (3.7%), and Lu. whitmani (0.8%); Leishmania (Leishmania) amazonensis (Lainson & Shaw, 1972) was found in Lu. whitmani (0.58%), while Leishmania (Viannia) lainsoni infected Lutzomyia evandroi (Costa Lima and Antunes, 1936) (3.4%), Lu. longipalpis (1.06%), and Lu. whitmani (0.29%). The occurrence of these parasites requires control measures to reduce the incidence of cutaneous leishmaniasis and to contain a possible epidemic of visceral leishmaniasis, the most severe form of the disease.

Detection of Leishmania amazonensis and Leishmania braziliensis in Culicoides (Diptera, Ceratopogonidae) in an endemic area of cutaneous leishmaniasis in the Brazilian Amazonia.

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Rebêlo, José Manuel Macário; Rodrigues, Bruno Leite; Bandeira, Maria da Conceição Abreu; Moraes, Jorge Luiz Pinto; Fonteles, Raquel Silva; Pereira, Silma Regina Ferreira

2016-12-01

Biting midges in the genus Culicoides act as vectors of arboviruses throughout the world and as vectors of filariasis in Latin America, the Caribbean, and parts of Africa. Although Culicoides spp. are currently not considered to be vectors of Leishmania protozoa, the high abundance of biting midges in areas with active cutaneous leishmaniasis transmission points to the possibility of Culicoides infection by these pathogens. We used PCR to test captured Culicoides species for natural infection with Leishmania spp. We tested 450 Culicoides females, divided into 30 pools of 15 individuals each, as follows: nine pools of C. foxi (135 specimens), seven pools of C. filariferus (105), seven pools of C. insignis (105), five pools of C. ignacioi (75), and two pools of C. flavivenula (30). PCR confirmed the presence of Leishmania braziliensis DNA in C. ignacioi (0.14%), C. insignis (0.14%), and C. foxi (0.11); and Le. amazonensis DNA in C. filariferus (0.14%) and C. flavivenula (0.50%). We conclude that these Culicoides species can be naturally infected, but vector competence and transmission capability must be confirmed in future studies. Our results warrant further investigation into the role of these biting midge species in the leishmaniasis epidemiological cycle. © 2016 The Society for Vector Ecology.

Polymerase chain reaction-based method for the identification of Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in mucosal tissues conserved in paraffin.

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Prestes, Suzane Ribeiro; Guerra, Jorge Augusto de Oliveira; Romero, Gustavo Adolfo Sierra; Magalhaes, Laylah Kelre Costa; Santana, Rosa Amelia Gonçalves; Maciel, Marcel Gonçalves; Custódio, Ana; Barbosa, Maria das Graças Vale; Silveira, Henrique

2015-01-01

In the Americas, mucosal leishmaniasis is primarily associated with infection by Leishmania (Viannia) braziliensis. However, Leishmania (Viannia) guyanensis is another important cause of this disease in the Brazilian Amazon. In this study, we aimed at detecting Leishmaniadeoxyribonucleic acid (DNA) within paraffin-embedded fragments of mucosal tissues, and characterizing the infecting parasite species. We evaluated samples collected from 114 patients treated at a reference center in the Brazilian Amazon by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses. Direct examination of biopsy imprints detected parasites in 10 of the 114 samples, while evaluation of hematoxylin and eosin-stained slides detected amastigotes in an additional 17 samples. Meanwhile, 31/114 samples (27.2%) were positive for Leishmania spp. kinetoplast deoxyribonucleic acid (kDNA) by PCR analysis. Of these, 17 (54.8%) yielded amplification of the mini-exon PCR target, thereby allowing for PCR-RFLP-based identification. Six of the samples were identified as L. (V.) braziliensis, while the remaining 11 were identified as L. (V.) guyanensis. The results of this study demonstrate the feasibility of applying molecular techniques for the diagnosis of human parasites within paraffin-embedded tissues. Moreover, our findings confirm that L. (V.) guyanensisis a relevant causative agent of mucosal leishmaniasis in the Brazilian Amazon.

A nitrous acid biosynthetic pathway for diazo group formation in bacteria.

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Sugai, Yoshinori; Katsuyama, Yohei; Ohnishi, Yasuo

2016-02-01

Although some diazo compounds have bioactivities of medicinal interest, little is known about diazo group formation in nature. Here we describe an unprecedented nitrous acid biosynthetic pathway responsible for the formation of a diazo group in the biosynthesis of the ortho-diazoquinone secondary metabolite cremeomycin in Streptomyces cremeus. This finding provides important insights into the biosynthetic pathways not only for diazo compounds but also for other naturally occurring compounds containing nitrogen-nitrogen bonds.

Leishmania (Viannia) braziliensis infection in wild small mammals in ecotourism area of Brazil.

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Tonelli, Gabriel Barbosa; Tanure, Aline; Rego, Felipe Dutra; Carvalho, Gustavo Mayr de Lima; Stumpp, Rodolfo; Ássimos, Gabriela Ribeiro; Campos, Aldenise Martins; Lima, Ana Cristina Viana Mariano da Rocha; Gontijo, Célia Maria Ferreira; Paz, Gustavo Fontes; Andrade Filho, José Dilermando

2017-01-01

Leishmaniases are parasitic diseases transmitted to mammalian hosts by sand fly vectors (Diptera: Psychodidae). Despite the increasing occurrence of visceral and cutaneous leishmaniasis cases in urban centers, their transmission still occur primarily in wild environments and may be associated with professional activities and recreation, such as ecotourism. The Reserva Particular do Patrimônio Natural Santuário do Caraça (RPPNSC) is one of the largest ecotourism attractions in the State of Minas Gerais, Brazil, and comprises an area of environmental preservation with 11,233 hectares presenting a transitional vegetation between Cerrado and Atlantic Forest. The present study describes the abundance of small mammals in RPPNSC, the isolation and identification of Leishmania in five wild animals. Small mammals were bimonthly trapped along 6 trails within the RPPNSC with 10 Tomahawk traps each. Two trails were located in peridomiciliary areas near tourist lodging facilities, and four trails were located at sites visited by tourists in forest areas. The most prevalent species were Akodon cursor, Cerradomys subflavus and Oligoryzomys nigripes. Six isolates of Leishmania were obtained from these animals and identified as Leishmania braziliensis through HSP70-PCR RFLP method. Leishmania spp. DNA was detected by kDNA-PCR method and isolated by biphasic culture. Studies point to some of the captured species as potential wild reservoirs of Leishmania, suggesting they may be involved in the transmission cycle in these wild environments.

Making Sense of the Yeast Sphingolipid Pathway.

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Megyeri, Márton; Riezman, Howard; Schuldiner, Maya; Futerman, Anthony H

2016-12-04

Sphingolipids (SL) and their metabolites play key roles both as structural components of membranes and as signaling molecules. Many of the key enzymes and regulators of SL metabolism were discovered using the yeast Saccharomyces cerevisiae, and based on the high degree of conservation, a number of mammalian homologs were identified. Although yeast continues to be an important tool for SL research, the complexity of SL structure and nomenclature often hampers the ability of new researchers to grasp the subtleties of yeast SL biology and discover new modulators of this intricate pathway. Moreover, the emergence of lipidomics by mass spectrometry has enabled the rapid identification of SL species in yeast and rendered the analysis of SL composition under various physiological and pathophysiological conditions readily amenable. However, the complex nomenclature of the identified species renders much of the data inaccessible to non-specialists. In this review, we focus on parsing both the classical SL nomenclature and the nomenclature normally used during mass spectrometry analysis, which should facilitate the understanding of yeast SL data and might shed light on biological processes in which SLs are involved. Finally, we discuss a number of putative roles of various yeast SL species. Copyright © 2016 Elsevier Ltd. All rights reserved.

Microbial stasis of Leishmania enriettii in activated guinea pig macrophages

International Nuclear Information System (INIS)

Groocock, C.M.; Soulsby, E.J.L.

1980-01-01

Peritoneal exudate cells (PEC) from Leishmania-sensitized guinea pigs were cultured in vitro in the presence (activated) or absence (non-activated) of leishmanial antigen for 24 or 48 hours. These were then labelled with 51 Cr and challenged with 125 I-labelled promastigotes. The changing relationship between the macrophage and the parasite was monitored by observing changes in the ratio of the cell-associated isotopes. Highly significant differences in the ratio change developed during culture. These differences were a result of the activated cultures showing a higher release of 51 Cr and a lower release of 125 I when compared with the non-activated cells, at 12 hours the percentage release of 125 I from the parasite within the activated macrophage was fourfold less than that released by parasites within non-activated cells (9.2% versus 38.3%) and tenfold less than that released from glutaraldehyde-killed organisms phagocytosed by activated macrophages (91.6%). These studies indicate that stasis rather than killing of leishmaniae occurs in the activated macrophage in vitro. Parallel experiments evaluated by the visual counting of leishmaniae within the macrophages support these data. PEC from tuberculin-sensitized guinea pigs activated in vitro by purified protein derivative showed little or no activity against leishmaniae, indicating a specific requirement for this microbial stasis by activated macrophages. As a corollary of this, peritoneal exudate lymphocytes separated from the same preparations of PEC were shown to be specifically reactive to leishmanial antigen by transformation and incorporation of 3 H-thymidine. (author)

Changes to cholesterol trafficking in macrophages by Leishmania parasites infection.

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Semini, Geo; Paape, Daniel; Paterou, Athina; Schroeder, Juliane; Barrios-Llerena, Martin; Aebischer, Toni

2017-08-01

Leishmania spp. are protozoan parasites that are transmitted by sandfly vectors during blood sucking to vertebrate hosts and cause a spectrum of diseases called leishmaniases. It has been demonstrated that host cholesterol plays an important role during Leishmania infection. Nevertheless, little is known about the intracellular distribution of this lipid early after internalization of the parasite. Here, pulse-chase experiments with radiolabeled cholesteryl esterified to fatty acids bound to low-density lipoproteins indicated that retention of this source of cholesterol is increased in parasite-containing subcellular fractions, while uptake is unaffected. This is correlated with a reduction or absence of detectable NPC1 (Niemann-Pick disease, type C1), a protein responsible for cholesterol efflux from endocytic compartments, in the Leishmania mexicana habitat and infected cells. Filipin staining revealed a halo around parasites within parasitophorous vacuoles (PV) likely representing free cholesterol accumulation. Labeling of host cell membranous cholesterol by fluorescent cholesterol species before infection revealed that this pool is also trafficked to the PV but becomes incorporated into the parasites' membranes and seems not to contribute to the halo detected by filipin. This cholesterol sequestration happened early after infection and was functionally significant as it correlated with the upregulation of mRNA-encoding proteins required for cholesterol biosynthesis. Thus, sequestration of cholesterol by Leishmania amastigotes early after infection provides a basis to understand perturbation of cholesterol-dependent processes in macrophages that were shown previously by others to be necessary for their proper function in innate and adaptive immune responses. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Arginine and Polyamines Fate in Leishmania Infection

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Muxel, Sandra M.; Aoki, Juliana I.; Fernandes, Juliane C. R.; Laranjeira-Silva, Maria F.; Zampieri, Ricardo A.; Acuña, Stephanie M.; Müller, Karl E.; Vanderlinde, Rubia H.; Floeter-Winter, Lucile M.

2018-01-01

Leishmania is a protozoan parasite that alternates its life cycle between the sand fly and the mammalian host macrophages, involving several environmental changes. The parasite responds to these changes by promoting a rapid metabolic adaptation through cellular signaling modifications that lead to transcriptional and post-transcriptional gene expression regulation and morphological modifications. Molecular approaches such as gene expression regulation, next-generation sequencing (NGS), microRNA (miRNA) expression profiling, in cell Western blot analyses and enzymatic activity profiling, have been used to characterize the infection of murine BALB/c and C57BL/6 macrophages, as well as the human monocytic cell-lineage THP-1, with Leishmania amazonensis wild type (La-WT) or arginase knockout (La-arg-). These models are being used to elucidate physiological roles of arginine and polyamines pathways and the importance of arginase for the establishment of the infection. In this review, we will describe the main aspects of Leishmania-host interaction, focusing on the arginine and polyamines pathways and pointing to possible targets to be used for prognosis and/or in the control of the infection. The parasite enzymes, arginase and nitric oxide synthase-like, have essential roles in the parasite survival and in the maintenance of infection. On the other hand, in mammalian macrophages, defense mechanisms are activated inducing alterations in the mRNA, miRNA and enzymatic profiles that lead to the control of infection. Furthermore, the genetic background of both parasite and host are also important to define the fate of infection. PMID:29379478

Characterization of a midgut mucin-like glycoconjugate of Lutzomyia longipalpis with a potential role in Leishmania attachment.

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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.

Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted-H-region amplification of methotrexate-resistant Leishmania major

International Nuclear Information System (INIS)

Petrillo-Peixoto, M.L.; Beverley, S.M.

1988-01-01

We describe the structure of amplified DNA that was discovered in two laboratory stocks of the protozoan parasite Leishmania tarentolae. Restriction mapping and molecular cloning revealed that a region of 42 kilobases was amplified 8- to 30-fold in these lines. Southern blot analyses of digested DNAs or chromosomes separated by pulsed-field electrophoresis showed that the amplified DNA corresponded to the H region, a locus defined originally by its amplification in methotrexate-resistant Leishmania major. Similarities between the amplified DNA of the two species included (i) extensive cross-hybridization; (ii) approximate conservation of sequence order; (iii) extrachromosomal localization; (iv) an overall inverted, head-to-head configuration as a circular 140-kilobase tetrameric molecule; (v) two regions of DNA sequence rearrangement, each of which was closely associated with the two centers of the inverted repeats; (vi) association with methotrexate resistance; and (vii) phenotypically conservative amplification, in which the wild-type chromosomal arrangement was retained without apparent modification. Our data showed that amplified DNA mediating drug resistance arose in unselected L. tarentolae, although the pressures leading to apparently spontaneous amplification and maintenance of the H region are not known. The simple structure and limited extent of DNA amplified in these and other Leishmania lines suggests that the study of gene amplification in Leishmania spp. offers an attractive model system for the study of amplification in cultured mammalian cells and tumors. We also introduced a method for measuring the size of large circular DNAs, using gamma-irradiation to introduce limited double-strand breaks followed by sizing of the linear DNAs by pulsed-field electrophoresis

[Advance in flavonoids biosynthetic pathway and synthetic biology].

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Zou, Li-Qiu; Wang, Cai-Xia; Kuang, Xue-Jun; Li, Ying; Sun, Chao

2016-11-01

Flavonoids are the valuable components in medicinal plants, which possess a variety of pharmacological activities, including anti-tumor, antioxidant and anti-inflammatory activities. There is an unambiguous understanding about flavonoids biosynthetic pathway, that is,2S-flavanones including naringenin and pinocembrin are the skeleton of other flavonoids and they can transform to other flavonoids through branched metabolic pathway. Elucidation of the flavonoids biosynthetic pathway lays a solid foundation for their synthetic biology. A few flavonoids have been produced in Escherichia coli or yeast with synthetic biological technologies, such as naringenin, pinocembrin and fisetin. Synthetic biology will provide a new way to get valuable flavonoids and promote the research and development of flavonoid drugs and health products, making flavonoids play more important roles in human diet and health. Copyright© by the Chinese Pharmaceutical Association.

Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples.

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Abbasi, Ibrahim; Kirstein, Oscar D; Hailu, Asrat; Warburg, Alon

2016-10-01

Visceral leishmaniasis (VL), one of the most important neglected tropical diseases, is caused by Leishmania donovani eukaryotic protozoan parasite of the genus Leishmania, the disease is prevalent mainly in the Indian sub-continent, East Africa and Brazil. VL can be diagnosed by PCR amplifying ITS1 and/or kDNA genes. The current study involved the optimization of Loop-mediated isothermal amplification (LAMP) for the detection of Leishmania DNA in human blood or tissue samples. Three LAMP systems were developed; in two of those the primers were designed based on shared regions of the ITS1 gene among different Leishmania species, while the primers for the third LAMP system were derived from a newly identified repeated region in the Leishmania genome. The LAMP tests were shown to be sufficiently sensitive to detect 0.1pg of DNA from most Leishmania species. The green nucleic acid stain SYTO16, was used here for the first time to allow real-time monitoring of LAMP amplification. The advantage of real time-LAMP using SYTO 16 over end-point LAMP product detection is discussed. The efficacy of the real time-LAMP tests for detecting Leishmania DNA in dried blood samples from volunteers living in endemic areas, was compared with that of qRT-kDNA PCR. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Functional conservation of coenzyme Q biosynthetic genes among yeasts, plants, and humans.

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Kazuhiro Hayashi

Full Text Available Coenzyme Q (CoQ is an essential factor for aerobic growth and oxidative phosphorylation in the electron transport system. The biosynthetic pathway for CoQ has been proposed mainly from biochemical and genetic analyses of Escherichia coli and Saccharomyces cerevisiae; however, the biosynthetic pathway in higher eukaryotes has been explored in only a limited number of studies. We previously reported the roles of several genes involved in CoQ synthesis in the fission yeast Schizosaccharomyces pombe. Here, we expand these findings by identifying ten genes (dps1, dlp1, ppt1, and coq3-9 that are required for CoQ synthesis. CoQ10-deficient S. pombe coq deletion strains were generated and characterized. All mutant fission yeast strains were sensitive to oxidative stress, produced a large amount of sulfide, required an antioxidant to grow on minimal medium, and did not survive at the stationary phase. To compare the biosynthetic pathway of CoQ in fission yeast with that in higher eukaryotes, the ability of CoQ biosynthetic genes from humans and plants (Arabidopsis thaliana to functionally complement the S. pombe coq deletion strains was determined. With the exception of COQ9, expression of all other human and plant COQ genes recovered CoQ10 production by the fission yeast coq deletion strains, although the addition of a mitochondrial targeting sequence was required for human COQ3 and COQ7, as well as A. thaliana COQ6. In summary, this study describes the functional conservation of CoQ biosynthetic genes between yeasts, humans, and plants.

Mitochondrial Proteomics of Antimony and Miltefosine Resistant Leishmania infantum

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Isabel M. Vincent

2015-10-01

Full Text Available Antimony (SbIII and miltefosine (MIL are important drugs for the treatment of Leishmania parasite infections. The mitochondrion is likely to play a central role in SbIII and MIL induced cell death in this parasite. Enriched mitochondrial samples from Leishmania promastigotes selected step by step for in vitro resistance to SbIII and MIL were subjected to differential proteomic analysis. A shared decrease in both mutants in the levels of pyruvate dehydrogenase, dihydrolipoamide dehydrogenase, and isocitrate dehydrogenase was observed, as well as a differential abundance in two calcium-binding proteins and the unique dynamin-1-like protein of the parasite. Both mutants presented a shared increase in the succinyl-CoA:3-ketoacid-coenzyme A transferase and the abundance of numerous hypothetical proteins was also altered in both mutants. In general, the proteomic changes observed in the MIL mutant were less pronounced than in the SbIII mutant, probably due to the early appearance of a mutation in the miltefosine transporter abrogating the need for a strong mitochondrial adaptation. This study is the first analysis of the Leishmania mitochondrial proteome and offers powerful insights into the adaptations to this organelle during SbIII and MIL drug resistance.

Fitness of Leishmania donovani parasites resistant to drug combinations.

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Raquel García-Hernández

2015-04-01

Full Text Available Drug resistance represents one of the main problems for the use of chemotherapy to treat leishmaniasis. Additionally, it could provide some advantages to Leishmania parasites, such as a higher capacity to survive in stress conditions. In this work, in mixed populations of Leishmania donovani parasites, we have analyzed whether experimentally resistant lines to one or two combined anti-leishmanial drugs better support the stress conditions than a susceptible line expressing luciferase (Luc line. In the absence of stress, none of the Leishmania lines showed growth advantage relative to the other when mixed at a 1:1 parasite ratio. However, when promastigotes from resistant lines and the Luc line were mixed and exposed to different stresses, we observed that the resistant lines are more tolerant of different stress conditions: nutrient starvation and heat shock-pH stress. Further to this, we observed that intracellular amastigotes from resistant lines present a higher capacity to survive inside the macrophages than those of the control line. These results suggest that resistant parasites acquire an overall fitness increase and that resistance to drug combinations presents significant differences in their fitness capacity versus single-drug resistant parasites, particularly in intracellular amastigotes. These results contribute to the assessment of the possible impact of drug resistance on leishmaniasis control programs.

Evaluating the Role of Host AMPK in Leishmania Burden.

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Moreira, Diana; Estaquier, Jérôme; Cordeiro-da-Silva, Anabela; Silvestre, Ricardo

2018-01-01

The study of host AMP-activated protein kinase (AMPK) activation during Leishmania infection imposes distinct types of techniques to measure protein expression and activation, as well as to quantify, at transcription and translational levels, its downstream targets. The investigation of host AMPK protein modulation during Leishmania infection should primarily be assessed during in vitro infections using as a host murine bone marrow-derived macrophages (BMMos). The infection outcome is assessed measuring the percentage of infected cells in the context of BMMos. To evaluate AMPK activity during infection, the expression of AMPK phosphorylated at Thr172 as well as the transcription and translational levels of its downstream targets are evaluated by quantitative PCR and immunoblotting. The modulation of AMPK activity in vivo is determined specifically in sorted splenic macrophages harboring Leishmania parasites recovered from infected mice using fluorescent-labeled parasites in the infectious inocolum. The modulation of AMPK activity was assessed by AMPK activators and inhibitors and also using AMPK, SIRT1, or LKB1 KO mice models. The infection outcome in BMMos and in vivo was further determined using these two different approaches. To finally understand the metabolic impact of AMPK during infection, in vitro metabolic assays in infected BMMos were measured in the bioenergetic profile using an extracellular flux analyzer.

Histochemical and molecular evaluation of the prevalence of Leishmania spp. in hematophagous insects

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Willian Marinho Dourado Coelho

2016-06-01

Full Text Available The prevalence study of Leishmania spp. in hematophagous insects captured from the environment in bat roosts and pigeon nests, or feeding their hosts (cattle, pigs, horses, dogs and humans in urban, peri-urban and rural areas, between 2012 and 2014. For this study, the amastigotes present in these insects were detected by histochemical and PCR techniques. Positive gene amplification for Leishmania was found in two horseflies of the species Tabanus importunus collected in the environment, and amastigote forms of Leishmania spp., as well as erythrocytes and leukocytes, were histochemically detected in one of that insect. The other analyzed insects were not positive by PCR our by direct parasitological examination. Only horseflies captured in urban and peri-urban areas were positive. During the collection, no phlebotomine sand flies were captured in rural areas far from the city limits. It can be concluded that the discovery of horseflies positive for Leishmania spp. in urban and peri-urban areas indicates the likelihood that urban areas and their surroundings provide vector parasites with an environment suitable for the spread and consequent perpetuation of the biological cycle of this protozoan.

Natural infection of Lutzomyia tortura with Leishmania (Viannia) naiffi in an Amazonian area of Ecuador.

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Kato, Hirotomo; Gomez, Eduardo A; Yamamoto, Yu-ichi; Calvopiña, Manuel; Guevara, Angel G; Marco, Jorge D; Barroso, Paola A; Iwata, Hiroyuki; Hashiguchi, Yoshihisa

2008-09-01

Natural infection of sand flies with Leishmania parasites was surveyed in an Amazonian area in Ecuador where leishmaniasis is endemic. Seventy-one female sand flies were dissected and one was positive for Leishmania protozoa. The species of this sand fly was identified as Lutzomyia (Lu.) tortura on the basis of morphologic characteristics. Analysis of the cytochrome b gene sequence identified the parasite as L. (Viannia) naiffi. We report the distribution of L. (V.) naiffi in Ecuador and detection of a naturally infected sand fly in the Ecuadorian Amazon and natural infection of Lu. tortura with Leishmania parasites in the New World.

Identification of Leishmania tropica from micro-foci of cutaneous leishmaniasis in the Kenyan Rift Valley.

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Odiwuor, Samwel; Muia, Alfred; Magiri, Charles; Maes, Ilse; Kirigi, George; Dujardin, Jean-Claude; Wasunna, Monique; Mbuchi, Margaret; Auwera, Gert Van der

2012-07-01

We performed diagnosis and species identification of parasites in lesion samples from suspected cutaneous leishmaniasis patients in four villages, three of which are in a known Leishmania tropica endemic region in Kenya. Samples were analyzed both by microscopy and PCR for Leishmania, and typed by an assay using four ribosomal DNA-based species-identification PCRs. The lesions were demonstrated to be caused by L. tropica, which confirms the re-emergence of cutaneous leishmaniasis from this species after a period of reduced incidence in the endemic zone. Our report highlights the importance of an intervention and sustained Leishmania control program.

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

DEFF Research Database (Denmark)

Chen, M; Christensen, S B; Blom, J

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...... 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...

Spatial distribution, Leishmania species and clinical traits of Cutaneous Leishmaniasis cases in the Colombian army.

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Patino, Luz H; Mendez, Claudia; Rodriguez, Omaira; Romero, Yanira; Velandia, Daniel; Alvarado, Maria; Pérez, Julie; Duque, Maria Clara; Ramírez, Juan David

2017-08-01

In Colombia, the cutaneous leishmaniasis (CL) is the most common manifestation across the army personnel. Hence, it is mandatory to determine the species associated with the disease as well as the association with the clinical traits. A total of 273 samples of male patients with CL were included in the study and clinical data of the patients was studied. PCR and sequencing analyses (Cytb and HSP70 genes) were performed to identify the species and the intra-specific genetic variability. A georeferenced database was constructed to identify the spatial distribution of Leishmania species isolated. The identification of five species of Leishmania that circulate in the areas where army personnel are deployed is described. Predominant infecting Leishmania species corresponds to L. braziliensis (61.1%), followed by Leishmania panamensis (33.5%), with a high distribution of both species at geographical and municipal level. The species L. guyanensis, L. mexicana and L. lainsoni were also detected at lower frequency. We also showed the identification of different genotypes within L. braziliensis and L. panamensis. In conclusion, we identified the Leishmania species circulating in the areas where Colombian army personnel are deployed, as well as the high intra-specific genetic variability of L. braziliensis and L. panamensis and how these genotypes are distributed at the geographic level.

Post-Genomics and Vaccine Improvement for Leishmania

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Seyed, Negar; Taheri, Tahereh; Rafati, Sima

2016-01-01

Leishmaniasis is a parasitic disease that primarily affects Asia, Africa, South America, and the Mediterranean basin. Despite extensive efforts to develop an effective prophylactic vaccine, no promising vaccine is available yet. However, recent advancements in computational vaccinology on the one hand and genome sequencing approaches on the other have generated new hopes in vaccine development. Computational genome mining for new vaccine candidates is known as reverse vaccinology and is believed to further extend the current list of Leishmania vaccine candidates. Reverse vaccinology can also reduce the intrinsic risks associated with live attenuated vaccines. Individual epitopes arranged in tandem as polytopes are also a possible outcome of reverse genome mining. Here, we will briefly compare reverse vaccinology with conventional vaccinology in respect to Leishmania vaccine, and we will discuss how it influences the aforementioned topics. We will also introduce new in vivo models that will bridge the gap between human and laboratory animal models in future studies. PMID:27092123

Assessment of PCR in the detection of Leishmania spp in experimentally infected individual phlebotomine sandflies (Diptera: Psychodidae: Phlebotominae

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MICHALSKY Érika M.

2002-01-01

Full Text Available DNA amplification by the polymerase chain reaction (PCR was applied in the investigation of the presence of Leishmania (Kinetoplastida: Trypanosomatidae parasites in single phlebotomine sandflies. Three phlebotomine/parasite pairs were used: Lutzomyia longipalpis/Leishmania chagasi, Lutzomyia migonei/Leishmania amazonensis and Lutzomyia migonei/Leishmania braziliensis, all of them incriminated in the transmission of visceral or cutaneous leishmaniasis. DNA extraction was performed with whole insects, with no need of previous digestive tract dissection or pooling specimens. The presence of either mouse blood in the digestive tract of the sandflies or the digestive tract itself did not interfere in the PCR. Infection by as few as 10 Leishmania sp. per individual were sufficient for DNA amplification with genus-specific primers. Using primers for L. braziliensis and L. mexicana complexes, respectively, it was possible to discriminate between L. braziliensis and L. amazonensis in experimentally infected vectors (L. migonei.

Prevalence of Leishmania species in rodents: A systematic review and meta-analysis in Iran.

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Foroutan, Masoud; Khademvatan, Shahram; Majidiani, Hamidreza; Khalkhali, Hamidreza; Hedayati-Rad, Faezeh; Khashaveh, Shahla; Mohammadzadeh, Habib

2017-08-01

Leishmaniasis are diverse group of diseases caused by numerous species of genus Leishmania. Herein we have contrived a systematic review and meta-analysis on the prevalence of Leishmania species in rodents of Iran. For this purpose, following the general methodology recommended for systematic reviews and meta-analysis, six English databases (PubMed, Science Direct, Scopus, Ovid, Web of Science and Google Scholar) and four Persian databases (Magiran, SID, Iran Doc and Iran Medex) were explored during January 1995 till June 2015. Papers were selected based on 8 pre-defined inclusion criteria. During the years, a total number of 4485 different rodents were captured; among which 1291 cases were Leishmania positive. The calculated weighted prevalence of Leishmania species in rodents was 23% (95% CI=18-28). Given geographical zones of Iran, the highest and lowest prevalence rate was belonged to North 50% (95% CI=40-61) and West 11% (95% CI=5-17), respectively. Rhombomys opimus (1766), Meriones lybicus (1258) and Tatera indica (488) were the three most abundant captured rodents, while the highest prevalence of Leishmania species was observed in Nesokia indica 48% (95% CI=42-54) and followed by R. opimus 39% (95% CI=30-47). Egger's regression test was performed to detect publication bias, which revealed it may not have a significant influence on overall weighted prevalence estimate (P=0.317). Meta-regression analysis demonstrated that there is no significant relationship between overall prevalence with sample size (P=0.1) and year of publication (P=0.7). The results showed remarkable prevalence of Leishmania species in rodent reservoirs. In future, adopting a suitable strategy for control and combat with rodents is necessary. Copyright © 2017 Elsevier B.V. All rights reserved.

Adenine phosphoribosyltransferase-deficient Leishmania donovani

International Nuclear Information System (INIS)

Kaur, K.; Iovannisci, D.M.; Ullman, B.

1986-01-01

To elucidate the relative roles of two routes for adenine salvage, the authors use biochemical genetic approaches to isolate clonal strains of Leishmania donovani promasatigotes genetically deficient in APRTase activity. The studies suggest that the metabolic rate of adenine in these organisms is initiated by deamination. The radiolabel incorporation experiments and biochemical experiments are described in which the rate of uptake of radiolabelled purine nucleobases (C 14) was determined. Results are presented

Role of calmodulin and calcineurin in regulating flagellar motility and wave polarity in Leishmania.

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Mukhopadhyay, Aakash Gautam; Dey, Chinmoy Sankar

2017-11-01

We have previously reported the involvement of cyclic AMP in regulating flagellar waveforms in Leishmania. Here, we investigated the roles of calcium, calmodulin, and calcineurin in flagellar motility regulation in L. donovani. Using high-speed videomicroscopy, we show that calcium-independent calmodulin and calcineurin activity is necessary for motility in Leishmania. Inhibition of calmodulin and calcineurin induced ciliary beats interrupting flagellar beating in both live (in vivo) and ATP-reactivated (in vitro) parasites. Our results indicate that signaling mediated by calmodulin and calcineurin operates antagonistically to cAMP signaling in regulating the waveforms of Leishmania flagellum. These two pathways are possibly involved in maintaining the balance between the two waveforms, essential for responding to environmental cues, survival, and infectivity.

Leishmania diagnostic and identification py using 32P labelled DNA probes

International Nuclear Information System (INIS)

Andrade, Antero Silva Ribeiro de; Melo, Maria Norma de

1999-10-01

P 32 labelled DNA probes are valious instruments for the parasitic diseases by using hybridization reaction. In this paper we describe the methodology and present the foundations for the radioactive probes production, based on the kinetoplast DNA (kDNA), for the Leishmania diagnostic an identification. We also describe the kDNA purification protocol from Leishmania reference cepa, the process of P 32 labelling of the kDNA by using the nick translation method, gathering, sample preparation and treatment, the optimum conditions for the hybridization reaction and the procedures for the autoradiography

Use of Recombinant Antigens for Sensitive Serodiagnosis of American Tegumentary Leishmaniasis Caused by Different Leishmania Species.

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Sato, Camila Massae; Sanchez, Maria Carmen Arroyo; Celeste, Beatriz Julieta; Duthie, Malcolm S; Guderian, Jeffrey; Reed, Steven G; de Brito, Maria Edileuza Felinto; Campos, Marliane Batista; de Souza Encarnação, Helia Valeria; Guerra, Jorge; de Mesquita, Tirza Gabrielle Ramos; Pinheiro, Suzana Kanawati; Ramasawmy, Rajendranath; Silveira, Fernando Tobias; de Assis Souza, Marina; Goto, Hiro

2017-02-01

American tegumentary leishmaniasis (ATL) (also known as cutaneous leishmaniasis [CL]) is caused by various species of protozoa of the genus Leishmania The diagnosis is achieved on a clinical, epidemiological, and pathological basis, supported by positive parasitological exams and demonstration of leishmanin delayed-type hypersensitivity. Serological assays are not routinely used in the diagnosis because many are considered to have low sensitivity and the particular Leishmania species causing the disease can lead to variable performance. In the present study, we generated recombinant versions of two highly conserved Leishmania proteins, Leishmania (Viannia) braziliensis-derived Lb8E and Lb6H, and evaluated both in enzyme-linked immunosorbent assays (ELISA). Recombinant Lb6H (rLb6H) had better performance and reacted with 100.0% of the ATL and 89.4% of the VL samples. These reactions with rLb6H were highly specific (98.5%) when compared against those for samples from healthy control individuals. We then assessed rLb6H against sera from ATL patients infected with different species of Leishmania prevalent in Brazil [Leishmania (Leishmania) amazonensis, L (Viannia) braziliensis, and L (V) guyanensis] and samples from patients with other infectious diseases. In analyses of 500 sera, ELISA using rLb6H detected all 219 ATL samples (sensitivity of 100.0%) with an overall specificity of 93.9% (considering healthy individuals and other infectious diseases patients). Only a minority of samples from Chagas disease patients possessed antibodies against rLb6H, and all of these responses were low (with a highest reactivity index of 2.2). Taken together, our data support further evaluation of rLb6H and the potential for its routine use in the serological diagnosis of ATL. Copyright © 2017 Sato et al.

Thrichomys laurentius (Rodentia; Echimyidae as a putative reservoir of Leishmania infantum and L. braziliensis: patterns of experimental infection.

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André Luiz Rodrigues Roque

Full Text Available The importance of the genus Thrichomys in the retention of infection and transmission of Leishmania species is supported by previous studies that describe an ancient interaction between caviomorphs and trypanosomatids and report the natural infection of Thrichomys spp. Moreover, these rodents are widely dispersed in Brazil and recognized as important hosts of other tripanosomatids. Our main purpose was to evaluate the putative role of Thrichomys laurentius in the retention of infection and amplification of the transmission cycle of Leishmania infantum and L. braziliensis. Male and female T. laurentius (n = 24 born in captivity were evaluated for the retention of infection with these Leishmania species and followed up by parasitological, serological, hematological, biochemical, histological, and molecular assays for 3, 6, 9, or 12 months post infection (mpi. T. laurentius showed its competence as maintenance host for the two inoculated Leishmania species. Four aspects should be highlighted: (i re-isolation of parasites 12 mpi; (ii the low parasitic burden displayed by T. laurentius tissues; (iii the early onset and maintenance of humoral response, and (iv the similar pattern of infection by the two Leishmania species. Both Leishmania species demonstrated the ability to invade and maintain itself in viscera and skin of T. laurentius, and no rodent displayed any lesion, histological changes, or clinical evidence of infection. We also wish to point out the irrelevance of the adjective dermotropic or viscerotropic to qualify L. braziliensis and L. infantum, respectively, when these species are hosted by nonhuman hosts. Our data suggest that T. laurentius may act at least as a maintenance host of both tested Leishmania species since it maintained long-lasting infections. Moreover, it cannot be discarded that Leishmania spp. infection in free-ranging T. laurentius could result in higher parasite burden due the more stressing conditions in the wild

Leishmania, Babesia and Ehrlichia in urban pet dogs: co-infection or cross-reaction in serological methods?

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Felipe da Silva Krawczak

2015-02-01

Full Text Available INTRODUCTION: The present study was designed to assess the occurrence of co-infection or cross-reaction in the serological techniques used for detecting the anti-Leishmania spp., -Babesia canis vogeli and -Ehrlichia canis antibodies in urban dogs from an area endemic to these parasites. METHODS: The serum samples from dogs were tested for the Babesia canis vogeli strain Belo Horizonte antigen and Ehrlichia canis strain São Paulo by immunofluorescence antibody test (IFAT and by anti-Leishmania immunoglobulin G (IgG antibody detection to assess Leishmania infection. We used the following four commercial kits for canine visceral leishmaniasis: ELISA, IFAT, Dual Path Platform (DPP (Bio Manguinhos(r/FIOCRUZ/MS and a rK39 RDT (Kalazar Detect Canine Rapid Test; Inbios. RESULTS : Of 96 serum samples submitted to serological assays, 4 (4.2% were positive for Leishmania as determined by ELISA; 12 (12.5%, by IFAT; 14 (14.6% by rK39 RDT; and 20 (20.8%, by DPP. Antibodies against Ehrlichia and Babesia were detected in 23/96 (23.9% and 30/96 (31.2% samples, respectively. No significant association was identified between the results of tests for detecting Babesia or Ehrlichia and those for detecting Leishmania (p-value>0.05. CONCLUSIONS: In the present study, we demonstrated co-infection with Ehrlichia or Babesia and Leishmania in dogs from Minas Gerais (Brazil; we also found that the serological tests that were used did not cross-react.

Fumonisin B₁ (FB₁) Induces Lamellar Separation and Alters Sphingolipid Metabolism of In Vitro Cultured Hoof Explants.

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Reisinger, Nicole; Dohnal, Ilse; Nagl, Veronika; Schaumberger, Simone; Schatzmayr, Gerd; Mayer, Elisabeth

2016-03-24

One of the most important hoof diseases is laminitis. Yet, the pathology of laminitis is not fully understood. Different bacterial toxins, e.g. endotoxins or exotoxins, seem to play an important role. Additionally, ingestion of mycotoxins, toxic secondary metabolites of fungi, might contribute to the onset of laminitis. In this respect, fumonsins are of special interest since horses are regarded as species most susceptible to this group of mycotoxins. The aim of our study was to investigate the influence of fumonisin B₁ (FB₁) on primary isolated epidermal and dermal hoof cells, as well as on the lamellar tissue integrity and sphingolipid metabolism of hoof explants in vitro. There was no effect of FB₁ at any concentration on dermal or epidermal cells. However, FB₁ significantly reduced the separation force of explants after 24 h of incubation. The Sa/So ratio was significantly increased in supernatants of explants incubated with FB₁ (2.5-10 µg/mL) after 24 h. Observed effects on Sa/So ratio were linked to significantly increased sphinganine concentrations. Our study showed that FB₁ impairs the sphingolipid metabolism of explants and reduces lamellar integrity at non-cytotoxic concentrations. FB₁ might, therefore, affect hoof health. Further in vitro and in vivo studies are necessary to elucidate the effects of FB₁ on the equine hoof in more detail.

Prostaglandin E2/leukotriene B4 balance induced by Lutzomyia longipalpis saliva favors Leishmania infantum infection.

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Araújo-Santos, Théo; Prates, Deboraci Brito; França-Costa, Jaqueline; Luz, Nívea F; Andrade, Bruno B; Miranda, José Carlos; Brodskyn, Claudia I; Barral, Aldina; Bozza, Patrícia T; Borges, Valéria Matos

2014-12-20

Eicosanoids and sand fly saliva have a critical role in the Leishmania infection. Here, we evaluated the effect of Lutzomyia longipalpis salivary gland sonicate (SGS) on neutrophil and monocyte recruitment and activation of eicosanoid production in a murine model of inflammation. C57BL/6 mice were inoculated intraperitonealy with Lutzomyia longipalpis SGS or Leishmania infantum or both, followed by analyses of cell recruitment, parasite load and eicosanoid production. Intraperitoneal injection of Lutzomyia longipalpis SGS together with Leishmania infantum induced an early increased parasite viability in monocytes and neutrophils. L. longipalpis SGS increased prostaglandin E2 (PGE2), but reduced leukotriene B4 (LTB4) production ex vivo in peritoneal leukocytes. In addition, the pharmacological inhibition of cyclooxygenase 2 (COX-2) with NS-398 decreased parasite viability inside macrophages during Leishmania infection in the presence of L. longipalpis SGS arguing that PGE2 production is associated with diminished parasite killing. These findings indicate that L. longipalpis SGS is a critical factor driving immune evasion of Leishmania through modulation of PGE2/LTB4 axis, which may represent an important mechanism on establishment of the infection.

Lipid Droplet Formation, Their Localization and Dynamics during Leishmania major Macrophage Infection.

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Sameh Rabhi

Full Text Available Leishmania, the causative agent of vector-borne diseases, known as leishmaniases, is an obligate intracellular parasite within mammalian hosts. The outcome of infection depends largely on the activation status of macrophages, the first line of mammalian defense and the major target cells for parasite replication. Understanding the strategies developed by the parasite to circumvent macrophage defense mechanisms and to survive within those cells help defining novel therapeutic approaches for leishmaniasis. We previously showed the formation of lipid droplets (LDs in L. major infected macrophages. Here, we provide novel insights on the origin of the formed LDs by determining their cellular distribution and to what extent these high-energy sources are directed to the proximity of Leishmania parasites. We show that the ability of L. major to trigger macrophage LD accumulation is independent of parasite viability and uptake and can also be observed in non-infected cells through paracrine stimuli suggesting that LD formation is from cellular origin. The accumulation of LDs is demonstrated using confocal microscopy and live-cell imagin in parasite-free cytoplasmic region of the host cell, but also promptly recruited to the proximity of Leishmania parasites. Indeed LDs are observed inside parasitophorous vacuole and in parasite cytoplasm suggesting that Leishmania parasites besides producing their own LDs, may take advantage of these high energy sources. Otherwise, these LDs may help cells defending against parasitic infection. These metabolic changes, rising as common features during the last years, occur in host cells infected by a large number of pathogens and seem to play an important role in pathogenesis. Understanding how Leishmania parasites and different pathogens exploit this LD accumulation will help us define the common mechanism used by these different pathogens to manipulate and/or take advantage of this high-energy source.

Leishmania promastigotes lack phosphatidylserine but bind annexin V upon permeabilization or miltefosine treatment.

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Adrien Weingärtner

Full Text Available The protozoan parasite Leishmania is an intracellular pathogen infecting and replicating inside vertebrate host macrophages. A recent model suggests that promastigote and amastigote forms of the parasite mimic mammalian apoptotic cells by exposing phosphatidylserine (PS at the cell surface to trigger their phagocytic uptake into host macrophages. PS presentation at the cell surface is typically analyzed using fluorescence-labeled annexin V. Here we show that Leishmania promastigotes can be stained by fluorescence-labeled annexin V upon permeabilization or miltefosine treatment. However, combined lipid analysis by thin-layer chromatography, mass spectrometry and (31P nuclear magnetic resonance (NMR spectroscopy revealed that Leishmania promastigotes lack any detectable amount of PS. Instead, we identified several other phospholipid classes such phosphatidic acid, phosphatidylethanolamine; phosphatidylglycerol and phosphatidylinositol as candidate lipids enabling annexin V staining.

Evaluación de la respuesta de isotipos de inmunoglobulina especifica a Leishmania en leishmaniasis tegumentaria Americana Evaluation of specific immunoglobin isotype response to Leishmania in American tegumentary leishmaniasis

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Maricel Labrada

1989-09-01

Full Text Available Con el fin de determinar las clases de anticuerpo producido contra el parásito y la cinética de los mismos en relación a la evolución de la infección, se estudiaron los sueros de 133 pacientes infectados con Leishmania del complejo braziliensis. Se utilizó la prueba de inmunofluorescencia indirecta y amastigotas de L. mexicana amazonensis como antígeno. En los sueros obtenidos al momento de consultar para el diagnóstico se encontró IgM en 54 de los sueros absorbidos con Straphylococcus aureus Cowan I y en 5 de los no absorbidos. La IgM sólo se encontro en los sueros de pacientes con tiempo devolución de las lesiones Leishmania-specific immunoglobulin subclass response was evaluated in 133 patients infected with Leishmania braziliensis. The indirect immunofluorescent antibody test (IFAT was employed with amastigotes of L. mexicana amazonensis as antigen. Among the 133 sera obtained at consultation fo rdiagnosis of active lesions, IgM was detected in 54 following absorption with Staphylococcus aureus Cowan strain I, and in 5 sera prior to absorption. IgM reactive with Leishmania antigen was only found in sera from patients whose lesions had envolved over past two months or less. Leishmania-specific IgG was detected in all sera prior to absorption. Sera obtained at the time of recurrence of after complete healing of lesions presented only specific IgC. The combined use of the Montenegro skin test and specific IgM increased the sensitivity of immunodiagnostic methods in patients with lesions of less than 2 months duration. Normal control volunteers were negative for specific IgM and unreactive to Montenegro skin testin. Among 16 patients with non-leishmanial lesion, 3 with sporotrichosis showed IgG reactive with Leishmania; none, including 4 with lesions of less than two months duration, showed specific IgM. We conclude that in patients infected with L. brasiliensis the presence of specific IgG and IgM is associated with the time of

Involvement of Leishmania donovani major surface glycoprotein ...

Indian Academy of Sciences (India)

The major surface glycoprotein gp63 of the kinetoplastid protozoal parasite Leishmania is implicated as a ligand mediating uptake of the parasite into, and survival within, the host macrophage. By expressing gp63 antisense RNA from an episomal vector in L. donovani promastigotes, gp63-deficient transfectants were ...

Transmission of Leishmania in coffee plantations of Minas Gerais, Brazil

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Bruce Alexander

2002-07-01

Full Text Available Transmission of Leishmania was studied in 27 coffee plantations in the Brazilian State of Minas Gerais. Eighteen females and six males (11.6% of the people tested, aged between 7-65 gave a positive response to the Montenegro skin test. Awareness of sand flies based on the ability of respondents to identify the insects using up to seven predetermined characteristics was significantly greater among inhabitants of houses occupied by at least one Mn+ve individual. Five species of phlebotomine sand fly, including three suspected Leishmania vectors, were collected within plantations under three different cultivation systems. Four of these species i.e., Lu. fischeri (Pinto 1926, Lu. migonei (França 1920, Lu. misionensis (Castro 1959 and Lutzomyia whitmani (Antunes & Coutinho 1939 were collected in an organic plantation and the last of these was also present in the other two plantation types. The remaining species, Lu. intermedia (Lutz & Neiva 1912, was collected in plantations under both the "adensado" and "convencional" systems. The results of this study indicate that transmission of Leishmania to man in coffee-growing areas of Minas Gerais may involve phlebotomine sand flies that inhabit plantations.

The past, present, and future of Leishmania genomics and transcriptomics

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Cantacessi, Cinzia; Dantas-Torres, Filipe; Nolan, Matthew J.; Otranto, Domenico

2015-01-01

It has been nearly 10 years since the completion of the first entire genome sequence of a Leishmania parasite. Genomic and transcriptomic analyses have advanced our understanding of the biology of Leishmania, and shed new light on the complex interactions occurring within the parasite–host–vector triangle. Here, we review these advances and examine potential avenues for translation of these discoveries into treatment and control programs. In addition, we argue for a strong need to explore how disease in dogs relates to that in humans, and how an improved understanding in line with the ‘One Health’ concept may open new avenues for the control of these devastating diseases. PMID:25638444

Cutaneous leishmaniasis with lymphadenopathy due to Leishmania donovani

NARCIS (Netherlands)

Faber, W. R.; Wonders, J.; Jensema, A. J.; Chocholova, E.; Kager, P. A.

2009-01-01

Summary We describe a case of cutaneous leishmaniasis with lymphadenopathy due to Leishmania donovani, which was successfully treated with oral miltefosine. Given the increased prevalence of travelling, patients presenting with lymph-node enlargement should have leishmaniasis included in the

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

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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.

Naturally infected Lutzomyia sand flies in a Leishmania-endemic area of Brazil.

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Carvalho, Gustavo M L; Andrade Filho, Jose D; Falcao, Alda L; Rocha Lima, Ana C V M; Gontijo, Celia M F

2008-06-01

In Brazil, Leishmania transmission involves several species of phlebotomine sand flies that are closely associated with different parasites and reservoirs, giving rise to different transmission cycles. The present study focused on naturally infected phlebotomines originating from Santa Luzia, a municipality near Belo Horizonte, capital of the Brazilian state of Minas Gerais, in which leishmaniasis are endemic. Systematic and non systematic approaches,involving the use of light traps and direct aspiration from resting sites, respectively, were used to collect females and flies. Identification of the captured insects and determination of natural infection by Leishmania spp. were performed using both conventional dissection methods and polymerase chain reaction (PCR). The dissection of 102 sand flies allowed five species of Lutzomyia to be identified, although no flagellate parasite forms were observed.In addition, 211 sand flies were identified, were separated according to species, and were combined into 11 pools of up to 20 individuals each. PCR analyses showed that two of these pools were infected with Leishmania:one pool of Lu. whitmani was infected with Le. (Viannia) spp. and another of Lu. cortelezzii was infected with Le. chagasi. This suggests that Lu. whitmani may be a possible vector of Leishmania in the study area, and more work needs to be performed to assess the role of Lu. cortelezzii as a vector.

Molecular screening of Leishmania spp. infection and bloodmeals in sandflies from a leishmaniasis focus in southwestern Turkey.

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Karaku Ş, M; Pekağ Irba Ş, M; Demir, S; Eren, H; Töz, S; Özbel, Y

2017-06-01

Leishmaniasis is an arthropod-borne disease that affects approximately 2 million people worldwide annually. The aims of this study were to detect the presence of Leishmania (Kinetoplastida: Trypanosomatidae) DNA and the feeding preferences of probable vector species in an endemic focus of Leishmania infantum in Turkey. Entomological sampling was performed in August and October 2015 in Aydın province, where cases of human and canine leishmaniasis have been reported previously. A total of 1059 sandfly specimens comprising nine species belonging to two genera, Phlebotomus and Sergentomyia (both: Diptera: Psychodidae), and five subgenera of the Phlebotomus genus (Phlebotomus, Paraphlebotomus, Larroussius, Adlerius and Transphlebotomus) were collected in five villages. Among all Phlebotomus specimens, Phlebotomus neglectus (39%) was noted as the most abundant species, followed by Phlebotomus tobbi (18%). Leishmania DNA was detected in pools from P. neglectus, P. tobbi and Sergentomyia dentata by kDNA polymerase chain reaction (PCR). Leishmania DNA from Phlebotomus specimens was identified as L. infantum, but Leishmania DNA from Sergentomyia spp. could not be identified to species level by ITS-1 real-time PCR. The detection of Leishmania DNA in wild-caught P. neglectus and the high percentage (24.2%) of human DNA in engorged specimens suggests that P. neglectus is probably an important vector species for L. infantum in Aydın province. © 2016 The Royal Entomological Society.

In vitro and in vivo efficacy of ether lipid edelfosine against Leishmania spp. and SbV-resistant parasites.

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Rubén E Varela-M

Full Text Available BACKGROUND: The leishmaniases are a complex of neglected tropical diseases caused by more than 20 Leishmania parasite species, for which available therapeutic arsenal is scarce and unsatisfactory. Pentavalent antimonials (SbV are currently the first-line pharmacologic therapy for leishmaniasis worldwide, but resistance to these compounds is increasingly reported. Alkyl-lysophospoholipid analogs (ALPs constitute a family of compounds with antileishmanial activity, and one of its members, miltefosine, has been approved as the first oral treatment for visceral and cutaneous leishmaniasis. However, its clinical use can be challenged by less impressive efficiency in patients infected with some Leishmania species, including L. braziliensis and L. mexicana, and by proneness to develop drug resistance in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We found that ALPs ranked edelfosine>perifosine>miltefosine>erucylphosphocholine for their antileishmanial activity and capacity to promote apoptosis-like parasitic cell death in promastigote and amastigote forms of distinct Leishmania spp., as assessed by proliferation and flow cytometry assays. Effective antileishmanial ALP concentrations were dependent on both the parasite species and their development stage. Edelfosine accumulated in and killed intracellular Leishmania parasites within macrophages. In vivo antileishmanial activity was demonstrated following oral treatment with edelfosine of mice and hamsters infected with L. major, L. panamensis or L. braziliensis, without any significant side-effect. Edelfosine also killed SbV-resistant Leishmania parasites in in vitro and in vivo assays, and required longer incubation times than miltefosine to generate drug resistance. CONCLUSIONS/SIGNIFICANCE: Our data reveal that edelfosine is the most potent ALP in killing different Leishmania spp., and it is less prone to lead to drug resistance development than miltefosine. Edelfosine is effective in killing Leishmania

Extensive sphingolipid depletion does not affect lipid raft integrity or lipid raft localization and efflux function of the ABC transporter MRP1

NARCIS (Netherlands)

Klappe, Karin; Dijkhuis, Anne-Jan; Hummel, Ina; van Dam, Annie; Ivanova, Pavlina T.; Milne, Stephen B.; Myers, David S.; Brown, H. Alex; Permentier, Hjalmar; Kok, Jan W.

2010-01-01

We show that highly efficient depletion of sphingolipids in two different cell lines does not abrogate the ability to isolate Lubrol-based DRMs (detergent-resistant membranes) or detergent-free lipid rafts from these cells. Compared with control, DRM/detergent-free lipid raft fractions contain equal

Can equids be a reservoir of Leishmania braziliensis in endemic areas?

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Jessé Henrique Truppel

Full Text Available In this study, we detected Leishmania (Viannia braziliensis infection in equids living in endemic regions of cutaneous leishmaniasis. To determine the role of these animals in the Leishmania cycle, we used two approaches: serological and molecular methods. Antibodies to the parasite were assayed using the Enzyme Linked Immunosorbent Assay (ELISA. Blood samples were collected and tested by polymerase chain reaction (PCR, and the positive products were sequenced. The results showed that 11.0% (25/227 of the equids were seropositive for Leishmania sp, and 16.3% (37/227 were PCR positive. Antibodies were detected in 20 horses, 3 donkeys, and 2 mules, and the parasite DNA was detected in 30 horses, 5 donkeys, and 2 mules. Sequencing the amplified DNA revealed 100% similarity with sequences for Viannia complex, corroborating the results of PCR for L. braziliensis. Our results show that equids are infected with L. braziliensis, which could be food sources for phlebotomines in the peridomiciliary environment and consequently play a role in the cutaneous leishmaniasis cycle.

Leishmania (V.) braziliensis infecting bats from Pantanal wetland, Brazil: First records for Platyrrhinus lineatus and Artibeus planirostris.

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de Castro Ferreira, Eduardo; Pereira, Agnes Antônio Sampaio; Silveira, Maurício; Margonari, Carina; Marcon, Glaucia Elisete Barbosa; de Oliveira França, Adriana; Castro, Ludiele Souza; Bordignon, Marcelo Oscar; Fischer, Erich; Tomas, Walfrido Moraes; Dorval, Maria Elizabeth Cavalheiros; Gontijo, Célia Maria Ferreira

2017-08-01

In the New World genus Leishmania parasites are etiological agents of neglected zoonoses known as leishmaniasis. Its epidemiology is very complex due to the participation of several species of sand fly vectors and mammalian hosts, and man is an accidental host. Control is very difficult because of the different epidemiological patterns of transmission observed. Studies about Leishmania spp. infection in bats are so scarce, which represents a large gap in knowledge about the role of these animals in the transmission cycle of these pathogens, especially when considering that Chiroptera is one of the most abundant and diverse orders among mammals. Leishmaniasis in Mato Grosso do Sul, Brazil are remarkably frequent, probably due to the abundance of its regional mastofauna. The recent record of L. braziliensis in bats from this state indicates the need to clarify the role of these mammals in the transmission cycle. In this study we evaluated the presence of Leishmania parasites in the skin of different species of bats, using PCR directed to Leishmania spp. kDNA for screening followed by PCR/RFLP analysis of the hsp70 gene for the identification of parasite species. Leishmania species identification was confirmed by PCR directed to the G6PD gene of L. braziliensis, followed by sequencing of the PCR product. Samples from 47 bats were processed, of which in three specimens (6.38%) was detected the presence of Leishmania sp. kDNA. PCR/RFLP and sequencing identified the species involved in the infection as L. braziliensis in all of them. This is the first report of Leishmania braziliensis in bats from Pantanal ecosystem and the first record of this species in Platyrrhinus lineatus and Artibeus planirostris, bats with a wide distribution in South America. These results reinforce the need to deepen the knowledge about the possibility of bats act as reservoirs of Leishmania spp. especially considering their ability of dispersion and occupation of anthropic environments

Arabidopsis dynamin-related protein 1E in sphingolipid-enriched plasma membrane domains is associated with the development of freezing tolerance.

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Minami, Anzu; Tominaga, Yoko; Furuto, Akari; Kondo, Mariko; Kawamura, Yukio; Uemura, Matsuo

2015-08-01

The freezing tolerance of Arabidopsis thaliana is enhanced by cold acclimation, resulting in changes in the compositions and function of the plasma membrane. Here, we show that a dynamin-related protein 1E (DRP1E), which is thought to function in the vesicle trafficking pathway in cells, is related to an increase in freezing tolerance during cold acclimation. DRP1E accumulated in sphingolipid and sterol-enriched plasma membrane domains after cold acclimation. Analysis of drp1e mutants clearly showed that DRP1E is required for full development of freezing tolerance after cold acclimation. DRP1E fused with green fluorescent protein was visible as small foci that overlapped with fluorescent dye-labelled plasma membrane, providing evidence that DRP1E localizes non-uniformly in specific areas of the plasma membrane. These results suggest that DRP1E accumulates in sphingolipid and sterol-enriched plasma membrane domains and plays a role in freezing tolerance development during cold acclimation. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

The prevalence of canine Leishmania infantum infection in western China detected by PCR and serological tests

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Chen Hai-Tang

2011-05-01

Full Text Available Abstract Background Canine leishmaniasis (CanL is endemic in western China, resulting in important public health problem. It is essential to evaluate the prevalence of canine Leishmania infantum infection for designing control policy. In the present study we report for the first time prevalence of Leishmania infection in dogs living in Jiuzhaigou County (Sichuan Provence, China, which is not only an important endemic area of CanL but also a tourism scenic spot, detected by PCR, ELISA and dipstick test. The results could provide key information for designing control programs against canine and human leishmaniasis. In addition, the complete sequence of the Leishmania isolate from Sichuan Province has not been reported to date and we present the sequences of 116 base-pair (bp fragment of the conserved region in the minicircle kinetoplast DNA (kDNA and the results of phylogenetic analyses based on the sequence of the amplified fragment. Results The proportion of dogs infected with Leishmania in Jiuzhaigou County was 36.79%, 9.43%, and 51.88% detected by ELISA, dipstick test, and PCR, respectively. The ELISA and PCR tests were more sensitive than dipstick test. The PCR method is the most sensitive way to detect dogs infected with Leishmania parasites. The total positive rate for infected dogs in the area was 59.43% by the three methods. The PCR products of 116-bp fragment amplified from the kDNA conserved region of dog blood samples and laboratory maintained L. infantum were DNA sequenced and the variation of the sequences was observed. The phylogenetic tree based on the sequences of 116-bp fragment reveals that L. infantum is more genetically related to visceralizing species L. donovani than to the Leishmania species associated with cutaneous disease. Conclusions More than half of dogs living in the endemic Jiuzhaigou County were infected by L. infantum. Control measures, such as treatment or eradication of infected dogs, or prohibition of

Dual biosynthetic pathways to phytosterol via cycloartenol and lanosterol in Arabidopsis.

Science.gov (United States)

Ohyama, Kiyoshi; Suzuki, Masashi; Kikuchi, Jun; Saito, Kazuki; Muranaka, Toshiya

2009-01-20

The differences between the biosynthesis of sterols in higher plants and yeast/mammals are believed to originate at the cyclization step of oxidosqualene, which is cyclized to cycloartenol in higher plants and lanosterol in yeast/mammals. Recently, lanosterol synthase genes were identified from dicotyledonous plant species including Arabidopsis, suggesting that higher plants possess dual biosynthetic pathways to phytosterols via lanosterol, and through cycloartenol. To identify the biosynthetic pathway to phytosterol via lanosterol, and to reveal the contributions to phytosterol biosynthesis via each cycloartenol and lanosterol, we performed feeding experiments by using [6-(13)C(2)H(3)]mevalonate with Arabidopsis seedlings. Applying (13)C-{(1)H}{(2)H} nuclear magnetic resonance (NMR) techniques, the elucidation of deuterium on C-19 behavior of phytosterol provided evidence that small amounts of phytosterol were biosynthesized via lanosterol. The levels of phytosterol increased on overexpression of LAS1, and phytosterols derived from lanosterol were not observed in a LAS1-knockout plant. This is direct evidence to indicate that the biosynthetic pathway for phytosterol via lanosterol exists in plant cells. We designate the biosynthetic pathway to phytosterols via lanosterol "the lanosterol pathway." LAS1 expression is reported to be induced by the application of jasmonate and is thought to have evolved from an ancestral cycloartenol synthase to a triterpenoid synthase, such as beta-amyrin synthase and lupeol synthase. Considering this background, the lanosterol pathway may contribute to the biosynthesis of not only phytosterols, but also steroids as secondary metabolites.

Polyketide synthase chemistry does not direct biosynthetic divergence between 9- and 10-membered enediynes

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Horsman, Geoff P.; Chen, Yihua; Thorson, Jon S.; Shen, Ben

2010-01-01

Enediynes are potent antitumor antibiotics that are classified as 9- or 10-membered according to the size of the enediyne core structure. However, almost nothing is known about enediyne core biosynthesis, and the determinants of 9- versus 10-membered enediyne core biosynthetic divergence remain elusive. Previous work identified enediyne-specific polyketide synthases (PKSEs) that can be phylogenetically distinguished as being involved in 9- versus 10-membered enediyne biosynthesis, suggesting that biosynthetic divergence might originate from differing PKSE chemistries. Recent in vitro studies have identified several compounds produced by the PKSE and associated thioesterase (TE), but condition-dependent product profiles make it difficult to ascertain a true catalytic difference between 9- and 10-membered PKSE-TE systems. Here we report that PKSE chemistry does not direct 9- versus 10-membered enediyne core biosynthetic divergence as revealed by comparing the products from three 9-membered and two 10-membered PKSE-TE systems under identical conditions using robust in vivo assays. Three independent experiments support a common catalytic function for 9- and 10-membered PKSEs by the production of a heptaene metabolite from: (i) all five cognate PKSE-TE pairs in Escherichia coli; (ii) the C-1027 and calicheamicin cognate PKSE-TEs in Streptomyces lividans K4-114; and (iii) selected native producers of both 9- and 10-membered enediynes. Furthermore, PKSEs and TEs from different 9- and 10-membered enediyne biosynthetic machineries are freely interchangeable, revealing that 9- versus 10-membered enediyne core biosynthetic divergence occurs beyond the PKSE-TE level. These findings establish a starting point for determining the origins of this biosynthetic divergence. PMID:20534556

Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay.

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de Almeida, Marcos E; Koru, Ozgur; Steurer, Francis; Herwaldt, Barbara L; da Silva, Alexandre J

2017-01-01

Leishmaniasis in humans is caused by Leishmania spp. in the subgenera Leishmania and Viannia Species identification often has clinical relevance. Until recently, our laboratory relied on conventional PCR amplification of the internal transcribed spacer 2 (ITS2) region (ITS2-PCR) followed by sequencing analysis of the PCR product to differentiate Leishmania spp. Here we describe a novel real-time quantitative PCR (qPCR) approach based on the SYBR green technology (LSG-qPCR), which uses genus-specific primers that target the ITS1 region and amplify DNA from at least 10 Leishmania spp., followed by analysis of the melting temperature (T m ) of the amplicons on qPCR platforms (the Mx3000P qPCR system [Stratagene-Agilent] and the 7500 real-time PCR system [ABI Life Technologies]). We initially evaluated the assay by testing reference Leishmania isolates and comparing the results with those from the conventional ITS2-PCR approach. Then we compared the results from the real-time and conventional molecular approaches for clinical specimens from 1,051 patients submitted to the reference laboratory of the Centers for Disease Control and Prevention for Leishmania diagnostic testing. Specimens from 477 patients tested positive for Leishmania spp. with the LSG-qPCR assay, specimens from 465 of these 477 patients also tested positive with the conventional ITS2-PCR approach, and specimens from 10 of these 465 patients had positive results because of retesting prompted by LSG-qPCR positivity. On the basis of the T m values of the LSG-qPCR amplicons from reference and clinical specimens, we were able to differentiate four groups of Leishmania parasites: the Viannia subgenus in aggregate; the Leishmania (Leishmania) donovani complex in aggregate; the species L (L) tropica; and the species L (L) mexicana, L (L) amazonensis, L (L) major, and L (L) aethiopica in aggregate. Copyright © 2016 American Society for Microbiology.

Effect of aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing amino acids on Leishmania spp. chemotaxis.

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Diaz, E; Zacarias, A K; Pérez, S; Vanegas, O; Köhidai, L; Padrón-Nieves, M; Ponte-Sucre, A

2015-11-01

In the sand-fly mid gut, Leishmania promastigotes are exposed to acute changes in nutrients, e.g. amino acids (AAs). These metabolites are the main energy sources for the parasite, crucial for its differentiation and motility. We analysed the migratory behaviour and morphological changes produced by aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing AAs in Leishmania amazonensis and Leishmania braziliensis and demonstrated that L-methionine (10-12 m), L-tryptophan (10-11 m), L-glutamine and L-glutamic acid (10-6 m), induced positive chemotactic responses, while L-alanine (10-7 m), L-methionine (10-11 and 10-7 m), L-tryptophan (10-11 m), L-glutamine (10-12 m) and L-glutamic acid (10-9 m) induced negative chemotactic responses. L-proline and L-cysteine did not change the migratory potential of Leishmania. The flagellum length of L. braziliensis, but not of L. amazonensis, decreased when incubated in hyperosmotic conditions. However, chemo-repellent concentrations of L-alanine (Hypo-/hyper-osmotic conditions) and L-glutamic acid (hypo-osmotic conditions) decreased L. braziliensis flagellum length and L-methionine (10-11 m, hypo-/hyper-osmotic conditions) decreased L. amazonensis flagellum length. This chemotactic responsiveness suggests that Leishmania discriminate between slight concentration differences of small and structurally closely related molecules and indicates that besides their metabolic effects, AAs play key roles linked to sensory mechanisms that might determine the parasite's behaviour.

Sterol 14α-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana.

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Roy Mwenechanya

2017-06-01

Full Text Available Amphotericin B has emerged as the therapy of choice for use against the leishmaniases. Administration of the drug in its liposomal formulation as a single injection is being promoted in a campaign to bring the leishmaniases under control. Understanding the risks and mechanisms of resistance is therefore of great importance. Here we select amphotericin B-resistant Leishmania mexicana parasites with relative ease. Metabolomic analysis demonstrated that ergosterol, the sterol known to bind the drug, is prevalent in wild-type cells, but diminished in the resistant line, where alternative sterols become prevalent. This indicates that the resistance phenotype is related to loss of drug binding. Comparing sequences of the parasites' genomes revealed a plethora of single nucleotide polymorphisms that distinguish wild-type and resistant cells, but only one of these was found to be homozygous and associated with a gene encoding an enzyme in the sterol biosynthetic pathway, sterol 14α-demethylase (CYP51. The mutation, N176I, is found outside of the enzyme's active site, consistent with the fact that the resistant line continues to produce the enzyme's product. Expression of wild-type sterol 14α-demethylase in the resistant cells caused reversion to drug sensitivity and a restoration of ergosterol synthesis, showing that the mutation is indeed responsible for resistance. The amphotericin B resistant parasites become hypersensitive to pentamidine and also agents that induce oxidative stress. This work reveals the power of combining polyomics approaches, to discover the mechanism underlying drug resistance as well as offering novel insights into the selection of resistance to amphotericin B itself.

Vitamin E γ-Tocotrienol Inhibits Cytokine-Stimulated NF-κB Activation by Induction of Anti-Inflammatory A20 via Stress Adaptive Response Due to Modulation of Sphingolipids.

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Wang, Yun; Park, Na-Young; Jang, Yumi; Ma, Averil; Jiang, Qing

2015-07-01

NF-κB plays a central role in pathogenesis of inflammation and cancer. Many phytochemicals, including γ-tocotrienol (γTE), a natural form of vitamin E, have been shown to inhibit NF-κB activation, but the underlying mechanism has not been identified. In this study, we show that γTE inhibited cytokine-triggered activation of NF-κB and its upstream regulator TGF-β-activated kinase-1 in murine RAW 264.7 macrophages and primary bone marrow-derived macrophages. In these cells, γTE induced upregulation of A20, an inhibitor of NF-κB. Knockout of A20 partially diminished γTE's anti-NF-κB effect, but γTE increased another NF-κB inhibitor, Cezanne, in A20(-/-) cells. In search of the reason for A20 upregulation, we found that γTE treatment increased phosphorylation of translation initiation factor 2, IκBα, and JNK, indicating induction of endoplasmic reticulum stress. Liquid chromatography-tandem mass spectrometry analyses revealed that γTE modulated sphingolipids, including enhancement of intracellular dihydroceramides, sphingoid bases in de novo synthesis of the sphingolipid pathway. Chemical inhibition of de novo sphingolipid synthesis partially reversed γTE's induction of A20 and the anti-NF-κB effect. The importance of dihydroceramide increase is further supported by the observation that C8-dihydroceramide mimicked γTE in upregulating A20, enhancing endoplasmic reticulum stress, and attenuating TNF-triggered NF-κB activation. Our study identifies a novel anti-NF-κB mechanism where A20 is induced by stress-induced adaptive response as a result of modulation of sphingolipids, and it demonstrates an immunomodulatory role of dihydrocermides. Copyright © 2015 by The American Association of Immunologists, Inc.

Emergent biosynthetic capacity in simple microbial communities.

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Hsuan-Chao Chiu

2014-07-01

Full Text Available Microbes have an astonishing capacity to transform their environments. Yet, the metabolic capacity of a single species is limited and the vast majority of microorganisms form complex communities and join forces to exhibit capabilities far exceeding those achieved by any single species. Such enhanced metabolic capacities represent a promising route to many medical, environmental, and industrial applications and call for the development of a predictive, systems-level understanding of synergistic microbial capacity. Here we present a comprehensive computational framework, integrating high-quality metabolic models of multiple species, temporal dynamics, and flux variability analysis, to study the metabolic capacity and dynamics of simple two-species microbial ecosystems. We specifically focus on detecting emergent biosynthetic capacity--instances in which a community growing on some medium produces and secretes metabolites that are not secreted by any member species when growing in isolation on that same medium. Using this framework to model a large collection of two-species communities on multiple media, we demonstrate that emergent biosynthetic capacity is highly prevalent. We identify commonly observed emergent metabolites and metabolic reprogramming patterns, characterizing typical mechanisms of emergent capacity. We further find that emergent secretion tends to occur in two waves, the first as soon as the two organisms are introduced, and the second when the medium is depleted and nutrients become limited. Finally, aiming to identify global community determinants of emergent capacity, we find a marked association between the level of emergent biosynthetic capacity and the functional/phylogenetic distance between community members. Specifically, we demonstrate a "Goldilocks" principle, where high levels of emergent capacity are observed when the species comprising the community are functionally neither too close, nor too distant. Taken together

Minimum Information about a Biosynthetic Gene cluster : commentary

NARCIS (Netherlands)

Medema, Marnix H; Kottmann, Renzo; Yilmaz, Pelin; Cummings, Matthew; Biggins, John B; Blin, Kai; de Bruijn, Irene; Chooi, Yit Heng; Claesen, Jan; Coates, R Cameron; Cruz-Morales, Pablo; Duddela, Srikanth; Dusterhus, Stephanie; Edwards, Daniel J; Fewer, David P; Garg, Neha; Geiger, Christoph; Gomez-Escribano, Juan Pablo; Greule, Anja; Hadjithomas, Michalis; Haines, Anthony S; Helfrich, Eric J N; Hillwig, Matthew L; Ishida, Keishi; Jones, Adam C; Jones, Carla S; Jungmann, Katrin; Kegler, Carsten; Kim, Hyun Uk; Kotter, Peter; Krug, Daniel; Masschelein, Joleen; Melnik, Alexey V; Mantovani, Simone M; Monroe, Emily A; Moore, Marcus; Moss, Nathan; Nutzmann, Hans-Wilhelm; Pan, Guohui; Pati, Amrita; Petras, Daniel; Reen, F Jerry; Rosconi, Federico; Rui, Zhe; Tian, Zhenhua; Tobias, Nicholas J; Tsunematsu, Yuta; Wiemann, Philipp; Wyckoff, Elizabeth; Yan, Xiaohui; Yim, Grace; Yu, Fengan; Xie, Yunchang; Aigle, Bertrand; Apel, Alexander K; Balibar, Carl J; Balskus, Emily P; Barona-Gomez, Francisco; Bechthold, Andreas; Bode, Helge B; Borriss, Rainer; Brady, Sean F; Brakhage, Axel A; Caffrey, Patrick; Cheng, Yi-Qiang; Clardy, Jon; Cox, Russell J; De Mot, Rene; Donadio, Stefano; Donia, Mohamed S; van der Donk, Wilfred A; Dorrestein, Pieter C; Doyle, Sean; Driessen, Arnold J M; Ehling-Schulz, Monika; Entian, Karl-Dieter; Fischbach, Michael A; Gerwick, Lena; Gerwick, William H; Gross, Harald; Gust, Bertolt; Hertweck, Christian; Hofte, Monica; Jensen, Susan E; Ju, Jianhua; Katz, Leonard; Kaysser, Leonard; Klassen, Jonathan L; Keller, Nancy P; Kormanec, Jan; Kuipers, Oscar P; Kuzuyama, Tomohisa; Kyrpides, Nikos C; Kwon, Hyung-Jin; Lautru, Sylvie; Lavigne, Rob; Lee, Chia Y; Linquan, Bai; Liu, Xinyu; Liu, Wen; Luzhetskyy, Andriy; Mahmud, Taifo; Mast, Yvonne; Mendez, Carmen; Metsa-Ketela, Mikko; Micklefield, Jason; Mitchell, Douglas A; Moore, Bradley S; Moreira, Leonilde M; Muller, Rolf; Neilan, Brett A; Nett, Markus; Nielsen, Jens; O'Gara, Fergal; Oikawa, Hideaki; Osbourn, Anne; Osburne, Marcia S; Ostash, Bohdan; Payne, Shelley M; Pernodet, Jean-Luc; Petricek, Miroslav; Piel, Jorn; Ploux, Olivier; Raaijmakers, Jos M; Salas, Jose A; Schmitt, Esther K; Scott, Barry; Seipke, Ryan F; Shen, Ben; Sherman, David H; Sivonen, Kaarina; Smanski, Michael J; Sosio, Margherita; Stegmann, Evi; Sussmuth, Roderich D; Tahlan, Kapil; Thomas, Christopher M; Tang, Yi; Truman, Andrew W; Viaud, Muriel; Walton, Jonathan D; Walsh, Christopher T; Weber, Tilmann; van Wezel, Gilles P; Wilkinson, Barrie; Willey, Joanne M; Wohlleben, Wolfgang; Wright, Gerard D; Ziemert, Nadine; Zhang, Changsheng; Zotchev, Sergey B; Breitling, Rainer; Takano, Eriko; Glockner, Frank Oliver

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit.

Assessment of nuclear and mitochondrial genes in precise identification and analysis of genetic polymorphisms for the evaluation of Leishmania parasites.

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Fotouhi-Ardakani, Reza; Dabiri, Shahriar; Ajdari, Soheila; Alimohammadian, Mohammad Hossein; AlaeeNovin, Elnaz; Taleshi, Neda; Parvizi, Parviz

2016-12-01

The polymorphism and genetic diversity of Leishmania genus has status under discussion depending on many items such as nuclear and/or mitochondrial genes, molecular tools, Leishmania species, geographical origin, condition of micro-environment of Leishmania parasites and isolation of Leishmania from clinical samples, reservoir host and vectors. The genetic variation of Leishmania species (L. major, L. tropica, L. tarentolae, L. mexicana, L. infantum) were analyzed and compared using mitochondrial (COII and Cyt b) and nuclear (nagt, ITS-rDNA and HSP70) genes. The role of each enzymatic (COII, Cyt b and nagt) or housekeeping (ITS-rDNA, HSP70) gene was employed for accurate identification of Leishmania parasites. After DNA extractions and amplifying of native, natural and reference strains of Leishmania parasites, polymerase chain reaction (PCR) products were sequenced and evaluation of genetic proximity and phylogenetic analysis were performed using MEGA6 and DnaSP5 software. Among the 72 sequences of the five genes, the number of polymorphic sites was significantly lower as compared to the monomorphic sites. Of the 72 sequences, 54 new haplotypes (five genes) of Leishmania species were submitted in GenBank (Access number: KU680818 - KU680871). Four genes had a remarkable number of informative sites (P=0.00), except HSP70 maybe because of its microsatellite regions. The non-synonymous (dN) variants of nagt gene were more than that of other expression genes (47.4%). The synonymous (dS)/dN ratio in three expression genes showed a significant variation between five Leishmania species (P=0.001). The highest and lowest levels of haplotype diversity were observed in L. tropica (81.35%) and L. major (28.38%) populations, respectively. Tajima's D index analyses showed that Cyt b gene in L. tropica species was significantly negative (Tajima's D=-2.2, PLeishmania parasites. Copyright © 2016 Elsevier B.V. All rights reserved.

Leishmania attachment in permissive vectors and the role of sand fly midgut proteins in parasite-vector interaction

OpenAIRE

Dostálová, Anna

2012-01-01

of PhD. thesis named "Leishmania attachment in permissive vectors and the role of sand fly midgut proteins in parasite-vector interaction", Anna Dostálová, 2011 This thesis focuses on the development of protozoan parasites of the genus Leishmania in their insect vectors, sand flies. It sums up results of three projects I was involved in during my PhD studies. Main emphasis was put on permissive sand fly species that support development of various species of Leishmania. Using a novel method of...

Detection and identification of Leishmania spp.: application of two hsp70-based PCR-RFLP protocols to clinical samples from the New World.

Science.gov (United States)

Montalvo, Ana M; Fraga, Jorge; Tirado, Dídier; Blandón, Gustavo; Alba, Annia; Van der Auwera, Gert; Vélez, Iván Darío; Muskus, Carlos

2017-07-01

Leishmaniasis is highly prevalent in New World countries, where several methods are available for detection and identification of Leishmania spp. Two hsp70-based PCR protocols (PCR-N and PCR-F) and their corresponding restriction fragment length polymorphisms (RFLP) were applied for detection and identification of Leishmania spp. in clinical samples recruited in Colombia, Guatemala, and Honduras. A total of 93 cases were studied. The samples were classified into positive or suspected of leishmaniasis according to parasitological criteria. Molecular amplification of two different hsp70 gene fragments and further RFLP analysis for identification of Leishmania species was done. The detection in parasitologically positive samples was higher using PCR-N than PCR-F. In the total of samples studied, the main species identified were Leishmania panamensis, Leishmania braziliensis, and Leishmania infantum (chagasi). Although RFLP-N was more efficient for the identification, RFLP-F is necessary for discrimination between L. panamensis and Leishmania guyanesis, of great importance in Colombia. Unexpectedly, one sample from this country revealed an RFLP pattern corresponding to Leishmania naiffi. Both molecular variants are applicable for the study of clinical samples originated in Colombia, Honduras, and Guatemala. Choosing the better tool for each setting depends on the species circulating. More studies are needed to confirm the presence of L. naiffi in Colombian territory.

Comparison of codon usage bias across Leishmania and Trypanosomatids to understand mRNA secondary structure, relative protein abundance and pathway functions.

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Subramanian, Abhishek; Sarkar, Ram Rup

2015-10-01

Understanding the variations in gene organization and its effect on the phenotype across different Leishmania species, and to study differential clinical manifestations of parasite within the host, we performed large scale analysis of codon usage patterns between Leishmania and other known Trypanosomatid species. We present the causes and consequences of codon usage bias in Leishmania genomes with respect to mutational pressure, translational selection and amino acid composition bias. We establish GC bias at wobble position that governs codon usage bias across Leishmania species, rather than amino acid composition bias. We found that, within Leishmania, homogenous codon context coding for less frequent amino acid pairs and codons avoiding formation of folding structures in mRNA are essentially chosen. We predicted putative differences in global expression between genes belonging to specific pathways across Leishmania. This explains the role of evolution in shaping the otherwise conserved genome to demonstrate species-specific function-level differences for efficient survival. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterization of the biosynthetic gene cluster for cryptic phthoxazolin A in Streptomyces avermitilis.

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Dian Anggraini Suroto

Full Text Available Phthoxazolin A, an oxazole-containing polyketide, has a broad spectrum of anti-oomycete activity and herbicidal activity. We recently identified phthoxazolin A as a cryptic metabolite of Streptomyces avermitilis that produces the important anthelmintic agent avermectin. Even though genome data of S. avermitilis is publicly available, no plausible biosynthetic gene cluster for phthoxazolin A is apparent in the sequence data. Here, we identified and characterized the phthoxazolin A (ptx biosynthetic gene cluster through genome sequencing, comparative genomic analysis, and gene disruption. Sequence analysis uncovered that the putative ptx biosynthetic genes are laid on an extra genomic region that is not found in the public database, and 8 open reading frames in the extra genomic region could be assigned roles in the biosynthesis of the oxazole ring, triene polyketide and carbamoyl moieties. Disruption of the ptxA gene encoding a discrete acyltransferase resulted in a complete loss of phthoxazolin A production, confirming that the trans-AT type I PKS system is responsible for the phthoxazolin A biosynthesis. Based on the predicted functional domains in the ptx assembly line, we propose the biosynthetic pathway of phthoxazolin A.

Computational prediction of protein-protein interactions in Leishmania predicted proteomes.

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Antonio M Rezende

Full Text Available The Trypanosomatids parasites Leishmania braziliensis, Leishmania major and Leishmania infantum are important human pathogens. Despite of years of study and genome availability, effective vaccine has not been developed yet, and the chemotherapy is highly toxic. Therefore, it is clear just interdisciplinary integrated studies will have success in trying to search new targets for developing of vaccines and drugs. An essential part of this rationale is related to protein-protein interaction network (PPI study which can provide a better understanding of complex protein interactions in biological system. Thus, we modeled PPIs for Trypanosomatids through computational methods using sequence comparison against public database of protein or domain interaction for interaction prediction (Interolog Mapping and developed a dedicated combined system score to address the predictions robustness. The confidence evaluation of network prediction approach was addressed using gold standard positive and negative datasets and the AUC value obtained was 0.94. As result, 39,420, 43,531 and 45,235 interactions were predicted for L. braziliensis, L. major and L. infantum respectively. For each predicted network the top 20 proteins were ranked by MCC topological index. In addition, information related with immunological potential, degree of protein sequence conservation among orthologs and degree of identity compared to proteins of potential parasite hosts was integrated. This information integration provides a better understanding and usefulness of the predicted networks that can be valuable to select new potential biological targets for drug and vaccine development. Network modularity which is a key when one is interested in destabilizing the PPIs for drug or vaccine purposes along with multiple alignments of the predicted PPIs were performed revealing patterns associated with protein turnover. In addition, around 50% of hypothetical protein present in the networks

Functional and phylogenetic evidence of a bacterial origin for the first enzyme in sphingolipid biosynthesis in a phylum of eukaryotic protozoan parasites.

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Mina, John G; Thye, Julie K; Alqaisi, Amjed Q I; Bird, Louise E; Dods, Robert H; Grøftehauge, Morten K; Mosely, Jackie A; Pratt, Steven; Shams-Eldin, Hosam; Schwarz, Ralph T; Pohl, Ehmke; Denny, Paul W

2017-07-21

Toxoplasma gondii is an obligate, intracellular eukaryotic apicomplexan protozoan parasite that can cause fetal damage and abortion in both animals and humans. Sphingolipids are essential and ubiquitous components of eukaryotic membranes that are both synthesized and scavenged by the Apicomplexa. Here we report the identification, isolation, and analyses of the Toxoplasma serine palmitoyltransferase, an enzyme catalyzing the first and rate-limiting step in sphingolipid biosynthesis: the condensation of serine and palmitoyl-CoA. In all eukaryotes analyzed to date, serine palmitoyltransferase is a highly conserved heterodimeric enzyme complex. However, biochemical and structural analyses demonstrated the apicomplexan orthologue to be a functional, homodimeric serine palmitoyltransferase localized to the endoplasmic reticulum. Furthermore, phylogenetic studies indicated that it was evolutionarily related to the prokaryotic serine palmitoyltransferase, identified in the Sphingomonadaceae as a soluble homodimeric enzyme. Therefore this enzyme, conserved throughout the Apicomplexa, is likely to have been obtained via lateral gene transfer from a prokaryote. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Reconstrucción quirúrgica tras destrucción nasal por Leishmania Panamensis Surgical reconstruction after nasal destruction by Leishmania Panamensis

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F. Vélez Bernal

2013-03-01

Full Text Available Algunas especies de Leishmania del subgénero Viannia, especialmente Leishmania braziliensis y Leishmania panamensis, pueden invadir la mucosa naso-orofaríngea al diseminarse por vía linfática y sanguínea a partir de una lesión cutánea y ocasionar lesiones en el tabique nasal, paladar blando, úvula, pilares amigdalinos, laringe, faringe, dorso nasal, labios y pómulos, que pueden conducir a la desfiguración. La mucosa más frecuentemente afectada es la del tabique nasal, principalmente en su parte anterior. La invasión de la mucosa puede ocurrir simultáneamente con lesiones cutáneas activas, aunque más frecuentemente aparecen 1 o 2 años después de la lesión en la piel; sin embargo, en el 16 % de los casos no hay antecedentes de lesiones cutáneas, lo que sugiere que con la picadura del insecto vector se produjo una infección primaria asintomática u oligosintomática y luego se produjo la diseminación del parasito a la mucosas. En este artículo presentamos 2 casos clínicos de leishmaniosis mucosa producidos por L. panamensis y los procedimientos quirúrgicos reconstructivos que se realizaron. Se hace además un recuento de los diagnósticos diferenciales en tejidos oronasales.Species of Leishmania of Viannia subgenus, mainly L. braziliensis and L. panamensis, may invade the nasooro-pharyngeal mucosal after spread from the skin lesion via lymph and blood, causing lesions in the nasal septum, soft palate, uvula, tonsillar pillars, larynx, pharynx, nasal dorsum, lips and cheeks. The mucosal membrane most frequently affected is the nasal septum, mainly in the anterior region. The invasion of mucosa may occur simultaneously with active skin lesions, but most often appear 1 or 2 years after the skin lesion; nevertheless, in 16 % of cases there is no history of skin lesions suggesting that the primary infection coursed with few symptoms and then was spread to mucosal membranes. In this article 2 cases of L panamensis mucosal

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

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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.

Expression profile of genes coding for carotenoid biosynthetic ...

Indian Academy of Sciences (India)

Expression profile of genes coding for carotenoid biosynthetic pathway during ripening and their association with accumulation of lycopene in tomato fruits. Shuchi Smita, Ravi Rajwanshi, Sangram Keshari Lenka, Amit Katiyar, Viswanathan Chinnusamy and. Kailash Chander Bansal. J. Genet. 92, 363–368. Table 1.

Effect of ionizing radiation on the morphology, physiology and growth of Leishmania ssp; Acao da radiacao ionizante sobre a morfologia, fisiologia e crescimento da Leishmania spp

Energy Technology Data Exchange (ETDEWEB)

Bonetti, Franco C.; Spencer, Patrick J.; Nascimento, Nanci do [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil); Junior A, Heitor F. [Sao Paulo Univ., SP (Brazil). Faculdade de Medicina. Instituto de Medicina Tropical

2000-07-01

The Leishmania spp is a pathogenic protozoan, which cause different diseases in man. The human diseases, in America, caused by this group of protozoa are divided in cutaneous or tegumentar and visceral, known as kala-azar. In this work, our principal study object was the specie that causes tegumentar leishmaniasis, in Brazil. Metabolic studies of cellular respiration and proteins and nucleic acids synthesis were accomplished using radiation as a form of sterilizing the parasites without however affecting their immunogenic capacity The promastigotes forms of irradiated Leishmania spp were totally sterilized with the dose of 1500 Gy, with their reproductive and nucleic acids, as well as protein synthesis capacity blocked. (author)

A Lipidomics Approach to Assess the Association Between Plasma Sphingolipids and Verbal Memory Performance in Coronary Artery Disease Patients Undertaking Cardiac Rehabilitation: A C18:0 Signature for Cognitive Response to Exercise.

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Saleem, Mahwesh; Herrmann, Nathan; Dinoff, Adam; Mielke, Michelle M; Oh, Paul I; Shammi, Prathiba; Cao, Xingshan; Venkata, Swarajya Lakshmi Vattem; Haughey, Norman J; Lanctôt, Krista L

2017-01-01

Early subtle deficits in verbal memory, which may indicate early neural risk, are common in patients with coronary artery disease (CAD). While exercise can improve cognition, cognitive response to exercise is heterogeneous. Sphingolipids have been associated with the development and progression of CAD, and impairments in sphingolipid metabolism may play roles in neurodegeneration and in the neural adaptation response to exercise. In this study, change in plasma concentrations of sphingolipids was assessed in relation to change in verbal memory performance and in other cognitive domains among CAD subjects undertaking a 6-month cardiac rehabilitation (CR) program. Patients with CAD (n = 120, mean age = 64±6 y, 84% male, years of education = 16±3) underwent CR with neuropsychological assessments and blood collected at baseline, 3-, and 6-months. Z-scores based on age, gender, and education were combined for verbal memory, visuospatial memory, processing speed, executive function, and global cognition tasks to calculate cognitive domain Z-scores. Plasma sphingolipid concentrations were measured from fasting blood samples using high performance liquid chromatography coupled electrospray ionization tandem mass spectrometry (LC/MS/MS). Mixed models were used to identify sphingolipids significantly associated with performance in verbal memory and other cognitive domains, adjusting for potential confounders. A decrease in ceramide C18:0 concentration was significantly associated with improvement in verbal memory performance (b[SE] = -0.51 [0.25], p = 0.04), visuospatial memory (b[SE] = -0.44 [0.22], p = 0.05), processing speed (b[SE] = -0.89 [0.32], p = 0.007), and global cognition (b[SE] = -1.47 [0.59], p = 0.01) over 6 months of CR. Plasma ceramide C18:0 concentrations may be a sensitive marker of cognitive response to exercise in patients with CAD.

Immunopathological Features of Canine Myocarditis Associated with Leishmania infantum Infection

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Alessandro Costagliola

2016-01-01

Full Text Available Myocarditis associated with infectious diseases may occur in dogs, including those caused by the protozoa Neospora caninum, Trypanosoma cruzi, Babesia canis, and Hepatozoon canis. However, although cardiac disease due to Leishmania infection has also been documented, the immunopathological features of myocarditis have not been reported so far. The aim of this study was to examine the types of cellular infiltrates and expression of MHC classes I and II in myocardial samples obtained at necropsy from 15 dogs with an established intravitam diagnosis of visceral leishmaniasis. Pathological features of myocardium were characterized by hyaline degeneration of cardiomyocytes, necrosis, and infiltration of mononuclear inflammatory cells consisting of lymphocytes and macrophages, sometimes with perivascular pattern; fibrosis was also present in various degrees. Immunophenotyping of inflammatory cells was performed by immunohistochemistry on cryostat sections obtained from the heart of the infected dogs. The predominant leukocyte population was CD8+ with a fewer number of CD4+ cells. Many cardiomyocytes expressed MHC classes I and II on the sarcolemma. Leishmania amastigote forms were not detected within macrophages or any other cell of the examined samples. Our study provided evidence that myocarditis in canine visceral leishmaniasis might be related to immunological alterations associated with Leishmania infection.

4-Hydroxynonenal Contributes to Angiogenesis through a Redox-Dependent Sphingolipid Pathway: Prevention by Hydralazine Derivatives

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Caroline Camaré

2017-01-01

Full Text Available The neovascularization of atherosclerotic lesions is involved in plaque development and may contribute to intraplaque hemorrhage and plaque fragilization and rupture. Among the various proangiogenic agents involved in the neovascularization process, proatherogenic oxidized LDLs (oxLDLs contribute to the formation of tubes via the generation of sphingosine 1-phosphate (S1P, a major mitogenic and proangiogenic sphingolipid mediator. In this study, we investigated whether 4-hydroxynonenal (4-HNE, an aldehydic lipid oxidation product abundantly present in oxLDLs, contributes to their proangiogenic properties. Immunofluorescence analysis of human atherosclerotic lesions from carotid endarterectomy showed the colocalization of HNE-adducts with CD31, a marker of endothelial cells, suggesting a close relationship between 4-HNE and neovessel formation. In vitro, low 4-HNE concentration (0.5–1 µM elicited the formation of tubes by human microvascular endothelial cells (HMEC-1, whereas higher concentrations were not angiogenic. The formation of tubes by 4-HNE involved the generation of reactive oxygen species and the activation of the sphingolipid pathway, namely, the neutral type 2 sphingomyelinase and sphingosine kinase-1 (nSMase2/SK-1 pathway, indicating a role for S1P in the angiogenic signaling of 4-HNE. Carbonyl scavengers hydralazine and bisvanillyl-hydralazone inhibited the nSMase2/SK1 pathway activation and the formation of tubes on Matrigel® evoked by 4-HNE. Altogether, these results emphasize the role of 4-HNE in the angiogenic effect of oxLDLs and point out the potential interest of pharmacological carbonyl scavengers to prevent the neovascularization process.

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

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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.

Recent development of antiSMASH and other computational approaches to mine secondary metabolite biosynthetic gene clusters

DEFF Research Database (Denmark)

Blin, Kai; Kim, Hyun Uk; Medema, Marnix H.

2017-01-01

Many drugs are derived from small molecules produced by microorganisms and plants, so-called natural products. Natural products have diverse chemical structures, but the biosynthetic pathways producing those compounds are often organized as biosynthetic gene clusters (BGCs) and follow a highly...... conserved biosynthetic logic. This allows for the identification of core biosynthetic enzymes using genome mining strategies that are based on the sequence similarity of the involved enzymes/genes. However, mining for a variety of BGCs quickly approaches a complexity level where manual analyses...... are no longer possible and require the use of automated genome mining pipelines, such as the antiSMASH software. In this review, we discuss the principles underlying the predictions of antiSMASH and other tools and provide practical advice for their application. Furthermore, we discuss important caveats...

Novel features of a PIWI-like protein homolog in the parasitic protozoan Leishmania.

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Prasad K Padmanabhan

Full Text Available 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.

[Leishmaniasis in Ecuador. 4. Natural infestation of the dog by Leishmania panamensis].

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Dereure, J; Espinel, I; Barrera, C; Guerrini, F; Martini, A; Echeverria, R; Guderian, R H; Le Pont, F

1994-03-01

In two endemic leishmaniasis foci of the Pacific coast of Ecuador 34 dogs suspected of having the disease have been surveyed clinically, serologically and parasitologically; immunofluorescence and electrosyneresis tests, lymph node aspirates, biopsies and smears have been performed. From two dogs with ulcers only one had ulcers on the muzzle and the scrotum infected by Leishmania (L. guyanensis complex). The isolated strain was identified as Leishmania panamensis. The disease was strictly cutaneous. In the study area the dog seems to be more a victim-host than a reservoir.

Profiling of Plasma Metabolites Suggests Altered Mitochondrial Fuel Usage and Remodeling of Sphingolipid Metabolism in Individuals With Type 2 Diabetes and Kidney Disease

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Jian-Jun Liu

2017-05-01

Discussion: DKD is associated with altered fuel substrate use and remodeling of sphingolipid metabolism in T2DM with DKD. Associations of albuminuria and impaired filtration function with distinct metabolomic signatures suggest different pathophysiology underlying these 2 manifestations of DKD.

Biosynthetic Pathway and Metabolic Engineering of Plant Dihydrochalcones.

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Ibdah, Mwafaq; Martens, Stefan; Gang, David R

2018-03-14

Dihydrochalcones are plant natural products containing the phenylpropanoid backbone and derived from the plant-specific phenylpropanoid pathway. Dihydrochalcone compounds are important in plant growth and response to stresses and, thus, can have large impacts on agricultural activity. In recent years, these compounds have also received increased attention from the biomedical community for their potential as anticancer treatments and other benefits for human health. However, they are typically produced at relatively low levels in plants. Therefore, an attractive alternative is to express the plant biosynthetic pathway genes in microbial hosts and to engineer the metabolic pathway/host to improve the production of these metabolites. In the present review, we discuss in detail the functions of genes and enzymes involved in the biosynthetic pathway of the dihydrochalcones and the recent strategies and achievements used in the reconstruction of multi-enzyme pathways in microorganisms in efforts to be able to attain higher amounts of desired dihydrochalcones.

Bibliography of Leishmania and Leishmanial Diseases. Volume 2

Science.gov (United States)

1980-08-01

Leishmaniosi visceizile latente in adulto rivelatasi PHOTINOS, P. B, (1930A) Formes atypiques nel corso di una, Infezione tifoidea . N infiltries en... fiebre recurrente y con gran flagelle’s au cours des leishmanioses cantidad de leishmanias en sangre penl- ’cliniques et expegrinientales. Ann.ferlca

Biosynthesis of silver nanoparticles by Leishmania tropica | Rahi ...

African Journals Online (AJOL)

A novel biosynthesis route for Silver Nanoparticles (Ag-NPs) was attempted in the present study using Leishmania tropica the causative agent of cutaneous leishmaniasis in different countries, particularly in Mediterranean region in Iraq. Silver nanoparticles were successfully synthesized from AgNO3 by reduction of ...

Identification of a RAC/AKT-like gene in Leishmania parasites as a putative therapeutic target in leishmaniasis.

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Varela-M, Rubén E; Ochoa, Rodrigo; Muskus, Carlos E; Muro, Antonio; Mollinedo, Faustino

2017-10-10

Leishmaniasis is one of the world's most neglected diseases caused by at least 20 different species of the protozoan parasite Leishmania. Although new drugs have become recently available, current therapy for leishmaniasis is still unsatisfactory. A subgroup of serine/threonine protein kinases named as related to A and C protein kinases (RAC), or protein kinase B (PKB)/AKT, has been identified in several organisms including Trypanosoma cruzi parasites. PKB/AKT plays a critical role in mammalian cell signaling promoting cell survival and is a major drug target in cancer therapy. However, the role of protozoan parasitic PKB/AKT remains to be elucidated. We have found that anti-human AKT antibodies recognized a protein of about 57 kDa in Leishmania spp. parasites. Anti-human phospho-AKT(Thr308) antibodies identified a protein in extracts from Leishmania spp. that was upregulated following parasite exposure to stressful conditions, such as nutrient deprivation or heat shock. Incubation of AKT inhibitor X with Leishmania spp. promastigotes under stressful conditions or with Leishmania-infected macrophages led to parasite cell death. We have identified and cloned a novel gene from Leishmania donovani named Ld-RAC/AKT-like gene, encoding a 510-amino acid protein of approximately 57.6 kDa that shows a 26.5% identity with mammalian AKT1. Ld-RAC/AKT-like protein contains major mammalian PKB/AKT hallmarks, including the typical pleckstrin, protein kinase and AGC kinase domains. Unlike mammalian AKT that contains key phosphorylation sites at Thr308 and Ser473 in the activation loop and hydrophobic motif, respectively, Ld-RAC/AKT-like protein has a Thr residue in both motifs. By domain sequence comparison, we classified AKT proteins from different origins in four major subcategories that included different parasites. Our data suggest that Ld-RAC/AKT-like protein represents a Leishmania orthologue of mammalian AKT involved in parasite stress response and survival, and

The Impact of Cholesterol, DHA, and Sphingolipids on Alzheimer’s Disease

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Marcus O. W. Grimm

2013-01-01

Full Text Available Alzheimer’s disease (AD is a devastating neurodegenerative disorder currently affecting over 35 million people worldwide. Pathological hallmarks of AD are massive amyloidosis, extracellular senile plaques, and intracellular neurofibrillary tangles accompanied by an excessive loss of synapses. Major constituents of senile plaques are 40–42 amino acid long peptides termed β-amyloid (Aβ. Aβ is produced by sequential proteolytic processing of the amyloid precursor protein (APP. APP processing and Aβ production have been one of the central scopes in AD research in the past. In the last years, lipids and lipid-related issues are more frequently discussed to contribute to the AD pathogenesis. This review summarizes lipid alterations found in AD postmortem brains, AD transgenic mouse models, and the current understanding of how lipids influence the molecular mechanisms leading to AD and Aβ generation, focusing especially on cholesterol, docosahexaenoic acid (DHA, and sphingolipids/glycosphingolipids.

Novel Leishmania and Malaria Potassium Channels: Candidate Therapeutic Targets

National Research Council Canada - National Science Library

McDonald, Thomas V

2005-01-01

.... major and T. cruzi). Using a combination of cultured mammalian cells and Xenopus oocytes for heterologous expression we have evidence that 2 channels from malaria [PFK1 & PFK22] and Leishmania [LMK1 & LMK2] generate K+...

On the biosynthetic origin of carminic acid

DEFF Research Database (Denmark)

Rasmussen, Silas A.; Kongstad, Kenneth T; Khorsand-Jamal, Paiman

2018-01-01

provides solid evidence of a polyketide, rather than a shikimate, origin of coccid pigments. Based on the newly identified compounds, we present a detailed biosynthetic scheme that accounts for the formation of carminic acid (CA) in D. coccus and all described coccid pigments which share a flavokermesic...... distribution suggests a common evolutionary origin for the trait in all coccid dye producing insect species....

Xenodiagnostico con Lutzomyia youngi en casos venezolanos de leishmaniasis cutaned por Leishmania braziliensis Xenodiagnosis with Lutzomyia youngi in Venezuelan cases of cutaneous leishmaniais due to Leishmania braziliensis

Directory of Open Access Journals (Sweden)

Elina Rojas

1989-03-01

Full Text Available Xenodiagnósticos con Lutzomya yungi aplicados en los bordes de las úlceras de pacientes infectados con Leishmania braziliensis antes y después del tratamiento con 10 dosis de antimonial pentavalente y un aminoglicósido, evidencian la condición reservoria de leishmanias del enfermo, para flebótomos endofágicos y la utilidad de un tratamiento específico-temprano que no solamente conduce a la curación clínica, sino a la eliminación del riesgo de una eventual transmisión intradomiciliar por insectos que pican dentro del domicilio durante la noche.Eight patients infected with Leishmania braziliensis were used for xenodiagnosis with Lutzomtyia youngi, before and after specific antileishmanial treatment with "glucantime" and "gabbromycin". All of them infected sandflies fed on the borders of the skin lesions before the treatment, suggesting that infected persons might act as reservoirs of infection for an indoor-bitting sandfly species. The negative results obtained by xenodiagnosis carried out after specific treatment of the same individuals indicated cure of the patients, and a reduction of risk for further intradomiciliary transmission.

Cross-protective immunity to Leishmania amazonensis is mediated by CD4+ and CD8+-epitopes of Leishmania donovani Nucleoside Hydrolase terminal domains

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Dirlei eNico

2014-05-01

Full Text Available The Nucleoside hydrolase of Leishmania donovani (NH36 is a phylogenetic marker of high homology among Leishmania parasites. In mice and dog vaccination NH36 induces a CD4+ T cell-driven protective response against Leishmania chagasi infection directed against its C-terminal domain (F3. The C-terminal and N-terminal domain vaccines also decreased the footpad lesion caused by Leishmania amazonensis. We studied the basis of the crossed immune response using recombinant generated peptides covering the whole NH36 sequence and saponin for mice prophylaxis against L. amazonensis. The F1 (amino acids 1-103 and F3 peptide (amino acids 199-314 vaccines enhanced the IgG and IgG2a anti-NH36 antibodies to similar levels. The F3 vaccine induced the strongest DTH response, the highest proportions of NH36-specific CD4+ and CD8+ T cells after challenge and the highest expression of IFN-γ and TNF-α. The F1 vaccine, on the other hand, induced a weaker but significant DTH response and a mild enhancement of IFN-γ and TNF-α levels. The in vivo depletion with anti-CD4 or CD8 monoclonal antibodies disclosed that cross-protection against L. amazonensis infection was mediated by a CD4+ T cell response directed against the C-terminal domain (75% of reduction of the size of footpad lesion followed by a CD8+ T cell response against the N-terminal domain of NH36 (57% of reduction of footpad lesions. Both vaccines were capable of inducing long-term cross-immunity. The amino acid sequence of NH36 showed 93% identity to the sequence of the NH A34480 of L. amazonensis which also showed the presence of completely conserved predicted epitopes for CD4+ and CD8+ T cells in F1 domain, and of CD4+ epitopes differing in a single amino acid, in F1 and F3 domains. The identification of the C-terminal and N-terminal domains as the targets of the immune response to NH36 in the model of L. amazonesis infection represents a basis for the rationale development of a bivalent vaccine

Expression of hsa Let-7a MicroRNA of Macrophages Infected by Leishmania Major

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Nooshin Hashemi

2016-10-01

Full Text Available Leishmaniasis is a vector-born disease caused by species of the genus Leishmania and is transmitted from host to host through the bite of an infected sandfly. MicroRNAs (miRNAs are non-coding small RNAs with 22-nucleotide length. They are involved in some biological and cellular processes. We aimed to evaluate the expression of let-7a in human macrophages miRNA when are infected by Leishmania major. We also evaluated the impact of Leishmania major infection on the expression of let-7a at two different times, 24 and 48 hours, after infection. Blood samples were collected from ten healthy volunteers with no history of leishmaniasis. Development of macrophages from peripheral monocytes and infection with stationary phase of Leishmania major promastigotes were done through serial cultures under 5% CO2 environment and 37C. To measure the expression levels of let-7a real-time PCR was performed with specific related primers using the SYBR® Green master mix Kit™. The real-time PCR showed let-7a was expressed in cells infected with parasites after 24 and 48h post-infection. Comparison of let-7a miRNA expression after 24 and 48 h revealed that let-7a miRNAs were down-regulated at 48 h post-infection more than 24h after infection. The results of this study suggest that according to the main function of miRNA in repression of mRNA translation it could be possible to manipulate host cells in order to alter miRNA levels and regulate macrophage functions after establishment of intracellular parasites such as Leishmania.

Studies on Stibanate unresponsive isolates of Leishmania donovani

Indian Academy of Sciences (India)

Unknown

503. Keywords. Drug resistance; Leishmania donovani; sodium stibogluconate; visceral leishmaniasis ... cases to less than 5% in treated ones (Zuckerman and. Lainson 1977). Later on .... Effect of Stibanate on in vitro growth of Ag83 promastigotes. (A) Virulent .... In rural areas where health care facilities are not adequate ...

Tetracycline-inducible gene expression system in Leishmania mexicana

Czech Academy of Sciences Publication Activity Database

Kraeva, N.; Ishemgulova, A.; Lukeš, Julius; Yurchenko, Vyacheslav

2014-01-01

Roč. 198, č. 1 (2014), s. 11-13 ISSN 0166-6851 R&D Projects: GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : Leishmania mexicana * Gene expression * Tet-inducible system Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.787, year: 2014

Structural Diversification of Lyngbyatoxin A by Host-Dependent Heterologous Expression of the tleABC Biosynthetic Gene Cluster.

Science.gov (United States)

Zhang, Lihan; Hoshino, Shotaro; Awakawa, Takayoshi; Wakimoto, Toshiyuki; Abe, Ikuro

2016-08-03

Natural products have enormous structural diversity, yet little is known about how such diversity is achieved in nature. Here we report the structural diversification of a cyanotoxin-lyngbyatoxin A-and its biosynthetic intermediates by heterologous expression of the Streptomyces-derived tleABC biosynthetic gene cluster in three different Streptomyces hosts: S. lividans, S. albus, and S. avermitilis. Notably, the isolated lyngbyatoxin derivatives, including four new natural products, were biosynthesized by crosstalk between the heterologous tleABC gene cluster and the endogenous host enzymes. The simple strategy described here has expanded the structural diversity of lyngbyatoxin A and its biosynthetic intermediates, and provides opportunities for investigation of the currently underestimated hidden biosynthetic crosstalk. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

MHC class II restricted innate-like double negative T cells contribute to optimal primary and secondary immunity to Leishmania major.

Science.gov (United States)

Mou, Zhirong; Liu, Dong; Okwor, Ifeoma; Jia, Ping; Orihara, Kanami; Uzonna, Jude Ezeh

2014-09-01

Although it is generally believed that CD4(+) T cells play important roles in anti-Leishmania immunity, some studies suggest that they may be dispensable, and that MHC II-restricted CD3(+)CD4(-)CD8(-) (double negative, DN) T cells may be more important in regulating primary anti-Leishmania immunity. In addition, while there are reports of increased numbers of DN T cells in Leishmania-infected patients, dogs and mice, concrete evidence implicating these cells in secondary anti-Leishmania immunity has not yet been documented. Here, we report that DN T cells extensively proliferate and produce effector cytokines (IFN-γ, TNF and IL-17) and granzyme B (GrzB) in the draining lymph nodes and spleens of mice following primary and secondary L. major infections. DN T cells from healed mice display functional characteristics of protective anti-Leishmania memory-like cells: rapid and extensive proliferation and effector cytokines production following L. major challenge in vitro and in vivo. DN T cells express predominantly (> 95%) alpha-beta T cell receptor (αβ TCR), are Leishmania-specific, restricted mostly by MHC class II molecules and display transcriptional profile of innate-like genes. Using in vivo depletion and adoptive transfer studies, we show that DN T cells contribute to optimal primary and secondary anti-Leishmania immunity in mice. These results directly identify DN T cells as important players in effective and protective primary and secondary anti-L. major immunity in experimental cutaneous leishmaniasis.

Expression of Recombinant Human Coagulation Factor VII by the Lizard Leishmania Expression System

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Sina Mirzaahmadi

2011-01-01

Full Text Available The variety of recombinant protein expression systems have been developed as a resource of FVII gene expression. In the current study, the authors used a novel protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host for expression of FVII. Plasmid containing cDNA encoding full-length human FVII was introduced into Lizard Leishmania and positive transfectants were analyzed by SDS-PAGE and Western blot analysis. Furthermore, biological activity of purified protein was detected by PT assay. The recombinant strain harboring a construct was analyzed for expression of FVII at the mRNA and protein level. Purified rFVII was obtained and in order to confirm the purified compound was in fact rFVII. Western blot analysis was carried out. Clotting time in PT assay was reduced about 30 seconds with the purified rFVII. In Conclusion, this study has demonstrated, for the first time, that Leishmania cells can be used as an expression system for producing recombinant FVII.

Importing statistical measures into Artemis enhances gene identification in the Leishmania genome project

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McDonagh Paul D

2003-06-01

Full Text Available Abstract Background Seattle Biomedical Research Institute (SBRI as part of the Leishmania Genome Network (LGN is sequencing chromosomes of the trypanosomatid protozoan species Leishmania major. At SBRI, chromosomal sequence is annotated using a combination of trained and untrained non-consensus gene-prediction algorithms with ARTEMIS, an annotation platform with rich and user-friendly interfaces. Results Here we describe a methodology used to import results from three different protein-coding gene-prediction algorithms (GLIMMER, TESTCODE and GENESCAN into the ARTEMIS sequence viewer and annotation tool. Comparison of these methods, along with the CODONUSAGE algorithm built into ARTEMIS, shows the importance of combining methods to more accurately annotate the L. major genomic sequence. Conclusion An improvised and powerful tool for gene prediction has been developed by importing data from widely-used algorithms into an existing annotation platform. This approach is especially fruitful in the Leishmania genome project where there is large proportion of novel genes requiring manual annotation.

Differentiation of Leishmania species by FT-IR spectroscopy

Science.gov (United States)

Aguiar, Josafá C.; Mittmann, Josane; Ferreira, Isabelle; Ferreira-Strixino, Juliana; Raniero, Leandro

2015-05-01

Leishmaniasis is a parasitic infectious disease caused by protozoa that belong to the genus Leishmania. It is transmitted by the bite of an infected female Sand fly. The disease is endemic in 88 countries Desjeux (2001) [1] (16 developed countries and 72 developing countries) on four continents. In Brazil, epidemiological data show the disease is present in all Brazilian regions, with the highest incidences in the North and Northeast. There are several methods used to diagnose leishmaniasis, but these procedures have many limitations, are time consuming, have low sensitivity, and are expensive. In this context, Fourier Transform Infrared Spectroscopy (FT-IR) analysis has the potential to provide rapid results and may be adapted for a clinical test with high sensitivity and specificity. In this work, FT-IR was used as a tool to investigate the promastigotes of Leishmaniaamazonensis, Leishmaniachagasi, and Leishmaniamajor species. The spectra were analyzed by cluster analysis and deconvolution procedure base on spectra second derivatives. Results: cluster analysis found four specific regions that are able to identify the Leishmania species. The dendrogram representation clearly indicates the heterogeneity among Leishmania species. The band deconvolution done by the curve fitting in these regions quantitatively differentiated the polysaccharides, amide III, phospholipids, proteins, and nucleic acids. L. chagasi and L. major showed a greater biochemistry similarity and have three bands that were not registered in L. amazonensis. The L. amazonensis presented three specific bands that were not recorded in the other two species. It is evident that the FT-IR method is an indispensable tool to discriminate these parasites. The high sensitivity and specificity of this technique opens up the possibilities for further studies about characterization of other microorganisms.

Epidemiologic relationship between Toscana virus infection and Leishmania infantum due to common exposure to Phlebotomus perniciosus sandfly vector.

Science.gov (United States)

Bichaud, Laurence; Souris, Marc; Mary, Charles; Ninove, Laëtitia; Thirion, Laurence; Piarroux, Raphaël P; Piarroux, Renaud; De Lamballerie, Xavier; Charrel, Rémi N

2011-09-01

Sand flies are recognised vectors of parasites in the genus Leishmania and a number of arthropod-borne viruses, in particular viruses within the genus Phlebovirus, family Bunyaviridae. In southern France, Toscana phlebovirus (TOSV) is recognized as a prominent cause of summer meningitis. Since Leishmania and TOSV have a common vector (Phlebotomus perniciosus), an epidemiologic link has been assumed for a long time. However, there is no scientific evidence of such a link between human leishmaniosis and phleboviral infections. To identify a possible link, we investigated the presence and distribution of antibodies against these two microorganisms (i) in individuals and (ii) at a spatial level in the city of Marseille (south-eastern France). Five hundred sera were selected randomly in the biobank of the Department of Parasitology of the Public Hospitals of Marseille. All sera were previously tested for IgG against Leishmania by Western Blotting, and TOSV IgG were detected by indirect immunofluorescence. The seropositivity rates were 21.4% for TOSV and 28% for Leishmania. Statistical analysis demonstrated that seropositivity for one pathogen was significantly associated with seropositivity to the other pathogen. This result provided the first robust evidence for the existence of an epidemiological relationship between Leishmania infantum and TOSV. Addresses of tested patients were geolocalized and integrated into Geographical Information System software, in order to test spatial relationship between the two pathogens. Spatial analysis did not allow to identify (i) specific patterns for the spatial distribution of positive serological results for TOSV or Leishmania, and (ii) a spatial relationship between Leishmania and TOSV positive serological results. This may reflect the fact that the sample studied was not powerful enough to demonstrate either a spatial clustering or co-location, i.e. that the actual risk exposure area is smaller than the mean of distance between

Epidemiologic relationship between Toscana virus infection and Leishmania infantum due to common exposure to Phlebotomus perniciosus sandfly vector.

Directory of Open Access Journals (Sweden)

Laurence Bichaud

2011-09-01

Full Text Available Sand flies are recognised vectors of parasites in the genus Leishmania and a number of arthropod-borne viruses, in particular viruses within the genus Phlebovirus, family Bunyaviridae. In southern France, Toscana phlebovirus (TOSV is recognized as a prominent cause of summer meningitis. Since Leishmania and TOSV have a common vector (Phlebotomus perniciosus, an epidemiologic link has been assumed for a long time. However, there is no scientific evidence of such a link between human leishmaniosis and phleboviral infections. To identify a possible link, we investigated the presence and distribution of antibodies against these two microorganisms (i in individuals and (ii at a spatial level in the city of Marseille (south-eastern France. Five hundred sera were selected randomly in the biobank of the Department of Parasitology of the Public Hospitals of Marseille. All sera were previously tested for IgG against Leishmania by Western Blotting, and TOSV IgG were detected by indirect immunofluorescence. The seropositivity rates were 21.4% for TOSV and 28% for Leishmania. Statistical analysis demonstrated that seropositivity for one pathogen was significantly associated with seropositivity to the other pathogen. This result provided the first robust evidence for the existence of an epidemiological relationship between Leishmania infantum and TOSV. Addresses of tested patients were geolocalized and integrated into Geographical Information System software, in order to test spatial relationship between the two pathogens. Spatial analysis did not allow to identify (i specific patterns for the spatial distribution of positive serological results for TOSV or Leishmania, and (ii a spatial relationship between Leishmania and TOSV positive serological results. This may reflect the fact that the sample studied was not powerful enough to demonstrate either a spatial clustering or co-location, i.e. that the actual risk exposure area is smaller than the mean of

Detection, molecular typing and phylogenetic analysis of Leishmania isolated from cases of leishmaniasis among Syrian refugees in Lebanon

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Tamara Salloum

2016-06-01

Two molecular typing methods of 39 FFPE Leishmania isolates were used: the ITS1-PCR RFLP and the nested ITS1-5.8S rDNA gene amplification followed by sequencing and phylogenetic analysis. The efficiency of these two techniques in Leishmania identification was compared and the phylogenetic relationships among these isolates were illustrated based on the neighbor-joining (NJ method. The results were statistically correlated with the parasitic index (PI. The DNA storage in formalin-fixed paraffin embedded (FFPE tissues was assessed as well. The parasites identified were all L. tropica as determined by both techniques. ITS1-5.8S rDNA gene based typing proved to be more sensitive in the detection of parasites (positive in 69.2% of the isolates as opposed to the ITS1-PCR RFLP method that was successful in identifying L. tropica in only 43.6% of the isolates. Sequencing and phylogenetic analysis revealed high levels of heterogeneity. A statistically significant correlation was observed between PI and the results of the nested ITS1-5.8S rDNA gene PCR. Genotyping at the species level is essential for monitoring the relative frequency of CL in the Mediterranean area that is correlated to three different Leishmania species (Leishmania infantum, Leishmania major and L. tropica, each characterized by distinct epidemiological features. The obtained results highlight the need to find a universally accepted diagnostic tool for Leishmania typing.

Molecular detection of Leishmania infection in sand flies in border line of Iran-Turkmenistan: restricted and permissive vectors.

Science.gov (United States)

Bakhshi, H; Oshaghi, M A; Abai, M R; Rassi, Y; Akhavan, A A; Sheikh, Z; Mohtarami, F; Saidi, Z; Mirzajani, H; Anjomruz, M

2013-10-01

A molecular study was carried out to incriminate sand fly vectors of cutaneous leishmaniasis (CL) in rural areas of Sarakhs district, Khorassane-Razavi Province, northeastern Iran, in 2011. Sand flies of Sergentomyia with three species and Phlebotomus with six species respectively comprised 73.3% and 26.7% of the specimens. Phlebotomus papatasi was the most common Phlebotomine species in outdoor and indoor resting places. Leishmania infection was found at least in 17 (22%) specimens including Ph. papatasi (n=9 pool samples), Phlebotomus caucasicus (n=6), Phlebotomus alexandri (n=1), and Sergentomyia sintoni (n=1). The parasites were found comprised Leishmania major (n=5), Leishmania turanica (n=10), and Leishmania gerbilli (n=4). Infection of Ph. papatasi with both L. major and L. turanica supporting the new suggestion indicating that it is not restricted only with L. major. Circulation of L. major by Ph. alexandri, and both L. gerbilli and L. turanica by Ph. caucasicus, in addition to previous data indicating the ability of Ph. alexandri to circulate Leishmania infantum and Leishmania donovani, and Ph. caucasicus to circulate L. major, suggests that these two species can be permissive vectors. The results suggest that Ph. papatasi and Ph. alexandri are the primary and secondary vectors of CL where circulating L. major between human and reservoirs, whereas Ph. caucasicus is circulating L. turanica and L. gerbilli between the rodents in the region. Copyright © 2013 Elsevier Inc. All rights reserved.

Enhancement of immune response induced by DNA vaccine cocktail expressing complete LACK and TSA genes against Leishmania major.

Science.gov (United States)

Ghaffarifar, Fatemeh; Jorjani, Ogholniaz; Sharifi, Zohreh; Dalimi, Abdolhossein; Hassan, Zuhair M; Tabatabaie, Fatemeh; Khoshzaban, Fariba; Hezarjaribi, Hajar Ziaei

2013-04-01

Leishmaniasis is an important disease in humans. Leishmania homologue of receptor for Activated C Kinase (LACK) and thiol specific antioxidant (TSA) as immuno-dominant antigens of Leishmania major are considered the most promising molecules for a DNA vaccine. We constructed a DNA cocktail, containing plasmids encoding LACK and TSA genes of Leishmania major and evaluated the immune response and survival rate in BALB/c mice. IgG and Interferon gamma values were noticeably increased in the immunized group with DNA cocktail vaccine, which were significantly higher than those in the single-gene vaccinated and control groups (p 0.05). The immunized mice with the cocktail DNA vaccine presented a considerable reduction in diameter of lesion compared to other groups and a significant difference was observed (p < 0.05) in this regard. The survival time of the immunized mice with the cocktail DNA vaccine was significantly higher than that in the other groups (p < 0.05) after their being challenged with Leishmania major. The findings of this study indicated that the cocktail DNA vaccine increased the cellular response and survival rate and induced protection against infection with Leishmania in the mice. © 2012 The Authors © 2012 APMIS.

Molecular Detection of Leishmania DNA in Wild-Caught Phlebotomine Sand Flies (Diptera: Psychodidae) From a Cave in the State of Minas Gerais, Brazil.

Science.gov (United States)

Carvalho, G M L; Brazil, R P; Rêgo, F D; Ramos, M C N F; Zenóbio, A P L A; Andrade Filho, J D

2017-01-01

Leishmania spp. are distributed throughout the world, and different species are associated with varying degrees of disease severity. In Brazil, Leishmania transmission involves several species of phlebotomine sand flies that are closely associated with different parasites and reservoirs, and thereby giving rise to different transmission cycles. Infection occurs during the bloodmeals of sand flies obtained from a variety of wild and domestic animals, and sometimes from humans. The present study focused on detection of Leishmania DNA in phlebotomine sand flies from a cave in the state of Minas Gerais. Detection of Leishmania in female sand flies was performed with ITS1 PCR-RFLP (internal transcribed spacer 1) using HaeIII enzyme and genetic sequencing for SSUrRNA target. The survey of Leishmania DNA was carried out on 232 pools and the parasite DNA was detected in four: one pool of Lutzomyia cavernicola (Costa Lima, 1932), infected with Le. infantum (ITS1 PCR-RFLP), two pools of Evandromyia sallesi (Galvão & Coutinho, 1939), both infected with Leishmania braziliensis complex (SSUrRNA genetic sequencing analysis), and one pool of Sciopemyia sordellii (Shannon & Del Ponte, 1927), infected with subgenus Leishmania (SSUrRNA genetic sequencing analysis). The present study identified the species for Leishmania DNA detected in four pools of sand flies, all of which were captured inside the cave. These results represent the first molecular detection of Lu cavernicola with Le infantum DNA, Sc sordellii with subgenus Leishmania DNA, and Ev sallesi with Leishmania braziliensis complex DNA. The infection rate in females captured for this study was 0.17%. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Genetic Manipulation of Leishmania donovani to Explore the Involvement of Argininosuccinate Synthase in Oxidative Stress Management

Science.gov (United States)

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

Canine antibody response to Phlebotomus perniciosus bites negatively correlates with the risk of Leishmania infantum transmission.

Directory of Open Access Journals (Sweden)

Michaela Vlkova

2011-10-01

Full Text Available BACKGROUND: Phlebotomine sand flies are blood-sucking insects that can transmit Leishmania parasites. Hosts bitten by sand flies develop an immune response against sand fly salivary antigens. Specific anti-saliva IgG indicate the exposure to the vector and may also help to estimate the risk of Leishmania spp. transmission. In this study, we examined the canine antibody response against the saliva of Phlebotomus perniciosus, the main vector of Leishmania infantum in the Mediterranean Basin, and characterized salivary antigens of this sand fly species. METHODOLOGY/PRINCIPAL FINDINGS: Sera of dogs bitten by P. perniciosus under experimental conditions and dogs naturally exposed to sand flies in a L. infantum focus were tested by ELISA for the presence of anti-P. perniciosus antibodies. Antibody levels positively correlated with the number of blood-fed P. perniciosus females. In naturally exposed dogs the increase of specific IgG, IgG1 and IgG2 was observed during sand fly season. Importantly, Leishmania-positive dogs revealed significantly lower anti-P. perniciosus IgG2 compared to Leishmania-negative ones. Major P. perniciosus antigens were identified by western blot and mass spectrometry as yellow proteins, apyrases and antigen 5-related proteins. CONCLUSIONS: Results suggest that monitoring canine antibody response to sand fly saliva in endemic foci could estimate the risk of L. infantum transmission. It may also help to control canine leishmaniasis by evaluating the effectiveness of anti-vector campaigns. Data from the field study where dogs from the Italian focus of L. infantum were naturally exposed to P. perniciosus bites indicates that the levels of anti-P. perniciosus saliva IgG2 negatively correlate with the risk of Leishmania transmission. Thus, specific IgG2 response is suggested as a risk marker of L. infantum transmission for dogs.

The polymerase chain reaction can reveal the occurrence of naturally mixed infections with Leishmania parasites

DEFF Research Database (Denmark)

Ibrahim, M E; Smyth, A J; Ali, M H

1994-01-01

On isolation and characterization of Leishmania parasites from Sudanese patients with visceral leishmaniasis (VL), four cases of mixed infections were found. Three of those cases were from the Eastern Sudan focus of VL. In one case the patient was found to be concomitantly infected with Leishmania...

Fabrication of biosynthetic vascular prostheses by 193-nm excimer laser radiation

Science.gov (United States)

Husinsky, Wolfgang; Csek, Ch.; Bartel, A.; Grabenwoeger, M.; Fitzal, F.; Wolner, Ernst

1998-05-01

This study was undertaken to investigate the feasibility of transmural capillary ingrowth into the inner surface of biosynthetic vascular prostheses (OmniflowTM) through perforations created by an excimer-laser, thus inducing an endothelial cell coverage. The biosynthetic vascular prostheses (10 cm length, 6 mm (phi) ) were perforated with an excimer laser ((phi) of the holes 50 - 100 micrometer, distance 4 mm) and implanted into the carotid arteries of 8 sheep. The laser tissue interaction process of 193 nm radiation ensures minimal thermal damage to the prostheses. They were compared to untreated OmniflowTM prostheses implanted at the contralateral side. Three months after implantation the prostheses were explanted and evaluated by gross morphology, histological examination and scanning electron microscopy. Scanning electron microscopy showed endothelial cells in the midgraft portion of all perforated prostheses, whereas collagen fibers, fibrin meshwork and activated platelets formed the inner layer in 6 out of 8 untreated OmniflowTM prostheses. It can be concluded, that spontaneous endothelialization of biosynthetic vascular prostheses can be achieved by transmural capillary ingrowth through perforations in the wall of the prostheses in an experimental sheep model.

Activation of human T lymphocytes by Leishmania lipophosphoglycan

DEFF Research Database (Denmark)

Kemp, M; Theander, T G; Handman, E

1991-01-01

This study describes Leishmania antigen-induced activation of lymphocytes isolated from Kenyan donors, previously treated for visceral leishmaniasis, and from Danish and Kenyan controls. Peripheral blood mononuclear cells (PBMC) from cured Kala-Azar patients proliferated and produced Interferon......, the results suggest that human T lymphocytes can respond to glycolipid antigens....

Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania amazonensis promastigotes

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José Andrés Morgado-Díaz

2005-07-01

Full Text Available Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME as substrate, phenylmethylsulphone fluoride (PMSF and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate, but also in a crude plasma membrane fraction (2.0-fold. Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP, with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani.

Science.gov (United States)

Zhang, Wen-Wei; Matlashewski, Greg

2015-07-21

The prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that L. donovani mainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent in L. donovani. With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker and GFP gene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in LdMT (M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool for L. donovani. Leishmania donovani is the causative agent of fatal visceral leishmaniasis. To understand Leishmania infection and pathogenesis and identify new drug targets for control of leishmaniasis, more-efficient ways to manipulate this parasite genome are required. In this

Passive transfer of leishmania lipopolysaccharide confers parasite survival in macrophages

International Nuclear Information System (INIS)

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

1986-01-01

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 identified 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

Detecção de DNA de Leishmania braziliensis em pacientes de leishmaniose tegumentar americana Detección de DNA de Leishmania braziliensis en pacientes de leishmaniose tegumentaria americana Detection of Leishmania braziliensis DNA in American tegumentary leishmaniasis patients

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Leila Martins

2010-06-01

Full Text Available Foi realizado diagnóstico para leishmaniose tegumentar americana a partir de sangue de pacientes residentes em dois municípios endêmicos do estado de Pernambuco. O DNA de 119 amostras de sangue foi extraído e submetido a reação em cadeia da polimerase. Utilizaram-se primers do minicírculo do DNA do cinetoplasto (kDNA de Leishmania braziliensis, circulante em Pernambuco, cuja seqüência-alvo gera um fragmento de 750 pares de bases. No total 58 (48,7% indivíduos apresentaram amplificação positiva e 61 (51,3% negativa. Das amostras positivas para a PCR, 37 (≅ 64% pertenciam a indivíduos tratados e sem lesão. Conclui-se que a técnica de PCR é eficaz para identificar o DNA de leishmânia em material de biópsias e em sangue venoso.Fue realizado diagnóstico para leishmaniosis tegumentaria americana a partir de sangre de pacientes residentes en dos municipios endémicos del estado de Pernambuco (Noreste de Brasil. El DNA de 119 muestras de sangre fue extraído y sometido a la reacción en cadena de la polimerasa. Se utilizaron primers del minicírculo del DNA del cinetoplasto (kDNA de Leishmania braziliensis, circulante en Pernambuco, cuya secuencia blanco genera un fragmento de 750 pares de bases. En total 58 (48,7% individuos presentaron amplificación positiva y 61 (51,3% negativa. De las muestras positivas para la PCR, 37 (≅64% pertenecían a individuos tratados y sin lesión. Se concluyó que la técnica de la PCR es eficaz para identificar el DNA de Leishmania en material de biopsias y en sangre venosa.Diagnostic tests for American tegumentary leishmaniasis were performed on blood samples of patients living in two endemic municipalities in the state of Pernambuco, Northeastern Brazil. DNA was extracted from 119 samples and used as template for polymerase chain reaction (PCR analysis. The tests used primers specific for the kinetoplast mini-circle DNA (kDNA of Leishmania braziliensis, a species circulating in Pernambuco, which

What is the evidence for the use of biologic or biosynthetic meshes in abdominal wall reconstruction?

Science.gov (United States)

Köckerling, F; Alam, N N; Antoniou, S A; Daniels, I R; Famiglietti, F; Fortelny, R H; Heiss, M M; Kallinowski, F; Kyle-Leinhase, I; Mayer, F; Miserez, M; Montgomery, A; Morales-Conde, S; Muysoms, F; Narang, S K; Petter-Puchner, A; Reinpold, W; Scheuerlein, H; Smietanski, M; Stechemesser, B; Strey, C; Woeste, G; Smart, N J

2018-04-01

Although many surgeons have adopted the use of biologic and biosynthetic meshes in complex abdominal wall hernia repair, others have questioned the use of these products. Criticism is addressed in several review articles on the poor standard of studies reporting on the use of biologic meshes for different abdominal wall repairs. The aim of this consensus review is to conduct an evidence-based analysis of the efficacy of biologic and biosynthetic meshes in predefined clinical situations. A European working group, "BioMesh Study Group", composed of invited surgeons with a special interest in surgical meshes, formulated key questions, and forwarded them for processing in subgroups. In January 2016, a workshop was held in Berlin where the findings were presented, discussed, and voted on for consensus. Findings were set out in writing by the subgroups followed by consensus being reached. For the review, 114 studies and background analyses were used. The cumulative data regarding biologic mesh under contaminated conditions do not support the claim that it is better than synthetic mesh. Biologic mesh use should be avoided when bridging is needed. In inguinal hernia repair biologic and biosynthetic meshes do not have a clear advantage over the synthetic meshes. For prevention of incisional or parastomal hernias, there is no evidence to support the use of biologic/biosynthetic meshes. In complex abdominal wall hernia repairs (incarcerated hernia, parastomal hernia, infected mesh, open abdomen, enterocutaneous fistula, and component separation technique), biologic and biosynthetic meshes do not provide a superior alternative to synthetic meshes. The routine use of biologic and biosynthetic meshes cannot be recommended.

Leishmania major infection in a dog with cutaneous manifestations.

Science.gov (United States)

Baneth, Gad; Nachum-Biala, Yaarit; Shabat Simon, Maytal; Brenner, Ori; Gaier, Sarit; Rojas, Alicia; Yasur-Landau, Daniel

2016-05-10

Leishmania major is a main cause of cutaneous leishmaniasis in humans in an area that stretches from India through Central Asia, the Middle East, to North and West Africa. In Israel, it is a common infection of humans with rodents as the reservoir hosts and Phlebotomus papatasi as its sand fly vector. A 6 months old spayed female mixed breed dog was referred to the Hebrew University Veterinary Teaching Hospital with a large ulcerative dermal lesion on the muzzle, and lesions in the foot pads and left hind leg. Histopathology of a skin biopsy found chronic lymphohistiocytic dermatitis with the presence of Leishmania spp. amastigotes in the muzzle. Physical examination indicated that the dog was overall in a good clinical condition and the main findings were the skin lesions and enlarged prescapular lymph nodes. Complete blood count and serum biochemistry profile were within reference ranges. Serology by ELISA was positive for Leishmania spp. and PCR of the prescapular lymph node was positive by an ITS1 region PCR-high resolution melt analysis. However, the melt curve and subsequent DNA sequencing indicated that infection was caused by L. major and not L. infantum, which is the main causative agent of canine leishmaniosis in the Mediterranean region. DNA was extracted from the paraffin embedded muzzle biopsy and PCR with sequencing also indicated L. major. The dog's young age and the absence of hyperglobulinemia and anemia were not typical of L. infantum infection. The dog was treated with allopurinol and the skin lesions improved and later disappeared when the dog was re-evaluated. This is the first molecularly-confirmed case of L. major infection in a dog. Two previous reports of L. major in dogs originated from Saudi-Arabia and Egypt in 1985 and 1987 were confirmed by enzymatic biochemical techniques. Serology for L. infantum was positive probably due to the well documented serological cross-reactivity between Leishmania spp. Although dogs and wild carnivores are

The efficacy of 2-nitrovinylfuran derivatives against Leishmania in vitro and in vivo

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Sergio Sifontes-Rodríguez

2015-04-01

Full Text Available Despite recent advances in the treatment of some forms of leishmaniasis, the available drugs are still far from ideal due to inefficacy, parasite resistance, toxicity and cost. The wide-spectrum antimicrobial activity of 2-nitrovinylfuran compounds has been described, as has their activity against Trichomonas vaginalis and other protozoa. Thus, the aim of this study was to test the antileishmanial activities of six 2-nitrovinylfurans in vitro and in a murine model of leishmaniasis. Minimum parasiticide concentration (MPC and 50% inhibitory concentration (IC50 values for these compounds against the promastigotes of Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis were determined, as were the efficacies of two selected compounds in an experimental model of cutaneous leishmaniasis (CL caused by L. amazonensis in BALB/c mice. All of the compounds were active against the promastigotes of the three Leishmania species tested. IC50 and MPC values were in the ranges of 0.8-4.7 µM and 1.7-32 µM, respectively. The compounds 2-bromo-5-(2-bromo-2-nitrovinyl-furan (furvina and 2-bromo-5-(2-methyl-2-nitrovinyl-furan (UC245 also reduced lesion growth in vivo at a magnitude comparable to or higher than that achieved by amphotericin B treatment. The results demonstrate the potential of this class of compounds as antileishmanial agents and support the clinical testing of Dermofural(r (a furvina-containing antifungal ointment for the treatment of CL.

Aislamiento de tres cepas de leishmania

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Florentino Rey

1942-08-01

Full Text Available Con el ánimo de estudiar el problema parasitológico de la Leishmaniasis cutánea en Colombia, emprendimos algunas experiencias al respecto. Este es un estudio de aislamiento de tres cepas de Leishmania obtenidas de tres enfermos de distintas regiones del país, Investigadores colombianos que se han ocupado con anterioridad de este problema, obtuvieron siempre resultados negativos. Comunicaciones posteriores informarán sobre estudios experimentales de las cepas mencionadas.

Evaluación del efecto del ácido nalidíxico, ampicilina, kanamicina, penicilina G y polimixina B en los cultivos de promastigotes de leishmania

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Sofía Duque Beltrán

1992-06-01

Full Text Available Se evaluó el efecto de diferentes concentraciones de ácido nalidíxico, ampicilina, kanarnicina, penicilina G y polimixina B, sobre la población de promastigotes de Leishmania braziliensis braziliensis , Leishmania donovani chagasi y Leishmania mexicana amazonensis in vitro. La penicilina G y la ampicilina se pueden utilizar hasta concentraciones de 1000 ug/ml y 500 ug/ml respectivamente en cultivo de promastigotes de cualquier cepa de Leishmania sin que éstos se afecten. La polimixina B disminuye la población de prosmastigotes por lo cual es preferible no usarse en cultivos de Leishmania. El ácido nalidíxico y kanamicina pueden ser utilizados in vitro pero teniéndose en cuenta la especie de Leishmania y la concentración de anfimicrobiano recomendado para la misma.

Differential Recruitment of Dendritic Cells Subsets to Lymph Nodes Correlates with a Protective or Permissive T-Cell Response during Leishmania (Viannia) Braziliensis or Leishmania (Leishmania) Amazonensis Infection.

Science.gov (United States)

Carvalho, A K; Carvalho, K; Passero, L F D; Sousa, M G T; da Matta, V L R; Gomes, C M C; Corbett, C E P; Kallas, G E; Silveira, F T; Laurenti, M D

2016-01-01

Leishmania (L.) amazonensis (La) and L. (V.) braziliensis (Lb) are responsible for a large clinical and immunopathological spectrum in human disease; while La may be responsible for anergic disease, Lb infection leads to cellular hypersensitivity. To better understand the dichotomy in the immune response caused by these Leishmania species, we evaluated subsets of dendritic cells (DCs) and T lymphocyte in draining lymph nodes during the course of La and Lb infection in BALB/c mice. Our results demonstrated a high involvement of DCs in La infection, which was characterized by the greater accumulation of Langerhans cells (LCs); conversely, Lb infection led to an increase in dermal DCs (dDCs) throughout the infection. Considering the T lymphocyte response, an increase of effector, activated, and memory CD4(+) T-cells was observed in Lb infection. Interleukin- (IL-) 4- and IL-10-producing CD4(+)and CD8(+) T-cells were present in both La and Lb infection; however, interferon- (IFN-) γ-producing CD4(+)and CD8(+) T-cells were detected only in Lb infection. The results suggest that during Lb infection, the dDCs were the predominant subset of DCs that in turn was associated with the development of Th1 immune response; in contrast La infection was associated with a preferential accumulation of LCs and total blockage of the development of Th1 immune response.

The Leishmania nicotinamidase is essential for NAD+ production and parasite proliferation.

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Gazanion, E; Garcia, D; Silvestre, R; Gérard, C; Guichou, J F; Labesse, G; Seveno, M; Cordeiro-Da-Silva, A; Ouaissi, A; Sereno, D; Vergnes, B

2011-10-01

NAD+ is a central cofactor that plays important roles in cellular metabolism and energy production in all living cells. Genomics-based reconstruction of NAD+ metabolism revealed that Leishmania protozoan parasites are NAD+ auxotrophs. Consequently, these parasites require assimilating NAD+ precursors (nicotinamide, nicotinic acid, nicotinamide riboside) from their host environment to synthesize NAD+ by a salvage pathway. Nicotinamidase is a key enzyme of this salvage pathway that catalyses conversion of nicotinamide (NAm) to nicotinic acid (Na), and that is absent in higher eukaryotes. We present here the biochemical and functional characterizations of the Leishmania infantum nicotinamidase (LiPNC1). Generation of Lipnc1 null mutants leads to a decrease in NAD+ content, associated with a metabolic shutdown-like phenotype with an extensive lag phase of growth. Both phenotypes could be rescued by an add-back construct or by addition of exogenous Na. In addition, Lipnc1 null mutants were unable to establish a sustained infection in a murine experimental model. Altogether, these results illustrate that NAD+ homeostasis is a fundamental component of Leishmania biology and virulence, and that NAm constitutes its main NAD+ source in the mammalian host. The crystal structure of LiPNC1 we solved allows now the design of rational inhibitors against this new promising therapeutic target. © 2011 Blackwell Publishing Ltd.

Andean cutaneous leishmaniasis (Andean-CL, uta) in Peru and Ecuador: the causative Leishmania parasites and clinico-epidemiological features.

Science.gov (United States)

Hashiguchi, Yoshihisa; Gomez, Eduardo A L; Cáceres, Abraham G; Velez, Lenin N; Villegas, Nancy V; Hashiguchi, Kazue; Mimori, Tatsuyuki; Uezato, Hiroshi; Kato, Hirotomo

2018-01-01

This study provides comprehensive information on the past and current status of the Andean cutaneous leishmaniasis (Andean-CL, uta) in Peru and Ecuador, mainly focusing on the causative Leishmania parasites and clinico-epidemiological features. Available information and data including our unpublished works were analyzed thoroughly. Endemic regions of the Andean-CL (uta) in Peru run from the north Piura/Cajamarca to the south Ayacucho at a wide range of the Pacific watersheds of the Andes through several departments, while in Ecuador those exist at limited and spotted areas in the country's mid-southwestern two provinces, Azuay and Chimborazo. The principal species of the genus Leishmania are completely different at subgenus level, L. (Viannia) peruviana in Peru, and L. (Leishmania) mexicana and L. (L.) major-like (infrequent occurrence) in Ecuador. The Peruvian uta is now prevalent in different age and sex groups, being not clearly defined as found in the past. The precise reasons are not known and should be elucidated further, though probable factors, such as emergence of other Leishmania parasites, non-immune peoples' migration into the areas, etc., were discussed briefly in the text. The Andean-CL cases in Ecuador are more rural than before, probably because of a rapid development of the Leishmania-positive communities and towns, and the change of life-styles of the inhabitants, including newly constructed houses and roads in the endemic areas. Such information is helpful for future management of the disease, not only for Leishmania-endemic areas in the Andes but also for other endemic areas. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of transformation growth factor beta1, interleukin-10, and interferon-gamma in male symptomatic and asymptomatic dogs naturally infected by Leishmania (Leishmania) chagasi.

Science.gov (United States)

Corrêa, Ana Paula Ferreira Lopes; Dossi, Ana Cláudia Silva; de Oliveira Vasconcelos, Rosemeri; Munari, Danísio Prado; de Lima, Valéria Marçal Felix

2007-02-28

The aims of this study were to evaluate the immunomodulatory role of TGF-beta1, IL-10, and INF-gamma in spleen and liver extracts and supernatant cultures of white spleen cells from male symptomatic and asymptomatic dogs, naturally infected by Leishmania (Leishmania) chagasi. Thirty dogs from Araçatuba, São Paulo, Brazil, an endemic leishmaniosis area, were selected by positive ELISA serological reaction for Leishmania sp. and divided into two groups: asymptomatic (n=15) and symptomatic (n=15) consisting of animals with at least three characteristic signs (fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachexia, locomotion problems, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy). After euthanasia, spleen and liver fragments were collected for ex vivo quantification of TGF-beta1, IL-10, and INF-gamma. Naturally active in vitro produced TGF-beta1 was also evaluated in spleen cell culture supernatant. Spleen and liver extract of asymptomatic dogs had higher mean TGF-beta1 levels than symptomatic dogs. High concentrations of IL-10 were found in spleen, and mainly in liver extract of both groups. Higher INF-gamma concentrations were found in spleen extracts of symptomatic dogs, and in liver extracts of asymptomatic dogs. Extract of this cytokine was lower in spleen extract. Although INF-gamma is being produced in canine infection, mean levels of TGF-beta1 and IL-10 from spleen and liver extracts were quantitatively much higher; suggesting that immune response in both asymptomatic and symptomatic dogs was predominantly type Th2.

Natural Sesquiterpene Lactones Induce Oxidative Stress in Leishmania mexicana

NARCIS (Netherlands)

Barrera, P.; Sulsen, V.P.; Lozano, E.; Rivera, M.; Beer, M.F.; Tonn, C.; Martino, V.S.; Sosa, M.A.

2013-01-01

Leishmaniasis is a worldwide parasitic disease, caused by monoflagellate parasites of the genus Leishmania. In the search for more effective agents against these parasites, the identification of molecular targets has been attempted to ensure the efficiency of drugs and to avoid collateral damages on

Generation of species-specific DNA probes for Leishmania aethiopica

NARCIS (Netherlands)

Laskay, T.; Kiessling, R.; Rinke deWit, T. F.; Wirth, D. F.

1991-01-01

We report here the cloning of kinetoplast DNA (kDNA) sequences from Leishmania aethiopica in order to develop a specific and sensitive method for the identification of the parasite. Analysis of the cloned kDNA sequences showed different taxonomic specificities demonstrating sequence diversity within

Evaluation of HIV-Leishmania co-infection in patients from the northwestern Paraná State, Brazil = Avaliação da co-infecção HIV-Leishmania em pacientes da região noroeste do Estado do Paraná, Brasil

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Élide Aparecida Oliveira

2011-01-01

Full Text Available Leishmaniasis occurs throughout the world and is one of the opportunistic infections that attack HIV-infected individuals. Few data are available on American cutaneous leishmaniasis (ACL in HIV-infected patients. Current research investigates the occurrence ofHIV-Leishmania co-infection in HIV-infected individuals in an endemic region in Southern of Brazil. A non-randomized transversal investigation, molecular and serum epidemiologic type, on the occurrence of ACL in 169 HIV-infected patients was undertaken. The patients were followed up at the Integrated Nucleus of Health of the city Maringá, Southern of Brazil. Results showed that 13 (7.7% of the HIV-infected patients also presented Leishmania (Viannia DNA, detectable in blood by PCR. Serology, direct research, culture and PCR in skin material produced negative results. PCR positiveness for Leishmania was not associated with CD4 T lymphocytes count, opportunistic disease, treatment, use of proteases inhibitors, tattooing/piercing or use of injectable drugs, residential environment or previous ACL history. Results show that HIVinfected patients who live in endemic areas may reveal Leishmania DNA in the blood without any ACL symptoms. Above findings may be attributed to anti-retrovirus medicine that controls viral replication and maintains the functionality of the immune system and to a possible anti- Leishmania activity of these drugs.As leishmanioses ocorrem em todo o mundo e são infecções oportunistas que afetam indivíduos portadores do vírus HIV. Este estudo investigou a ocorrência da co-infecção HIV-Leishmania em portadores do HIV numa região endêmica para LTA do Sul do Brasil. Foi realizado estudo transversal, não randomizado, utilizando metodologia molecular e sorológica, sobre a ocorrência de LTA em 169 portadores do HIV. Foram estudados pacientes atendidos no Núcleo Integrado de Saúde de Maringá, Paraná, Sul do Brasil. Observou-se que 13 (7,7% dos pacientes infectados

Natural infection of the opossum Didelphis albiventris (Marsupialia, Didelphidae with Leishmania donovani, in Brazil

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Ítalo A. Sherlock

1984-12-01

Full Text Available An opossum, Didelphis albiventris, from Jacobina, bahia State, was found naturally infected with Leishmania donovani, being the first non-canid wild mammal to be detected with agent of kala-azar in the New World.Um gambá, Didelphis albiventris, de Jacobina, Bahia, foi encontrado com infecção natural pela Leishmania donovani, sendo o primeiro mamífero silvestre não-canídeo a ser achado com o agente do calazar nas Américas.

Reproduction in Leishmania: A focus on genetic exchange.

Science.gov (United States)

Rougeron, V; De Meeûs, T; Bañuls, A-L

2017-06-01

One key process of the life cycle of pathogens is their mode of reproduction. Indeed, this fundamental biological process conditions the multiplication and the transmission of genes and thus the propagation of diseases in the environment. Reproductive strategies of protozoan parasites have been a subject of debate for many years, principally due to the difficulty in making direct observations of sexual reproduction (i.e. genetic recombination). Traditionally, these parasites were considered as characterized by a preeminent clonal structure. Nevertheless, with the development of elaborate culture experiments, population genetics and evolutionary and population genomics, several studies suggested that most of these pathogens were also characterized by constitutive genetic recombination events. In this opinion, we focused on Leishmania parasites, pathogens responsible of leishmaniases, a major public health issue. We first discuss the evolutionary advantages of a mixed mating reproductive strategy, then we review the evidence of genetic exchange, and finally we detail available tools to detect naturally occurring genetic recombination in Leishmania parasites and more generally in protozoan parasites. Copyright © 2016. Published by Elsevier B.V.

Effect of Kelussia odoratissima Mozaff essential oil on promastigot form of Leishmania major (in vitro

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Pirali Kheirabadi Khodadad

2015-01-01

Full Text Available Introduction: Leishmaniasis is a zoonotic disease caused by a protozoan of the genus Leishmania. In this study, the effects of Kelussia odoratissima Mozaff essential oil on the promastigot form of Leishmania major were studied. Methods: In this study, the effects of Kelussia odoratissima Mozaff essential oil on the promastigot form of Leishmania major were assessed by calculating the average number of surviving promastigots after exposure to different concentrations of essential oil, relative to the control Glucantime, at different time intervals. To achieve this, various essential oil concentrations (7.5 μl, 15 μl, 25 μl, 35.25 μl, 50 μl were added to parasites. Different groups in this study were kept in a 26°C incubator under identical conditions. 24, 48 and 72 hours after incubation, living promastigots were counted. Results: The effect of the essential oil of Kelussia odoratissima Mozaff differed from the negative and positive controls and depended on the concentration: higher concentrations (35.25 μl, 50 μl had a stronger effect on promastigots, causing total mortality. Conclusion: This study showed that Kelussia odoratissima Mozaff essential oil had effects on promastigot form of Leishmania major. So it might be possible to use the essential oil of Kelussia odoratissima instead of chemical drugs.

Crystal structures of Leishmania mexicana arginase complexed with α,α-disubstituted boronic amino-acid inhibitors

Energy Technology Data Exchange (ETDEWEB)

Hai, Yang; Christianson, David W.

2016-03-16

Leishmaniaarginase is a potential drug target for the treatment of leishmaniasis because this binuclear manganese metalloenzyme initiatesde novopolyamine biosynthesis by catalyzing the hydrolysis of L-arginine to generate L-ornithine and urea. The product L-ornithine subsequently undergoes decarboxylation to yield putrescine, which in turn is utilized for spermidine biosynthesis. Polyamines such as spermidine are essential for the growth and survival of the parasite, so inhibition of enzymes in the polyamine-biosynthetic pathway comprises an effective strategy for treating parasitic infections. To this end, two X-ray crystal structures ofL. mexicanaarginase complexed with α,α-disubstituted boronic amino-acid inhibitors based on the molecular scaffold of 2-(S)-amino-6-boronohexanoic acid are now reported. Structural comparisons with human and parasitic arginase complexes reveal interesting differences in the binding modes of the additional α-substituents,i.e.the D side chains, of these inhibitors. Subtle differences in the three-dimensional contours of the outer active-site rims among arginases from different species lead to different conformations of the D side chains and thus different inhibitor-affinity trends. The structures suggest that it is possible to maintain affinity while fine-tuning intermolecular interactions of the D side chain of α,α-disubstituted boronic amino-acid inhibitors in the search for isozyme-specific and species-specific arginase inhibitors.

Leishmania OligoC-TesT as a simple, rapid, and standardized tool for molecular diagnosis of cutaneous leishmaniasis in Peru.

Science.gov (United States)

Espinosa, Diego; Boggild, Andrea K; Deborggraeve, Stijn; Laurent, Thierry; Valencia, Cristian; Pacheco, Rosa; Miranda-Verástegui, César; Llanos-Cuentas, Alejandro; Leclipteux, Thierry; Dujardin, Jean-Claude; Büscher, Philippe; Arévalo, Jorge

2009-08-01

Molecular methods such as PCR have become attractive tools for diagnosis of cutaneous leishmaniasis (CL), both for their high sensitivity and for their specificity. However, their practical use in routine diagnosis is limited due to the infrastructural requirements and the lack of any standardization. Recently, a simplified and standardized PCR format for molecular detection of Leishmania was developed. The Leishmania OligoC-TesT is based on simple and rapid detection using a dipstick with PCR-amplified Leishmania DNA. In this study, we estimated the diagnostic accuracy of the Leishmania OligoC-TesT for 61 specimens from 44 CL-suspected patients presenting at the leishmaniasis clinic of the Instituto de Medicina Tropical Alexander von Humboldt, Peru. On the basis of parasitological detection and the leishmanin skin test (LST), patients were classified as (i) confirmed CL cases, (ii) LST-positive cases, and (iii) LST-negative cases. The sensitivities of the Leishmania OligoC-TesT was 74% (95% confidence interval (CI), 60.5% to 84.1%) for lesion aspirates and 92% (95% CI, 81.2% to 96.9%) for scrapings. A significantly higher sensitivity was observed with a conventional PCR targeting the kinetoplast DNA on the aspirates (94%) (P = 0.001), while there was no significant difference in sensitivity for the lesion scrapings (88%) (P = 0.317). In addition, the Leishmania OligoC-TesT was evaluated for 13 CL-suspected patients in two different peripheral health centers in the central jungle of Peru. Our findings clearly indicate the high accuracy of the Leishmania OligoC-TesT for lesion scrapings for simple and rapid molecular diagnosis of CL in Peru.

Leishmania OligoC-TesT as a Simple, Rapid, and Standardized Tool for Molecular Diagnosis of Cutaneous Leishmaniasis in Peru▿

Science.gov (United States)

Espinosa, Diego; Boggild, Andrea K.; Deborggraeve, Stijn; Laurent, Thierry; Valencia, Cristian; Pacheco, Rosa; Miranda-Verástegui, César; Llanos-Cuentas, Alejandro; Leclipteux, Thierry; Dujardin, Jean-Claude; Büscher, Philippe; Arévalo, Jorge

2009-01-01

Molecular methods such as PCR have become attractive tools for diagnosis of cutaneous leishmaniasis (CL), both for their high sensitivity and for their specificity. However, their practical use in routine diagnosis is limited due to the infrastructural requirements and the lack of any standardization. Recently, a simplified and standardized PCR format for molecular detection of Leishmania was developed. The Leishmania OligoC-TesT is based on simple and rapid detection using a dipstick with PCR-amplified Leishmania DNA. In this study, we estimated the diagnostic accuracy of the Leishmania OligoC-TesT for 61 specimens from 44 CL-suspected patients presenting at the leishmaniasis clinic of the Instituto de Medicina Tropical Alexander von Humboldt, Peru. On the basis of parasitological detection and the leishmanin skin test (LST), patients were classified as (i) confirmed CL cases, (ii) LST-positive cases, and (iii) LST-negative cases. The sensitivities of the Leishmania OligoC-TesT was 74% (95% confidence interval (CI), 60.5% to 84.1%) for lesion aspirates and 92% (95% CI, 81.2% to 96.9%) for scrapings. A significantly higher sensitivity was observed with a conventional PCR targeting the kinetoplast DNA on the aspirates (94%) (P = 0.001), while there was no significant difference in sensitivity for the lesion scrapings (88%) (P = 0.317). In addition, the Leishmania OligoC-TesT was evaluated for 13 CL-suspected patients in two different peripheral health centers in the central jungle of Peru. Our findings clearly indicate the high accuracy of the Leishmania OligoC-TesT for lesion scrapings for simple and rapid molecular diagnosis of CL in Peru. PMID:19553579

Studies on Using Cattle and Sheep Hydatid Cyst Fluid Instead of the Fetal Calf Serum in Leishmania Culture

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Hossein Rezvan

2013-12-01

Full Text Available Background: Leishmania is a single cell parasite causing leishmaniasis, which is a common disease between humans and animals. Due to the importance of in-vitro culture of the parasite in leishmania research, developing new methods for in-vitro cultivation of the parasite has always been a goal for leishmania researchers. The main objective of7T 5T7Tthis study was to use sheep and bovine hydatid cyst fluids as alternatives for fetal calf serum (FCS in leishmania in-vitro5T culture5T. Materials and Methods: 7TA total of 5T7T1 million leishmania promastigotes were added to 4 flasks as follow5T7T. A f5T7Tlask containing DMEM medium with 105T7T% 5T7Tfetal bovine serum5T7T, a f5T7Tlask containing DMEM and 10% sheep hydatid cyst fluid5T7T, a f5T7Tlask containing DMEM medium with 105T7T% 5T7Tbovine hydatid cyst fluid and a5T7T f5T7Tlask containing DMEM medium alone. After 2, 45T7T, 5T7T7, 95T7T, 11, 5T7T21 and 24 days, the number of parasites were counted and compared5T7T. Results: The result of this study showed that, DMEM medium enriched with 10% sheep hydatid cyst fluid in 168 hours and medium enriched with 10% bovine hydatid cyst fluid in 96 hours can act as a good alternative for fetal bovine serum in the culture Leishmania major. Conclusion: 5TThe results showed that sheep and bovine hydatid cyst fluid can be used as alternatives to FCS for dense cultivation of leishmania. The results also showed that5T, 5Tthe growth of promastigotes in medium enriched with bovine cyst fluid is more rapid than the medium enriched with sheep5T c5Tyst fluid5T in5T the beginning of cultivation.

Comparison of small mammal prevalence of Leishmania (Leishmania) mexicana in five foci of cutaneous leishmaniasis in the State of Campeche, Mexico.

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Van Wynsberghe, N R; Canto-Lara, S B; Sosa-Bibiano, E I; Rivero-Cárdenas, N A; Andrade-Narváez, F J

2009-01-01

In the Yucatan Peninsula of Mexico, 95% of the human cases of Cutaneous Leishmaniasis are caused by Leishmania (Leishmania) mexicana with an incidence rate of 5.08 per 100,000 inhabitants. Transmission is limited to the winter months (November to March). One study on wild rodents has incriminated Ototylomys phyllotis and Peromyscus yucatanicus as primary reservoirs of L. (L.) mexicana in the focus of La Libertad, Campeche. In the present study, the prevalence of both infection and disease caused by L. (L.) mexicana in small terrestrial mammals were documented during five transmission seasons (1994-2004) in five foci of Leishmaniasis in the state of Campeche. Foci separated by only 100 km, with similar relative abundances of small mammals, were found to differ significantly in their prevalence of both symptoms and infection. Transmission rates and reservoir species seemed to change in space as well as in time which limited the implementation of effective control measures of the disease even in a small endemic area such as the south of the Yucatan Peninsula.

Bacterial natural product biosynthetic domain composition in soil correlates with changes in latitude on a continent-wide scale.

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Lemetre, Christophe; Maniko, Jeffrey; Charlop-Powers, Zachary; Sparrow, Ben; Lowe, Andrew J; Brady, Sean F

2017-10-31

Although bacterial bioactive metabolites have been one of the most prolific sources of lead structures for the development of small-molecule therapeutics, very little is known about the environmental factors associated with changes in secondary metabolism across natural environments. Large-scale sequencing of environmental microbiomes has the potential to shed light on the richness of bacterial biosynthetic diversity hidden in the environment, how it varies from one environment to the next, and what environmental factors correlate with changes in biosynthetic diversity. In this study, the sequencing of PCR amplicons generated using primers targeting either ketosynthase domains from polyketide biosynthesis or adenylation domains from nonribosomal peptide biosynthesis was used to assess biosynthetic domain composition and richness in soils collected across the Australian continent. Using environmental variables collected at each soil site, we looked for environmental factors that correlated with either high overall domain richness or changes in the domain composition. Among the environmental variables we measured, changes in biosynthetic domain composition correlate most closely with changes in latitude and to a lesser extent changes in pH. Although it is unclear at this time the exact mix of factors that may drive the relationship between biosynthetic domain composition and latitude, from a practical perspective the identification of a latitudinal basis for differences in soil metagenome biosynthetic domain compositions should help guide future natural product discovery efforts. Published under the PNAS license.

Leishmania parasite detection and quantification using PCR-ELISA

Czech Academy of Sciences Publication Activity Database

Kobets, Tetyana; Badalová, Jana; Grekov, Igor; Havelková, Helena; Lipoldová, Marie

2010-01-01

Roč. 5, č. 6 (2010), s. 1074-1080 ISSN 1754-2189 R&D Projects: GA ČR GA310/08/1697; GA MŠk(CZ) LC06009 Institutional research plan: CEZ:AV0Z50520514 Keywords : polymerase chain reaction * Leishmania major infection * parasite quantification Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.362, year: 2010

Leishmaniose cutânea na Amazônia: registro do primeiro caso humano de infecção mista, determinado por duas espécies distintas de Leishmnias: Leishmania brasiliensis e Leishmania mexicana amazonensis

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F. T. Silveira

1984-10-01

Full Text Available Fez-se o registro, na Amazônia, do primeiro caso humano de infecção cutânea mista determinada por duas espécies distintas de Leishmania: a Leishmania braziliensis braziliensis e a Leishmania mexicana amazonensis. As duas amostras, em questão, foram isoladas de lesões distintas de um mesmo paciente, e a caracterização das espécies foi feita com base em observações de infecção experimental em hamsters, comportamento em meios artificiais de cultura, desenvolvimento de infecção experimental em Lutzomyia longipalpis, e eletroforese de isoenzimas em gel de amido. Conclui-se ser de interesse o achado que, combinado com o fato já conhecido de ausência de imunidade cruzada entre a maioria das leishmânias, sugere a necessidade do emprego de uma vacina polivalente para a região.

Accumulation of Rutin and Betulinic Acid and Expression of Phenylpropanoid and Triterpenoid Biosynthetic Genes in Mulberry (Morus alba L.).

Science.gov (United States)

Zhao, Shicheng; Park, Chang Ha; Li, Xiaohua; Kim, Yeon Bok; Yang, Jingli; Sung, Gyoo Byung; Park, Nam Il; Kim, Soonok; Park, Sang Un

2015-09-30

Mulberry (Morus alba L.) is used in traditional Chinese medicine and is the sole food source of the silkworm. Here, 21 cDNAs encoding phenylpropanoid biosynthetic genes and 21 cDNAs encoding triterpene biosynthetic genes were isolated from mulberry. The expression levels of genes involved in these biosynthetic pathways and the accumulation of rutin, betulin, and betulinic acid, important secondary metabolites, were investigated in different plant organs. Most phenylpropanoid and triterpene biosynthetic genes were highly expressed in leaves and/or fruit, and most genes were downregulated during fruit ripening. The accumulation of rutin was more than fivefold higher in leaves than in other organs, and higher levels of betulin and betulinic acid were found in roots and leaves than in fruit. By comparing the contents of these compounds with gene expression levels, we speculate that MaUGT78D1 and MaLUS play important regulatory roles in the rutin and betulin biosynthetic pathways.

Cyclosporin A treatment of Leishmania donovani reveals stage-specific functions of cyclophilins in parasite proliferation and viability.

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Wai-Lok Yau

Full Text Available BACKGROUND: Cyclosporin A (CsA has important anti-microbial activity against parasites of the genus Leishmania, suggesting CsA-binding cyclophilins (CyPs as potential drug targets. However, no information is available on the genetic diversity of this important protein family, and the mechanisms underlying the cytotoxic effects of CsA on intracellular amastigotes are only poorly understood. Here, we performed a first genome-wide analysis of Leishmania CyPs and investigated the effects of CsA on host-free L. donovani amastigotes in order to elucidate the relevance of these parasite proteins for drug development. METHODOLOGY/PRINCIPAL FINDINGS: Multiple sequence alignment and cluster analysis identified 17 Leishmania CyPs with significant sequence differences to human CyPs, but with highly conserved functional residues implicated in PPIase function and CsA binding. CsA treatment of promastigotes resulted in a dose-dependent inhibition of cell growth with an IC50 between 15 and 20 microM as demonstrated by proliferation assay and cell cycle analysis. Scanning electron microscopy revealed striking morphological changes in CsA treated promastigotes reminiscent to developing amastigotes, suggesting a role for parasite CyPs in Leishmania differentiation. In contrast to promastigotes, CsA was highly toxic to amastigotes with an IC50 between 5 and 10 microM, revealing for the first time a direct lethal effect of CsA on the pathogenic mammalian stage linked to parasite thermotolerance, independent from host CyPs. Structural modeling, enrichment of CsA-binding proteins from parasite extracts by FPLC, and PPIase activity assays revealed direct interaction of the inhibitor with LmaCyP40, a bifunctional cyclophilin with potential co-chaperone function. CONCLUSIONS/SIGNIFICANCE: The evolutionary expansion of the Leishmania CyP protein family and the toxicity of CsA on host-free amastigotes suggest important roles of PPIases in parasite biology and implicate

Genetic Manipulation of Leishmania donovani to Explore the Involvement of Argininosuccinate Synthase in Oxidative Stress Management.

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Abul Hasan Sardar

2016-03-01

Full Text Available 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

Molecular characterization and functional analysis of pteridine reductase in wild-type and antimony-resistant Leishmania lines.

Science.gov (United States)

de Souza Moreira, Douglas; Ferreira, Rafael Fernandes; Murta, Silvane M F

2016-01-01

Pteridine reductase (PTR1) is an NADPH-dependent reductase that participates in the salvage of pteridines, which are essential to maintain growth of Leishmania. In this study, we performed the molecular characterization of ptr1 gene in wild-type (WTS) and SbIII-resistant (SbR) lines from Leishmania guyanensis (Lg), Leishmania amazonensis (La), Leishmania braziliensis (Lb) and Leishmania infantum (Li), evaluating the chromosomal location, mRNA levels of the ptr1 gene and PTR1 protein expression. PFGE results showed that the ptr1 gene is located in a 797 kb chromosomal band in all Leishmania lines analyzed. Interestingly, an additional chromosomal band of 1070 kb was observed only in LbSbR line. Northern blot results showed that the levels of ptr1 mRNA are increased in the LgSbR, LaSbR and LbSbR lines. Western blot assays using the polyclonal anti-LmPTR1 antibody demonstrated that PTR1 protein is more expressed in the LgSbR, LaSbR and LbSbR lines compared to their respective WTS counterparts. Nevertheless, no difference in the level of mRNA and protein was observed between the LiWTS and LiSbR lines. Functional analysis of PTR1 enzyme was performed to determine whether the overexpression of ptr1 gene in the WTS L. braziliensis and L. infantum lines would change the SbIII-resistance phenotype of transfected parasites. Western blot results showed that the expression level of PTR1 protein was increased in the transfected parasites compared to the non-transfected ones. IC50 analysis revealed that the overexpression of ptr1 gene in the WTS L. braziliensis line increased 2-fold the SbIII-resistance phenotype compared to the non-transfected counterpart. Furthermore, the overexpression of ptr1 gene in the WTS L. infantum line did not change the SbIII-resistance phenotype. These results suggest that the PTR1 enzyme may be implicated in the SbIII-resistance phenotype in L. braziliensis line. Copyright © 2015 Elsevier Inc. All rights reserved.

Estudo, ao microscópio óptico e eletrônico, do rim de caes natural e experimentalmente infectados com Leishmania (Leishmania chagasi

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Washington Luiz Tafuri

1989-06-01

Full Text Available Os autores estudam os rins de 4 cães infectados com Leishmania (Leishmania chagasi. Dois animais (um macho e uma fêmea naturalmente infectados foram sacrificados 18 meses após sua permanência no laboratório. Dois machos foram inoculados por via endovenosa, com lxlO6 promastigotas da cepa MHO/BR/70/BH46 e sacrificados após 18 meses e 2 anos, respectivamente. Em todos os animais os rins estavam lesados. As alterações encontradas foram: (1 glomerulonefrite mesangioproliferativa focal ou difusa, com pronunciada hipertrofia e hiperplasia das células mesangiais e com alargamento da matriz; (2 espessamento da membrana basal com depósitos eletrondensos; (3 nefrite intersticial intertubular crônica com exsudação plasmocitária intensa. (4 degeneração albuminosa dos túbulos renais. Baseados nos achados os autores discutem os prováveis mecanismos patogenéticos.

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

DEFF Research Database (Denmark)

Chen, M; Zhai, L; Christensen, S B

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...

Gentamicin-attenuated Leishmania infantum: a clinicopathological study in dogs.

Science.gov (United States)

Daneshvar, Hamid; Molaei, Mohammad M; Afshar, Reza Malekpour; Kamiabi, Hosein; Burchmore, Richard; Hagan, Paul; Phillips, R Stephen

2009-05-15

The clinicopathological changes following infection with an attenuated line of Leishmania infantum (L. infantum H-line) were evaluated in mixed breed dogs. Two groups of dogs were infected intravenously (i.v.) or intradermally (i.d.) with L. infantum H-line and two control groups were infected i.v. or i.d. with L. infantum wild-type (L. infantum WT). None of the dogs, which were infected i.v. or i.d. with L. infantum H-line, showed any abnormalities during the observation period. In contrast, two out of three dogs, which were infected i.v. with L. infantum WT, developed clinical signs of disease. In addition, no histopathological changes were seen in the liver and spleen of the dogs infected with the attenuated line of parasite, whereas the histopathological changes in the two dogs infected i.v. with L. infantum WT were severe in form and manifested by infiltration of high numbers of inflammatory cells. No promastigotes were found in cultures set up from spleens and livers of dogs infected with L. infantum H-line at 12 months post-infection, whereas promastigotes were seen in the spleen and liver cultures from 2 dogs infected i.v. with L. infantum WT. Serum levels of total IgG anti-Leishmania antibody were raised in all dogs. The antibody level in the serum of dogs infected i.v. with L. infantum WT was higher than that in dogs infected with L. infantum H-line. These results show no clinicopathological abnormalities in the dogs infected with gentamicin-attenuated L. infantum H-line. Moreover, L. infantum H-line induced IgG anti-Leishmania antibody in the dogs.

Linking metabolic QTLs with network and cis-eQTLs controlling biosynthetic pathways.

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Adam M Wentzell

2007-09-01

Full Text Available Phenotypic variation between individuals of a species is often under quantitative genetic control. Genomic analysis of gene expression polymorphisms between individuals is rapidly gaining popularity as a way to query the underlying mechanistic causes of variation between individuals. However, there is little direct evidence of a linkage between global gene expression polymorphisms and phenotypic consequences. In this report, we have mapped quantitative trait loci (QTLs-controlling glucosinolate content in a population of 403 Arabidopsis Bay x Sha recombinant inbred lines, 211 of which were previously used to identify expression QTLs controlling the transcript levels of biosynthetic genes. In a comparative study, we have directly tested two plant biosynthetic pathways for association between polymorphisms controlling biosynthetic gene transcripts and the resulting metabolites within the Arabidopsis Bay x Sha recombinant inbred line population. In this analysis, all loci controlling expression variation also affected the accumulation of the resulting metabolites. In addition, epistasis was detected more frequently for metabolic traits compared to transcript traits, even when both traits showed similar distributions. An analysis of candidate genes for QTL-controlling networks of transcripts and metabolites suggested that the controlling factors are a mix of enzymes and regulatory factors. This analysis showed that regulatory connections can feedback from metabolism to transcripts. Surprisingly, the most likely major regulator of both transcript level for nearly the entire pathway and aliphatic glucosinolate accumulation is variation in the last enzyme in the biosynthetic pathway, AOP2. This suggests that natural variation in transcripts may significantly impact phenotypic variation, but that natural variation in metabolites or their enzymatic loci can feed back to affect the transcripts.

Pharmacological activities of cilantro’s aliphatic Aldehydes against leishmania donovani

Science.gov (United States)

Leishmaniasis is a chronic infectious disease caused by different Leishmania species. Global occurrences of this disease are primarily limited to tropical and subtropical regions. Treatments are available; however, patients complain of side effects. Different species of plants have been screened as ...

L-Asparaginase of Leishmania donovani: Metabolic target and its role in Amphotericin B resistance

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Jasdeep Singh

2017-12-01

Full Text Available Emergence of Amphotericin B (AmB resistant Leishmania donovani has posed major therapeutic challenge against the parasite. Consequently, combination therapy aimed at multiple molecular targets, based on proteome wise network analysis has been recommended. In this regard we had earlier identified and proposed L-asparaginase of Leishmania donovani (LdAI as a crucial metabolic target. Here we report that both LdAI overexpressing axenic amastigote and promastigote forms of L. donovani survives better when challenged with AmB as compared to wild type strain. Conversely, qRT-PCR analysis showed an upregulation of LdAI in both forms upon AmB treatment. Our data demonstrates the importance of LdAI in imparting immediate protective response to the parasite upon AmB treatment. In the absence of structural and functional information, we modeled LdAI and validated its solution structure through small angle X-ray scattering (SAXS analysis. We identified its specific inhibitors through ligand and structure-based approach and characterized their effects on enzymatic properties (Km, Vmax, Kcat of LdAI. We show that in presence of two of the inhibitors L1 and L2, the survival of L. donovani is compromised whereas overexpression of LdAI in these cells restores viability. Taken together, our results conclusively prove that LdAI is a crucial metabolic enzyme conferring early counter measure against AmB treatment by Leishmania. Keywords: Leishmania donovani, L-asparaginase, Amphotericin B resistance, Metabolic target

Geographic Distribution of Leishmania Species in Ecuador Based on the Cytochrome B Gene Sequence Analysis.

Science.gov (United States)

Kato, Hirotomo; Gomez, Eduardo A; Martini-Robles, Luiggi; Muzzio, Jenny; Velez, Lenin; Calvopiña, Manuel; Romero-Alvarez, Daniel; Mimori, Tatsuyuki; Uezato, Hiroshi; Hashiguchi, Yoshihisa

2016-07-01

A countrywide epidemiological study was performed to elucidate the current geographic distribution of causative species of cutaneous leishmaniasis (CL) in Ecuador by using FTA card-spotted samples and smear slides as DNA sources. Putative Leishmania in 165 samples collected from patients with CL in 16 provinces of Ecuador were examined at the species level based on the cytochrome b gene sequence analysis. Of these, 125 samples were successfully identified as Leishmania (Viannia) guyanensis, L. (V.) braziliensis, L. (V.) naiffi, L. (V.) lainsoni, and L. (Leishmania) mexicana. Two dominant species, L. (V.) guyanensis and L. (V.) braziliensis, were widely distributed in Pacific coast subtropical and Amazonian tropical areas, respectively. Recently reported L. (V.) naiffi and L. (V.) lainsoni were identified in Amazonian areas, and L. (L.) mexicana was identified in an Andean highland area. Importantly, the present study demonstrated that cases of L. (V.) braziliensis infection are increasing in Pacific coast areas.

Development and evaluation of zinc phthalocyanine nanoemulsions for use in photodynamic therapy for Leishmania spp.

Science.gov (United States)

Betzler de Oliveira de Siqueira, Luciana; da Silva Cardoso, Verônica; Almeida Rodrigues, Igor; Lúcia Vazquez-Villa, Ana; Pereira dos Santos, Elisabete; da Costa Leal Ribeiro Guimarães, Bruno; dos Santos Cerqueira Coutinho, Cristal; Vermelho, Alane Beatriz; Ricci Junior, Eduardo

2017-02-01

Photodynamic therapy (PDT) combines light with photosensitizers (PS) for production of reactive oxygen species (ROS) that can kill infectious microorganisms such as bacteria, fungi and protozoa. The application of nanotechnology has enabled the advancement of PDT because many PS are insoluble in water, necessitating a nanocarrier as a physiologically acceptable carrier. Nanoemulsions are efficient nanocarriers for solubilizing liposoluble drugs, like the PS, in water. Cutaneous (CL) and mucocutaneous leishmaniasis (ML) are caused by different species of the genus Leishmania, transmitted to humans by sandfly bites. Parasites are hosted in skin macrophages producing ulcerative lesions. Thus, a topical treatment, effective and inexpensive, for CL and ML is preferable to systemic interventions. There are topical treatments like paromomycin and amphotericin B, but they have many local side effects or a very high cost, limiting their use. This work aimed to develop a zinc phthalocyanine (photosensitizer) oil-in-water nanoemulsion, essential clove oil and polymeric surfactant (Pluronic® F127) for the formulation of a topical delivery system for use in PDT against Leishmania amazonensis and Leishmania infantum. The nanoemulsion was produced by a high-energy method and characterized by size, polydispersity, morphology, pH, content and stability studies. The toxicity in the dark and the photobiological activity of the formulations were evaluated in vitro for Leishmania and macrophages. The formulation presented was pH compatible with topical use, approximately 30 nm in size, with a polydispersity index ≤0.1 and remained stable at room and refrigerator temperature during the stability study (60 days). The zinc phthalocyanine nanoemulsion is effective in PDT against Leishmania spp.; use against skin infections can be a future application of this topical formulation, avoiding the use of oral or injectable medications, decreasing systemic adverse effects.

Histopathological and parasitological study of the gastrointestinal tract of dogs naturally infected with Leishmania infantum

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Pinto Aldair JW

2011-12-01

Full Text Available Abstract Background The aim of this study was to provide a systematic pathological and parasitological overview of the gastrointestinal tract (GIT, including the stomach, duodenum, jejunum, ileum, caecum and colon, of dogs naturally infected with Leishmania. Methods Twenty mongrel dogs naturally infected with Leishmania (Leishmania infantum and obtained from the Control Zoonosis Center of the Municipality of Ribeirão das Neves, Belo Horizonte Metropolitan area, Minas Gerais (MG state, Brazil, were analyzed. The dogs were divided into two groups: Group 1 comprised nine clinically normal dogs and group 2 comprised 11 clinically affected dogs. After necropsy, one sample was collected from each GIT segment, namely the stomach, duodenum, jejunum, ileum, caecum and colon. Furthermore, paraffin-embedded samples were used for histological and parasitological (immunohistochemistry evaluation and a morphometrical study were carried out to determine the parasite load (immunolabeled amastigote forms of Leishmania. The Friedman and the Mann Whitney tests were used for statistical analysis. The Friedman test was used to analyze each segment of the GIT within each group of dogs and the Mann Whitney test was used to compare the GIT segments between clinically unaffected and affected dogs. Results The infected dogs had an increased number of macrophages, plasma cells and lymphocytes, but lesions were generally mild. Parasite distribution in the GIT was evident in all intestinal segments and layers of the intestinal wall (mucosal, muscular and submucosal irrespective of the clinical status of the dogs. However, the parasite load was statistically higher in the caecum and colon than in other segments of the GIT. Conclusion The high parasite burden evident throughout the GIT mucosa with only mild pathological alterations led us to consider whether Leishmania gains an advantage from the intestinal immunoregulatory response (immunological tolerance.

Bacterial feeding, Leishmania infection and distinct infection routes induce differential defensin expression in Lutzomyia longipalpis.

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Telleria, Erich L; Sant'Anna, Maurício R Viana; Alkurbi, Mohammad O; Pitaluga, André N; Dillon, Rod J; Traub-Csekö, Yara M

2013-01-11

Phlebotomine insects harbor bacterial, viral and parasitic pathogens that can cause diseases of public health importance. Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the New World. Insects can mount a powerful innate immune response to pathogens. Defensin peptides take part in this response and are known to be active against Gram-positive and Gram-negative bacteria, and some parasites. We studied the expression of a defensin gene from Lutzomyia longipalpis to understand its role in sand fly immune response. We identified, sequenced and evaluated the expression of a L. longipalpis defensin gene by semi-quantitative RT-PCR. The gene sequence was compared to other vectors defensins and expression was determined along developmental stages and after exposure of adult female L. longipalpis to bacteria and Leishmania. Phylogenetic analysis showed that the L. longipalpis defensin is closely related to a defensin from the Old World sand fly Phlebotomus duboscqi. Expression was high in late L4 larvae and pupae in comparison to early larval stages and newly emerged flies. Defensin expression was modulated by oral infection with bacteria. The Gram-positive Micrococcus luteus induced early high defensin expression, whilst the Gram-negative entomopathogenic Serratia marcescens induced a later response. Bacterial injection also induced defensin expression in adult insects. Female sand flies infected orally with Leishmania mexicana showed no significant difference in defensin expression compared to blood fed insects apart from a lower defensin expression 5 days post Leishmania infection. When Leishmania was introduced into the hemolymph by injection there was no induction of defensin expression until 72 h later. Our results suggest that L. longipalpis modulates defensin expression upon bacterial and Leishmania infection, with patterns of expression that are distinct among bacterial species and routes of infection.

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.

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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.

Antileishmanial activity and tubulin polymerization inhibition of podophyllotoxin derivatives on Leishmania infantum

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José Miguel Escudero-Martínez

2017-12-01

Full Text Available Leishmania microtubules play an important role not only in cell division, but also in keeping the shape of the parasite and motility of its free-living stages. Microtubules result from the self-assembly of alpha and beta tubulins, two phylogenetically conserved and very abundant eukaryotic proteins in kinetoplastids. The colchicine binding domain has inspired the discovery and development of several drugs currently in clinical use against parasites. However, this domain is less conserved in kinetoplastids and may be selectively targeted by new compounds. This report shows the antileishmanial effect of several series of compounds (53, derived from podophyllotoxin (a natural cyclolignan isolated from rhizomes of Podophyllum spp. and podophyllic aldehyde, on a transgenic, fluorescence-emitting strain of Leishmania infantum. These compounds were tested on both promastigotes and amastigote-infected mouse splenocytes, and in mammalian – mouse non-infected splenocytes and liver HepG2 cells – in order to determine selective indexes of the drugs. Results obtained with podophyllotoxin derivatives showed that the hydroxyl group at position C-7α was a structural requisite to kill the parasites. On regards podophyllic aldehyde, derivatives with C9-aldehyde group integrated into a bicyclic heterostructure displayed more potent antileishmanial effects and were relatively safe for host cells. Docking studies of podophyllotoxin and podophyllic aldehyde derivatives showed that these compounds share a similar pattern of interaction at the colchicine site of Leishmania tubulin, thus pointing to a common mechanism of action. However, the results obtained suggested that despite tubulin is a remarkable target against leishmaniasis, there is a poor correlation between inhibition of tubulin polymerization and antileishmanial effect of many of the compounds tested, fact that points to alternative pathways to kill the parasites. Keywords: Leishmania, Tubulin, DNA

Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

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Klemm, Robin W; Ejsing, Christer S.; Surma, Michal A

2009-01-01

The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane...... trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN...... than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery....

Association of pro-inflammatory cytokines and iron regulatory protein 2 (IRP2 with Leishmania burden in canine visceral leishmaniasis.

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Paulo Ricardo Porfírio do Nascimento

Full Text Available Leishmania infantum infection in humans and dogs can evolve with a wide range of clinical presentations, varying from asymptomatic infections to visceral leishmaniasis. We hypothesized that the immune response elicited by L. infantum infection could modulate whether the host will remain asymptomatic or progress to disease. A total of 44 dogs naturally infected with L. infantum were studied. Leishmania burden was estimated in the blood and spleen by qPCR. The expression of IFN-γ, TNF-α, IL-10 and Iron Regulatory Protein 2 (IRP2 were determined in the spleen by quantitative PCR. Sera cytokines were evaluated by ELISA. Dogs were grouped in quartiles according parasite burden. Increased expression of IFN-γ and TNF-α was associated with reduced Leishmania burden, whereas increased IL-10 and IRP2 expressions were associated with higher Leishmania load. Increased plasma albumin and IFN-γ expression explained 22.8% of the decrease in parasite burden in the spleen. These data confirm that lower IFN-γ response and higher IL-10 correlated with increased parasite load and severity of the visceral leishmaniasis in dogs. The balance between the branches of immune response and the intracellular iron availability could determine, in part, the course of Leishmania infection.

A combined fluorescence spectroscopy, confocal and 2-photon microscopy approach to re-evaluate the properties of sphingolipid domains.

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Pinto, Sandra N; Fernandes, Fábio; Fedorov, Alexander; Futerman, Anthony H; Silva, Liana C; Prieto, Manuel

2013-09-01

The aim of this study is to provide further insight about the interplay between important signaling lipids and to characterize the properties of the lipid domains formed by those lipids in membranes containing distinct composition. To this end, we have used a combination of fluorescence spectroscopy, confocal and two-photon microscopy and a stepwise approach to re-evaluate the biophysical properties of sphingolipid domains, particularly lipid rafts and ceramide (Cer)-platforms. By using this strategy we were able to show that, in binary mixtures, sphingolipids (Cer and sphingomyelin, SM) form more tightly packed gel domains than those formed by phospholipids with similar acyl chain length. In more complex lipid mixtures, the interaction between the different lipids is intricate and is strongly dictated by the Cer-to-Chol ratio. The results show that in quaternary phospholipid/SM/Chol/Cer mixtures, Cer forms gel domains that become less packed as Chol is increased. Moreover, the extent of gel phase formation is strongly reduced in these mixtures, even though Cer molar fraction is increased. These results suggest that in biological membranes, lipid domains such as rafts and ceramide platforms, might display distinctive biophysical properties depending on the local lipid composition at the site of the membrane where they are formed, further highlighting the potential role of membrane biophysical properties as an underlying mechanism for mediating specific biological processes. Copyright © 2013 Elsevier B.V. All rights reserved.

Leishmania amazonensis DNA in wild females of Lutzomyia cruzi (Diptera: Psychodidae) in the state of Mato Grosso do Sul, Brazil.

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Oliveira, Everton Falcão de; Casaril, Aline Etelvina; Mateus, Nathália Lopes Fontoura; Murat, Paula Guerra; Fernandes, Wagner Souza; Oshiro, Elisa Teruya; Oliveira, Alessandra Gutierrez de; Galati, Eunice Aparecida Bianchi

2015-12-01

Studies on natural infection by Leishmania spp of sandflies collected in endemic and nonendemic areas can provide important information on the distribution and intensity of the transmission of these parasites. This study sought to investigate the natural infection by Leishmaniain wild female sandflies. The specimens were caught in the city of Corumbá, state of Mato Grosso do Sul (Brazil) between October 2012-March 2014, and dissected to investigate flagellates and/or submitted to molecular analysis to detect Leishmania DNA. A total of 1,164 females (77.56% of which were Lutzomyia cruzi) representing 11 species were investigated using molecular analysis; 126 specimens of Lu. cruziwere dissected and also submitted to molecular analysis. The infection rate based on the presence of Leishmania DNA considering all the sandfly species analysed was 0.69%; only Leishmania (Leishmania) amazonensis was identified in Lu. cruzi by the molecular analysis. The dissections were negative for flagellates. This is the first record of the presence of L. (L.) amazonensis DNA in Lu. cruzi, and the first record of this parasite in this area. These findings point to the need for further investigation into the possible role of this sandfly as vector of this parasite.

The Leishmania HSP20 Is Antigenic during Natural Infections, but, as DNA Vaccine, It does not Protect BALB/c Mice against Experimental L. amazonensis Infection

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Ana M. Montalvo-Álvarez

2008-01-01

Full Text Available Protozoa of the genus Leishmania are causative agents of leishmaniasis, an important health problem in both human and veterinary medicine. Here, we describe a new heat shock protein (HSP in Leishmania, belonging to the small HSP (sHSP family in kinetoplastids. The protein is highly conserved in different Leishmania species, showing instead significant divergence with sHSP's from other organisms. The humoral response elicited against this protein during Leishmania infection has been investigated in natural infected humans and dogs, and in experimentally infected hamsters. Leishmania HSP20 is a prominent antigen for canine hosts; on the contrary, the protein seems to be a poor antigen for human immune system. Time-course analysis of appearance of anti-HSP20 antibodies in golden hamsters indicated that these antibodies are produced at late stages of the infection, when clinical symptoms of disease are patent. Finally, the protective efficacy of HSP20 was assessed in mice using a DNA vaccine approach prior to challenge with Leishmania amazonensis.

Downregulation of host tryptophan-aspartate containing coat (TACO gene restricts the entry and survival of Leishmania donovani in human macrophage model

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Venkateswara Reddy Gogulamudi

2015-10-01

Full Text Available Leishmania are obligate intracellular protozoan parasites of mammalian hosts. Promastigotes of Leishmania are internalized by macrophages and transformed into amastigotes in phagosomes, and replicate in phagolysosomes. Phagosomal maturation arrest is known to play a central role in the survival of pathogenic Leishmania within activated macrophages. Recently, tryptophan-aspartate containing coat (TACO gene has been recognized as playing a crucial role in the survival of Mycobacterium tuberculosis within human macrophages by arresting the phagosome maturation process. We postulated that a similar association of TACO gene with phagosomes would prevent the vacuole from maturation in the case of Leishmania. In this study we attempted to define the effect of TACO gene downregulation on the uptake/survival of Leishmania donovani intracellularly, by treatment with Vitamin D3/Retinoic acid (RA & Chenodeoxycholic acid (CDCA/Retinoic acid (RA combinations in human THP-1 macrophages (in vitro. Treatment with these molecules downregulated the TACO gene in macrophages, resulting in reduced parasite load and marked reduction of disease progression in L. donovani infected macrophages. Taken together, these results suggest that TACO gene downregulation may play a role in subverting macrophage machinery in establishing the L.donovani replicative niche inside the host. Our study is the first to highlight the importantrole of the TACO gene in Leishmania entry, and to identify TACO gene downregulation as potential drug target against leishmaniasis.

Correlation between presence of Leishmania RNA virus 1 and clinical characteristics of nasal mucosal leishmaniosis.

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Ito, Marcos Massayuki; Catanhêde, Lilian Motta; Katsuragawa, Tony Hiroshi; Silva Junior, Cipriano Ferreira da; Camargo, Luis Marcelo Aranha; Mattos, Ricardo de Godoi; Vilallobos-Salcedo, Juan Miguel

2015-01-01

Mucosal leishmaniosis (ML) is a severe clinical form of leishmaniosis. Complex factors related to the parasite and the host are attributed to the development of mucosal lesions. Leishmania RNA virus 1 (LRV1) can disrupt immune response, and may be the main determinant of severity of the disease; it should be investigated. To study the existence of clinical differences between patients with ML with endosymbiosis by LRV1 and. those without it. A cross-sectional cohort study with clinical evaluation, polymerase chain reaction (PCR) detection of Leishmania, species classification, and search of LRV1 was performed. Only patients with confirmed diagnosis of ML by positive PCR and with nasal mucosa injuries were included in this analysis. Out of 37 patients, 30 (81.1%) were diagnosed with Leishmania braziliensis, five (13.5%) with Leishmania guyanensis, and two (5.4%) with mixed infection of L. braziliensis and L. guyanensis. LVR1 virus was present in 26 (70.3%) of the cases. Correlation between clinical phenotype and presence of LRV1 was not observed, although the frequency of the virus is two-fold higher in mucosal lesions than that found in the literature on skin lesions in the same geographical area. Copyright © 2015 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. All rights reserved.

Design and Validation of Real-Time PCR: Quantitative Diagnosis of Common Leishmania Species in Iran.

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Fekri Soofi Abadi, Maryam; Dabiri, Shahriar; Fotouhi Ardakani, Reza; Fani Malaki, Lina; Amirpoor Rostami, Sahar; Ziasistani, Mahsa; Dabiri, Donya

2016-07-01

Design and validation of Real-time PCR on the protected gene region ITS2 to quantify the parasite load in common leishmania (L) species. Probe and primer were designed from the ITS2 region between the rRNA genes with minimum gene variation in three common leishmania species followed by a Real-time PCR using the Taq man probe method in the form of absolute quantification. A series of different concentrations of leishmania were analyzed. After the purified PCR product was successfully placed in a PTG19-T plasmid vector, specialized ITS2 region was cloned in this plasmid. In the last phase, the cloned gene was transferred to the Ecoli.Top10F bacteria. The standard plasmid was provided in 10(7) to 10(1) copies/rxn concentrations. The specification and clinical sensitivity of the data was analyzed using inter and intra scales. The probe and primer were designed using three species, including L. infantum, L. major, and L.tropica. Seven concentrations of purified parasite in culture media showed that the selected region for quantifying the parasite is suitable. Clinical and analytical specificity and sensitivity were both 100%, respectively. The Taq man method for the ITS2 region in leishmania is one the most sensitive diagnostic test for identifying the parasite load and is suggested as a tool for fast identification and quantification of species.

Nitric oxide-sphingolipid interplays in plant signalling: a new enigma from the Sphinx?

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Guillas, Isabelle; Puyaubert, Juliette; Baudouin, Emmanuel

2013-09-12

Nitric oxide (NO) emerged as one of the major signaling molecules operating during plant development and plant responses to its environment. Beyond the identification of the direct molecular targets of NO, a series of studies considered its interplay with other actors of signal transduction and the integration of NO into complex signaling networks. Beside the close relationships between NO and calcium or phosphatidic acid signaling pathways that are now well-established, recent reports paved the way for interplays between NO and sphingolipids (SLs). This mini-review summarizes our current knowledge of the influence NO and SLs might exert on each other in plant physiology. Based on comparisons with examples from the animal field, it further indicates that, although SL-NO interplays are common features in signaling networks of eukaryotic cells, the underlying mechanisms and molecular targets significantly differ.

Recognition of Leishmania antigens by T lymphocytes from nonexposed individuals

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Kemp, M; Hansen, M B; Theander, T G

1992-01-01

Crude antigen preparations of Leishmania promastigote sonicates were found to induce in vitro proliferation and gamma interferon production in peripheral blood mononuclear cells (PBMC) from individuals without known exposure to the parasite. The proliferating cells were mainly CD2-positive T cell...

Leishmania Infection: Laboratory Diagnosing in the Absence of a “Gold Standard”

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Rodríguez-Cortés, Alhelí; Ojeda, Ana; Francino, Olga; López-Fuertes, Laura; Timón, Marcos; Alberola, Jordi

2010-01-01

There is no gold standard for diagnosing leishmaniases. Our aim was to assess the operative validity of tests used in detecting Leishmania infection using samples from experimental infections, a reliable equivalent to the classic definition of gold standard. Without statistical differences, the highest sensitivity was achieved by protein A (ProtA), immunoglobulin (Ig)G2, indirect fluorescenece antibody test (IFAT), lymphocyte proliferation assay, quantitative real-time polymerase chain reaction of bone marrow (qPCR-BM), qPCR-Blood, and IgG; and the highest specificity by IgG1, IgM, IgA, qPCR-Blood, IgG, IgG2, and qPCR-BM. Maximum positive predictive value was obtained simultaneously by IgG2, qPCR-Blood, and IgG; and maximum negative predictive value by qPCR-BM. Best positive and negative likelihood ratios were obtained by IgG2. The test having the greatest, statistically significant, area under the receiver operating characteristics curve was IgG2 enzyme-linked immunosorbent assay (ELISA). Thus, according to the gold standard used, IFAT and qPCR are far from fulfilling the requirements to be considered gold standards, and the test showing the highest potential to detect Leishmania infection is Leishmania-specific ELISA IgG2. PMID:20134001

Sandflies (Diptera: Psychodidae associated with opossum nests at urban sites in southeastern Brazil: a risk factor for urban and periurban zoonotic Leishmania transmission?

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Andre Antonio Cutolo

2014-06-01

Full Text Available Sandflies associated with opossum nests are reported for the first time in the yards of residences located in the urban area of the municipality of Monte Mor, situated in the metropolitan region of Campinas, state of São Paulo, Brazil. Eleven specimens of Evandromyia cortelezzii and one of Evandromyia lenti were captured in two Didelphis albiventris nests. Ev. cortelezzii is considered a secondary vector species for the transmission of Leishmania (Viannia braziliensis and Leishmania (Leishmania infantum in the Neotropics. This association may contribute to the introduction, establishment and maintenance of urban and periurban zoonotic transmission outbreaks of Leishmania and should therefore be investigated further.

Calcium and magnesium ions modulate the oligomeric state and function of mitochondrial 2-Cys peroxiredoxins in Leishmania parasites.

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Morais, Mariana A B; Giuseppe, Priscila O; Souza, Tatiana A C B; Castro, Helena; Honorato, Rodrigo V; Oliveira, Paulo S L; Netto, Luis E S; Tomas, Ana M; Murakami, Mario T

2017-04-28

Leishmania parasites have evolved a number of strategies to cope with the harsh environmental changes during mammalian infection. One of these mechanisms involves the functional gain that allows mitochondrial 2-Cys peroxiredoxins to act as molecular chaperones when forming decamers. This function is critical for parasite infectivity in mammals, and its activation has been considered to be controlled exclusively by the enzyme redox state under physiological conditions. Herein, we have revealed that magnesium and calcium ions play a major role in modulating the ability of these enzymes to act as molecular chaperones, surpassing the redox effect. These ions are directly involved in mitochondrial metabolism and participate in a novel mechanism to stabilize the decameric form of 2-Cys peroxiredoxins in Leishmania mitochondria. Moreover, we have demonstrated that a constitutively dimeric Prx1m mutant impairs the survival of Leishmania under heat stress, supporting the central role of the chaperone function of Prx1m for Leishmania parasites during the transition from insect to mammalian hosts. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.