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Sample records for leishmania major nucleoside

  1. Production, purification, crystallization and preliminary X-ray diffraction studies of the nucleoside diphosphate kinase b from Leishmania major

    International Nuclear Information System (INIS)

    Tonoli, Celisa Caldana Costa; Vieira, Plinio Salmazo; Ward, Richard John; Arni, Raghuvir Krishnaswamy; Oliveira, Arthur Henrique Cavalcante de; Murakami, Mario Tyago

    2009-01-01

    Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the nucleoside diphosphate kinase b from Leishmania major are reported. The crystals belonged to the trigonal space group P3 2 21 and diffracted to 2.18 Å resolution. Nucleoside diphosphate kinases (NDKs; EC 2.7.4.6) play an essential role in the synthesis of nucleotides from intermediates in the salvage pathway in all parasitic trypanosomatids and their structural studies will be instrumental in shedding light on the biochemical machinery involved in the parasite life cycle and host–parasite interactions. In this work, NDKb from Leishmania major was overexpressed in Escherichia coli, purified to homogeneity and crystallized using the sitting-drop vapour-diffusion method. The NDK crystal diffracted to 2.2 Å resolution and belonged to the trigonal crystal system, with unit-cell parameters a = 114.2, c = 93.9 Å. Translation-function calculations yielded an unambiguous solution in the enantiomorphic space group P3 2 21

  2. 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...

  3. Leishmania donovani Nucleoside Hydrolase terminal domains in cross-protective immunotherapy against Leishmania amazonensis murine infection

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    Dirlei eNico

    2014-06-01

    Full Text Available Nucleoside hydrolases of the Leishmania genus are vital enzymes for the replication of the DNA and conserved phylogenetic markers of the parasites. Leishmania donovani Nucleoside hydrolase (NH36 induced a main CD4+ T cell driven protective response against Leishmania chagasi infection in mice which is directed against its C-terminal domain. In this study, we used the three recombinant domains of NH36: N-terminal domain (F1, amino acids 1-103, central domain (F2 aminoacids 104-198 and C-terminal domain (F3 amino acids 199-314 in combination with saponin and assayed their immunotherapeutic effect on Balb/c mice previously infected with L. amazonensis. We identified that the F1 and F3 peptides determined strong cross-immunotherapeutic effects, reducing the size of footpad lesions to 48% and 64%, and the parasite load in footpads to 82.6% and 81%, respectively. The F3 peptide induced the strongest anti-NH36 antibody response and intradermal response (IDR against L. amazonenis and a high secretion of IFN-γ and TNF-α with reduced levels of IL-10. The F1 vaccine, induced similar increases of IgG2b antibodies and IFN-γ and TNF-α levels, but no IDR and no reduction of IL-10. The multiparameter flow cytometry analysis was used to assess the immune response after immunotherapy and disclosed that the degree of the immunotherapeutic effect is predicted by the frequencies of the CD4+ and CD8+ T cells producing IL-2 or TNF-α or both. Total frequencies and frequencies of double-cytokine CD4 T cell producers were enhanced by F1 and F3 vaccines. Collectively, our multifunctional analysis disclosed that immunotherapeutic protection improved as the CD4 responses progressed from 1+ to 2+, in the case of the F1 and F3 vaccines, and as the CD8 responses changed qualitatively from 1+ to 3+, mainly in the case of the F1 vaccine, providing new correlates of immunotherapeutic protection against cutaneous leishmaniasis in mice based on T-helper TH1 and CD8+ mediated

  4. 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 ...

  5. Developmentally regulated sphingolipid degradation in Leishmania major.

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    Ou Zhang

    Full Text Available Leishmania parasites alternate between extracellular promastigotes in sandflies and intracellular amastigotes in mammals. These protozoans acquire sphingolipids (SLs through de novo synthesis (to produce inositol phosphorylceramide and salvage (to obtain sphingomyelin from the host. A single ISCL (Inositol phosphoSphingolipid phospholipase C-Like enzyme is responsible for the degradation of both inositol phosphorylceramide (the IPC hydrolase or IPCase activity and sphingomyelin (the SMase activity. Recent studies of a L. major ISCL-null mutant (iscl(- indicate that SL degradation is required for promastigote survival in stationary phase, especially under acidic pH. ISCL is also essential for L. major proliferation in mammals. To further understand the role of ISCL in Leishmania growth and virulence, we introduced a sole IPCase or a sole SMase into the iscl(- mutant. Results showed that restoration of IPCase only complemented the acid resistance defect in iscl(- promastigotes and improved their survival in macrophages, but failed to recover virulence in mice. In contrast, a sole SMase fully restored parasite infectivity in mice but was unable to reverse the promastigote defects in iscl(-. These findings suggest that SL degradation in Leishmania possesses separate roles in different stages: while the IPCase activity is important for promastigote survival and acid tolerance, the SMase activity is required for amastigote proliferation in mammals. Consistent with these findings, ISCL was preferentially expressed in stationary phase promastigotes and amastigotes. Together, our results indicate that SL degradation by Leishmania is critical for parasites to establish and sustain infection in the mammalian host.

  6. The SNARE protein family of Leishmania major

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    Mottram Jeremy C

    2006-10-01

    Full Text Available Abstract Background Leishmania major is a protozoan parasite with a highly polarised cell shape that depends upon endocytosis and exocytosis from a single area of the plasma membrane, the flagellar pocket. SNAREs (soluble N-ethylmaleimide-sensitive factor adaptor proteins receptors are key components of the intracellular vesicle-mediated transports that take place in all eukaryotic cells. They are membrane-bound proteins that facilitate the docking and fusion of vesicles with organelles. The recent availability of the genome sequence of L. major has allowed us to assess the complement of SNAREs in the parasite and to investigate their location in comparison with metazoans. Results Bioinformatic searches of the L. major genome revealed a total of 27 SNARE domain-containing proteins that could be classified in structural groups by phylogenetic analysis. 25 of these possessed the expected features of functional SNAREs, whereas the other two could represent kinetoplastid-specific proteins that might act as regulators of the SNARE complexes. Other differences of Leishmania SNAREs were the absence of double SNARE domain-containing and of the brevin classes of these proteins. Members of the Qa group of Leishmania SNAREs showed differential expressions profiles in the two main parasite forms whereas their GFP-tagging and in vivo expression revealed localisations in the Golgi, late endosome/lysosome and near the flagellar pocket. Conclusion The early-branching eukaryote L. major apparently possess a SNARE repertoire that equals in number the one of metazoans such as Drosophila, showing that the machinery for vesicle fusion is well conserved throughout the eukaryotes. However, the analysis revealed the absence of certain types of SNAREs found in metazoans and yeast, while suggesting the presence of original SNAREs as well as others with unusual localisation. This study also presented the intracellular localisation of the L. major SNAREs from the Qa group

  7. 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

  8. leishmania major-phlebotomus duboscqi interactions

    African Journals Online (AJOL)

    hi-tech

    2004-02-01

    Feb 1, 2004 ... Leishmaniasis is a vector-borne disease in which. Leishmania parasites are transmitted by the bite of phlebotomine sand flies. In the vertebrate host,. Leishmania parasites survive and multiply intracellularly in mononuclear phagocytes as a flagellated amastigotes, about 2 to 54 µm in diameter. These cells ...

  9. Discovery of novel inhibitors for Leishmania nucleoside diphosphatase kinase (NDK) based on its structural and functional characterization

    Science.gov (United States)

    Mishra, Arjun K.; Singh, Nidhi; Agnihotri, Pragati; Mishra, Shikha; Singh, Saurabh P.; Kolli, Bala K.; Chang, Kwang Poo; Sahasrabuddhe, Amogh A.; Siddiqi, M. I.; Pratap, J. Venkatesh

    2017-06-01

    Nucleoside diphosphate kinases (NDKs) are ubiquitous enzymes that catalyze the transfer of the γ-phosphate moiety from an NTP donor to an NDP acceptor, crucial for maintaining the cellular level of nucleoside triphosphates (NTPs). The inability of trypanosomatids to synthesize purines de novo and their dependence on the salvage pathway makes NDK an attractive target to develop drugs for the diseases they cause. Here we report the discovery of novel inhibitors for Leishmania NDK based on the structural and functional characterization of purified recombinant NDK from Leishmania amazonensis. Recombinant LaNDK possesses auto-phosphorylation, phosphotransferase and kinase activities with Histidine 117 playing an essential role. LaNDK crystals were grown by hanging drop vapour diffusion method in a solution containing 18% PEG-MME 500, 100 mM Bis-Tris propane pH 6.0 and 50 mM MgCl2. It belongs to the hexagonal space group P6322 with unit cell parameters a = b = 115.18, c = 62.18 Å and α = β = 90°, γ = 120°. The structure solved by molecular replacement methods was refined to crystallographic R-factor and Rfree values of 22.54 and 26.52%, respectively. Molecular docking and dynamics simulation -based virtual screening identified putative binding compounds. Protein inhibition studies of selected hits identified five inhibitors effective at micromolar concentrations. One of the compounds showed 45% inhibition of Leishmania promastigotes proliferation. Analysis of inhibitor-NDK complexes reveals the mode of their binding, facilitating design of new compounds for optimization of activities as drugs against leishmaniasis.

  10. Vaccinations with live-attenuated Leishmania major promastigotes ...

    African Journals Online (AJOL)

    Background: Currently there is no vaccine available in use against any form of leishmaniases worldwide. Objective: To assess potential of a live-attenuated Leishmania major promastigates, for protection against a challenge infection with L. major in BALB/c mice. Design. A laboratory based study. Setting: Study was carried ...

  11. [Importance of amastigote forms morphology to differentiate Leishmania infantum and Leishmania major species].

    Science.gov (United States)

    Aoun, K; Chahed, M K; Mokni, M; Harrat, Z; Bouratbine, A

    2003-01-01

    The microscopic study of the dermal smears of 62 cases of cutaneous leishmaniose, 27 infected by Leishmania (L.) infantum and 35 by L. major, showed that the amastigotes of L. infantum are meaningfully smaller (p < 0.001). This criteria is a simple pary alternative to distinguish these 2 species which have completely different epidemiology, recovery delay and prophylactic dispositions.

  12. Refractoriness to Leishmania donovani and L. major in ...

    African Journals Online (AJOL)

    In the first of a series of experiments, a wild rock pigeon (Columba guinea), a wild guinea fowl (Numidia meleagris), a domestic chicken (Gallus gallus) and a domestic feral pigeon (Columbia livia) were subcutaneously challenged each with 1´107 culture-derived stationary phase Leishmania major (NLB-144) promastigotes ...

  13. 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].

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

    Science.gov (United States)

    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.

  15. 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.

  16. Unusual forms of cutaneous leishmaniasis due to Leishmania major.

    Science.gov (United States)

    Solomon, M; Greenberger, S; Baum, S; Pavlotsky, F; Barzilai, A; Schwartz, E

    2016-07-01

    Cutaneous leishmaniasis (CL) due to Leishmania major (L. major) is common in the Middle East; however, this skin infection may be under-diagnosed when it presents atypically. To highlight the occurrence of uncommon presentations of CL that may elude diagnosis. A retrospective study was performed among patients who presented at The Sheba Medical Center between 2005 and 2014 with atypical clinical presentations of CL due to L. major. Twelve patients with unusual clinical presentations of L. major CL were identified. All infections were acquired in L. major - endemic areas of Israel. The average age was 37 years. The average number of lesions was 2. Nine patients presented with a form that mimicked other forms of CL, such as lupoid, giant ulcer, sporotrichoid and recidivans, and three had a variant resembling other infectious skin diseases, such as erysipeloid and verruciform. All patients required systemic therapy. Cutaneous leishmaniasis due to L. major can masquerade as many other infectious and inflammatory diseases. In addition, it can mimic clinical forms of New World CL. We suggest that in endemic countries or in travellers returning from countries where L. major is endemic, polymerase chain reaction (PCR) for Leishmania-specific DNA should be performed routinely in cases of unusual presentations of dermatitis with a single or a few lesions, even if a diagnosis of CL was not considered by the referring clinician. © 2015 European Academy of Dermatology and Venereology.

  17. 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

  18. Gene Cloning of Iranian Leishmania major Mannose-1-Phosphate Guanyltransferase

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    R Salehi

    2009-07-01

    Full Text Available "nBackground: Leishmania is an obligatory intracellular protozoan parasite, which infects human be­ings when infected sand fly vector takes a blood meal.  Most efforts are towards designing an effective vaccine to prevent leishmaniasis. In this way, development of candidate antigen for vaccine has spe­cial im­portant. In this study, we cloned mannose-1-phosphate guanyltransferase gene of Iranian L .major in pET32a expression vector. "nMethods: Primers based on L. major mannose-1-phosphate guanyltransferase sequence gene was de­signed and synthesized. DNA of Leishmania promastigotes was extracted and PCR reaction was done. PCR product was cloned into pTZ57R and sub cloned into pET32a expression vector. "nResults: Recombinant plasmid containing 1140 bp as L. major mannose-1-phosphate guanyltrans­ferase gene was extracted and confirmed by restriction analysis. PCR product was sequenced and de­posited to GenBank. There were some differences in amino acid sequences between Iranian L. major mannose-1-phosphate guanyltransferase and others previously accepted in GenBank "nConclusion: We amplified and cloned Iranian L. major mannose-1-phosphate guanyltransferase successfully.

  19. INTRACELLULAR Leishmania amazonensis KILLING INDUCED BY THE GUANINE NUCLEOSIDE 8-BROMOGUANOSINE

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    GIORGIO Selma

    1998-01-01

    Full Text Available In this study we investigated the effect of 8-Bromoguanosine, an immunostimulatory compound, on the cytotoxicity of macrophages against Leishmania amazonensis in an in vitro system. The results showed that macrophages treated with 8-Bromoguanosine before or after infection are capable to reduce parasite load, as monitored by the number of amastigotes per macrophage and the percentage of infected cells (i.e. phagocytic index. Since 8-Bromoguanosine was not directly toxic to the promastigotes, it was concluded that the ribonucleoside induced macrophage activation. Presumably, 8-Bromoguanosine primed macrophages by inducing interferon alpha and beta which ultimately led to L. amazonensis amastigote killing. The results suggest that guanine ribonucleosides may be useful to treat infections with intracellular pathogens.

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

    OpenAIRE

    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...

  1. Aurapten, a coumarin with growth inhibition against Leishmania major promastigotes

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    Napolitano H.B.

    2004-01-01

    Full Text Available Several natural compounds have been identified for the treatment of leishmaniasis. Among them are some alkaloids, chalcones, lactones, tetralones, and saponins. The new compound reported here, 7-geranyloxycoumarin, called aurapten, belongs to the chemical class of the coumarins and has a molecular weight of 298.37. The compund was extracted from the Rutaceae species Esenbeckia febrifuga and was purified from a hexane extract starting from 407.7 g of dried leaves and followed by four silica gel chromatographic fractionation steps using different solvents as the mobile phase. The resulting compound (47 mg of shows significant growth inhibition with an LD50 of 30 µM against the tropical parasite Leishmania major, which causes severe clinical manifestations in humans and is endemic in the tropical and subtropical regions. In the present study, we investigated the atomic structure of aurapten in order to determine the existence of common structural motifs that might be related to other coumarins and potentially to other identified inhibitors of Leishmania growth and viability. This compound has a comparable inhibitory activity of other isolated molecules. The aurapten is a planar molecule constituted of an aromatic system with electron delocalization. A hydrophobic side chain consisting of ten carbon atoms with two double bonds and negative density has been identified and may be relevant for further compound synthesis.

  2. Leishmania major and Trypanosoma cruzi present distinct DNA damage responses.

    Science.gov (United States)

    Garcia, Juliana B F; Rocha, João P Vieira da; Costa-Silva, Héllida M; Alves, Ceres L; Machado, Carlos R; Cruz, Angela K

    2016-05-01

    Leishmania major and Trypanosoma cruzi are medically relevant parasites and interesting model organisms, as they present unique biological processes. Despite increasing data regarding the mechanisms of gene expression regulation, there is little information on how the DNA damage response (DDR) occurs in trypanosomatids. We found that L. major presented a higher radiosensitivity than T. cruzi. L. major showed G1 arrest and displayed high mortality in response to ionizing radiation as a result of the inefficient repair of double-strand breaks (DSBs). Conversely, T. cruzi exhibited arrest in the S/G2 cell cycle phase, was able to efficiently repair DSBs and did not display high rates of cell death after exposure to gamma irradiation. L. major showed higher resistance to alkylating DNA damage, and only L. major was able to promote DNA repair and growth recovery in the presence of MMS. ASF1c overexpression did not interfere with the efficiency of DNA repair in either of the parasites but did accentuate the DNA damage checkpoint response, thereby delaying cell fate after damage. The observed differences in the DNA damage responses of T. cruzi and L. major may originate from the distinct preferred routes of genetic plasticity of the two parasites, i.e., DNA recombination versus amplification. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Expression, purification, immunogenicity and protective efficacy of a recombinant nucleoside hydrolase from Leishmania donovani, a vaccine candidate for preventing cutaneous leishmaniasis.

    Science.gov (United States)

    McAtee, C Patrick; Seid, Christopher A; Hammond, Molly; Hudspeth, Elissa; Keegan, Brian P; Liu, Zhuyun; Wei, Junfei; Zhan, Bin; Arjona-Sabido, Raul; Cruz-Chan, Vladimir; Dumonteil, Eric; Hotez, Peter J; Bottazzi, Maria Elena

    2017-02-01

    The nucleoside hydrolase gene from Leishmania donovani was cloned and expressed in Escherichia coli as a full length 36-kDa protein (LdNH36). Following lysis and extraction, the protein was purified by anion exchange and gel filtration chromatography. The purified protein had a molecular mass of approximately 36-kDa and was confirmed to be >99% pure. Using a nucleoside hydrolase assay, the protein was found to exhibit a Km of 741 ± 246 μM. Protein integrity was confirmed by lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE), mass spectrometry (MS), and enzymatic assay. Analysis of antibody levels from immunized mice indicated that LdNH36 alone or in a stable emulsion with the Toll-like receptor-4 ligand glucopyranosyl lipid adjuvant (GLA-SE) as immunostimulant induced high levels of antigen-specific IgG antibodies. The cellular immune response indicated a T h 1 response in mice immunized with LdNH36, but only when formulated with GLA-SE. Mice immunized with the LdNH36 antigen in combination with the GLA-SE adjuvant and challenged with Leishmania mexicana showed significant reductions (>20 fold) in parasite burden, confirming the protective efficacy of this vaccine candidate. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. In silico and in vitro comparative activity of novel experimental derivatives against Leishmania major and Leishmania infantum promastigotes.

    Science.gov (United States)

    Khademvatan, Shahram; Adibpour, Neda; Eskandari, Alborz; Rezaee, Saeed; Hashemitabar, Mahmoud; Rahim, Fakher

    2013-10-01

    This in silico and in vitro comparative study was designed to evaluate the effectiveness of some biurets (K1 to K8) and glucantime against Leishmania major and Leishmania infantum promastigotes. Overall, eight experimental ligands and glucantime were docked using AutoDock 4.3 program into the active sites of Leishmania major and Leishmania infantum pteridine reductase 1, which were modeled using homology modeling programs. The colorimetric MTT assay was used to find L. major and L. infantum promastigotes viability at different concentrations of biuret derivatives in a concentration and time-dependent manner and the obtained results were expressed as 50% and 90% of inhibitory concentration (IC₅₀ and IC₉₀). In silico method showed that out of eight experimental ligands, four compounds were more active on pteridine reductase 1. K3 was the most active against L. major promastigotes with an IC₅₀ of 6.8 μM and an IC₉₀ of 40.2 μM, whereas for L. infantum promastigotes was K8 with IC₅₀ of 7.8 μM. The phenylethyl derivative (K7) showed less toxicity (IC₅₀s>60 μM) in both Leishmania strains. Glucantime displayed less growth inhibition in concentration of about 20 μM. In silico and especially docking results in a recent study were in accordance with the in vitro activity of these compounds in presented study and compound K3, K2 and K8 showed reasonable levels of selectivity for the Leishmania pteridine reductase 1. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. The activity of ozonated olive oil against Leishmania major promastigotes

    Directory of Open Access Journals (Sweden)

    Omid Rajabi

    2015-09-01

    Full Text Available Objective(s:Cutaneous Leishmaniasis is a common and endemic disease in Khorasan province in North-East of Iran. The pentavalant antimony (Sb V is the mainstay of treatment that has many side effects and resistance to the drug has been reported. The microbicidal effect of ozone was proven in different microorganisms. Since there is no study in this respect and to achieve a low cost and effective treatment, we decided to evaluate the efficacy of ozone against promastigotes of Leishmania major,in vitro. Materials and Methods: Ozonated olive oil was prepared after production of ozone by bubbling ozone-oxygen gas produced by ozone generator through olive oil until it solidified. Promastigotes of L. major were cultivated in two phasic media. After calculation of the number of promastigotes, they were incubated with ozonated olive oil (0, 0.626, 0.938, 1.25, 2.5, 5, 10 mcg/ml at 28 °c for 24 hr. Parasites survival percentage was evaluated using MTS and microscopic assay, and then compared with Glucantime and non-ozonated olive oil. Results:According to the results, there were significant differences in parasites survival percentage between ozonated olive oil and non-ozonated olive oil, at similar concentrations (P

  6. The activity of ozonated olive oil against Leishmania major promastigotes.

    Science.gov (United States)

    Rajabi, Omid; Sazgarnia, Ameneh; Abbasi, Fatemeh; Layegh, Pouran

    2015-09-01

    Cutaneous Leishmaniasis is a common and endemic disease in Khorasan province in North-East of Iran. The pentavalant antimony (Sb V) is the mainstay of treatment that has many side effects and resistance to the drug has been reported. The microbicidal effect of ozone was proven in different microorganisms. Since there is no study in this respect and to achieve a low cost and effective treatment, we decided to evaluate the efficacy of ozone against promastigotes of Leishmania major, in vitro. Ozonated olive oil was prepared after production of ozone by bubbling ozone-oxygen gas produced by ozone generator through olive oil until it solidified. Promastigotes of L. major were cultivated in two phasic media. After calculation of the number of promastigotes, they were incubated with ozonated olive oil (0, 0.626, 0.938, 1.25, 2.5, 5, 10 mcg/ml) at 28 °c for 24 hr. Parasites survival percentage was evaluated using MTS and microscopic assay, and then compared with Glucantime and non-ozonated olive oil. According to the results, there were significant differences in parasites survival percentage between ozonated olive oil and non-ozonated olive oil, at similar concentrations (POzonated olive oil was more effective than Glucantime. According to MTS results, Glucantime and ozonated olive oil gel concentrations that are required to inhibit the growth of L. major promastigotes by 50% (IC50), were 165 and 0.002 mg/ml, respectively. Ozonated olive oil has in vitro activity against the promastigotes of L. major and this effect is dose dependent.

  7. First report of naturally infected Sergentomyia minuta with Leishmania major in Tunisia.

    Science.gov (United States)

    Jaouadi, Kaouther; Ghawar, Wissem; Salem, Sadok; Gharbi, Mohamed; Bettaieb, Jihene; Yazidi, Rihab; Harrabi, Mariem; Hamarsheh, Omar; Ben Salah, Afif

    2015-12-21

    Many sand fly species are implicated in the transmission cycle of Leishmania parasites around the world. Incriminating new sand flies species, as vectors of Leishmania is crucial to understanding the parasite-vector transmission cycle in different areas in Tunisia and surrounding countries. Seventy-four unfed females belonging to the genera Sergentomyia and Phlebotomus were collected in South Tunisia between June and November 2014, using sticky papers. PCR-RFLP (Restriction Fragment Length Polymorphism) analysis of the internal transcribed spacer 1 (ITS1) was used for Leishmania parasites detection and identification. Leishmania (L.) major (Yakimoff & Shokkor, 1914) was identified within two Sergentomyia (S.) minuta (Rondani, 1843) and one Phlebotomus papatasi (Scopoli, 1786). This is the first report of L. major identified from S. minuta in Tunisia. This novel finding enhances the understanding of the transmission cycle of L. major parasites of cutaneous leishmaniasis in an endemic area in South Tunisia.

  8. 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...... compared with values for untreated control animals. The [3H]thymidine uptake by the parasites isolated from the treated hamsters was only about 1% of that observed in parasites isolated from the controls. Oral administration of licochalcone A at concentrations of 5 to 150 mg/kg of body weight per day for 6...

  9. Structure, substrate recognition and reactivity of Leishmania major mevalonate kinase

    Directory of Open Access Journals (Sweden)

    Hunter William N

    2007-03-01

    Full Text Available Abstract Background Isoprenoid precursor synthesis via the mevalonate route in humans and pathogenic trypanosomatids is an important metabolic pathway. There is however, only limited information available on the structure and reactivity of the component enzymes in trypanosomatids. Since isoprenoid biosynthesis is essential for trypanosomatid viability and may provide new targets for therapeutic intervention it is important to characterize the pathway components. Results Putative mevalonate kinase encoding genes from Leishmania major (LmMK and Trypanosoma brucei (TbMK have been cloned, over-expressed in and proteins isolated from procyclic-form T. brucei. A highly sensitive radioactive assay was developed and shows ATP-dependent phosphorylation of mevalonate. Apo and (R-mevalonate bound crystal structures of LmMK, from a bacterial expression system, have been determined to high resolution providing, for the first time, information concerning binding of mevalonate to an MK. The mevalonate binds in a deep cavity lined by highly conserved residues. His25 is key for binding and for discrimination of (R- over (S-mevalonate, with the main chain amide interacting with the C3 hydroxyl group of (R-mevalonate, and the side chain contributing, together with Val202 and Thr283, to the construction of a hydrophobic binding site for the C3 methyl substituent. The C5 hydroxyl, where phosphorylation occurs, points towards catalytic residues, Lys18 and Asp155. The activity of LmMK was significantly reduced compared to MK from other species and we were unable to obtain ATP-binding data. Comparisons with the rat MK:ATP complex were used to investigate how this substrate might bind. In LmMK, helix α2 and the preceding polypeptide adopt a conformation, not seen in related kinase structures, impeding access to the nucleotide triphosphate binding site suggesting that a conformational rearrangement is required to allow ATP binding. Conclusion Our new structural

  10. Molecular epidemiology of cutaneous leishmaniasis and heterogeneity of Leishmania major strains in Iran.

    Science.gov (United States)

    Mahmoudzadeh-Niknam, Hamid; Ajdary, Soheila; Riazi-Rad, Farhad; Mirzadegan, Ebrahim; Rezaeian, Abdolhossein; Khaze, Vahid; Djadid, Navid D; Alimohammadian, Mohammad H

    2012-11-01

    To determine the geographical distribution of Leishmania species causing cutaneous leishmaniasis (CL) and to study the genetic heterogeneity of Leishmania major isolates from different endemic areas of Iran. A total of 341 isolates from lesions of patients living in 11 provinces of Iran were grown in culture medium and inoculated to BALB/c mice to detect possible visceralisation. The species were identified by isoenzyme analysis using a battery of six enzymes and kinetoplast (k) DNA-PCR technique. Genetic variation among L. major isolates was analysed by random amplified polymorphic DNA (RAPD) technique. Of the total 341 isolates, 283 isolates were L. major and 58 isolates were Leishmania tropica. In rural areas, the causative agent of CL was mainly L. major (95%L. major vs. 5%L. tropica), in urban areas it was L. tropica (65%L. tropica vs. 35%L. major). All isolates of L. major and 8.6% of L. tropica isolates showed visceralisation in BALB/c mice. There is considerable genetic diversity between L. major strains from different endemic areas and even between some isolates of the same endemic area. Leishmania major is the most frequent species in the endemic areas of CL in eleven provinces of Iran, and genetic diversity is a common feature of L. major in the country. © 2012 Blackwell Publishing Ltd.

  11. Molecular Cloning and Expression of Iranian Leishmania major Pteridine Reductase 1

    Directory of Open Access Journals (Sweden)

    N Mosaffa

    2008-04-01

    Full Text Available Background: Leishmaniasis is an endemic disease in 88 countries. Reports on Leishmania drug resistance are growing in number. The mechanism of unresponsiveness against glucantime in Iranian cutaneous leishmaniasis has not yet been characterized. To begin the first step in finding an anti-Leishmania chemotherapy, we prepared recombinant L. major PTR1 enzyme and characterized its activity by enzymatic assay. Methods: Leishmania promastigote DNA was extracted and the ptr1 gene amplified using specific primers. The PCR product was cloned in pQE30 expression vector, transformed into E.coli and expressed. The recombinant protein was purified, its enzymatic activity was assayed and anti-PTR1 antibody prepared in rabbit. Results: The PCR product of ptr1 gene was sequenced and deposited in GenBank. The amino acid sequence of Iranian L.major PTR1 was compared with other Leishmania PTR1 and showed some identities and diversities. Purified protein was reacted by anti PTR1 antibody in gel diffusion and western blot assy. Enzyme activity of purified recombinant PTR1 was 38 nmol/min per 0.4 mg of protein and it showed pteridine reduction by PTR1 Conclusion: We cloned and expressed Iranian L. major ptr1 gene and assayed its enzymatic activity. This enzyme will be used for further investigation about Leishmania antifolate therapy that is effective against PTR1

  12. Th1-like human T-cell clones recognizing Leishmania gp63 inhibit Leishmania major in human macrophages

    DEFF Research Database (Denmark)

    Kemp, M; Hey, A S; Bendtzen, K

    1994-01-01

    The major surface protease of Leishmania major, gp63, has been suggested as a vaccine candidate for cutaneous leishmaniasis. In this study gp63 was purified from L. major promastigotes. A panel of human T-cell clones recognizing this protein were generated from individuals who had previously had...... self-healing cutaneous leishmaniasis. The T-cell clones expressed CD4, and the alpha chain of the T-cell antigen receptor. GP63 reactive T-cell clones activated by antigen or by immobilized anti-CD3 antibody released relative large amounts of interferon-gamma and no or little interleukin-4, thereby...... resembling Th1 cells. Autologous mononuclear cells and Epstein-Barr virus-transformed B cell lines were equally efficient in presenting the antigen to the T cells. The gp63 reactive T cells induced resistance to infection in cultured human macrophages by L. major. The data confirm that human CD4+ T cells...

  13. First detection of Leishmania major DNA in Sergentomyia (Spelaeomyia) darlingi from cutaneous leishmaniasis foci in Mali.

    Science.gov (United States)

    Berdjane-Brouk, Zohra; Koné, Abdoulaye K; Djimdé, Abdoulaye A; Charrel, Rémi N; Ravel, Christophe; Delaunay, Pascal; del Giudice, Pascal; Diarra, Adama Z; Doumbo, Siala; Goita, Siaka; Thera, Mahamadou A; Depaquit, Jérôme; Marty, Pierre; Doumbo, Ogobara K; Izri, Arezki

    2012-01-01

    Leishmania major complex is the main causative agent of zoonotic cutaneous leishmaniasis (ZCL) in the Old World. Phlebotomus papatasi and Phlebotomus duboscqi are recognized vectors of L. major complex in Northern and Southern Sahara, respectively. In Mali, ZCL due to L. major is an emerging public health problem, with several cases reported from different parts of the country. The main objective of the present study was to identify the vectors of Leishmania major in the Bandiagara area, in Mali. An entomological survey was carried out in the ZCL foci of Bandiagara area. Sandflies were collected using CDC miniature light traps and sticky papers. In the field, live female Phlebotomine sandflies were identified and examined for the presence of promastigotes. The remaining sandflies were identified morphologically and tested for Leishmania by PCR in the ITS2 gene. The source of blood meal of the engorged females was determined using the cyt-b sequence. Out of the 3,259 collected sandflies, 1,324 were identified morphologically, and consisted of 20 species, of which four belonged to the genus Phlebotomus and 16 to the genus Sergentomyia. Leishmania major DNA was detected by PCR in 7 of the 446 females (1.6%), specifically 2 out of 115 Phlebotomus duboscqi specimens, and 5 from 198 Sergentomyia darlingi specimens. Human DNA was detected in one blood-fed female S. darlingi positive for L. major DNA. Our data suggest the possible involvement of P. duboscqi and potentially S. darlingi in the transmission of ZCL in Mali.

  14. The pathology of cutaneous leishmaniasis due to Leishmania major in Sudan

    DEFF Research Database (Denmark)

    Gaafar, A; el Kadaro, A Y; Theander, T G

    1995-01-01

    The pathology of cutaneous leishmaniasis in Sudan, where the disease is caused by Leishmania major, was studied by light and electron microscopy. Lesions were classified into four distinct groups based on the ratio of different cell types, especially lymphocytes, macrophages, and plasma cells...

  15. Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase

    DEFF Research Database (Denmark)

    Montalvetti, A; Pena Diaz, Javier; Hurtado, R

    2000-01-01

    In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from L...

  16. Antigen-presenting cells in human cutaneous leishmaniasis due to Leishmania major

    DEFF Research Database (Denmark)

    ElHassan, A M; Gaafar, A; Theander, T G

    1995-01-01

    In this study biopsies from skin lesions and draining lymph nodes of patients suffering from cutaneous leishmaniasis caused by Leishmania major were examined by immunohistochemistry, and by light and electron microscopy to identify the types of antigen-presenting cells (APC) and their location. APC...

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

    Czech Academy of Sciences Publication Activity Database

    Chajbullinova, A.; Votýpka, Jan; Sádlová, J.; Kvapilová, K.; Seblova, V.; Kreisinger, J.; Jirků, Milan; Sanjoba, C.; Gantuya, S.; Matsumoto, Y.; Volf, P.

    2012-01-01

    Roč. 5, OCT 2 (2012) ISSN 1756-3305 Institutional support: RVO:60077344 Keywords : Leishmania turanica * L. major * mixed infections * competition * genetic exchange * vector competence * Phlebotomus Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.246, year: 2012 http://www.parasitesandvectors.com/content/5/1/219

  18. A radioattenuated Leishmania major vaccine markedly increases the resistance of CBA mice to subsequent infection with Leishmania mexicana mexicana

    International Nuclear Information System (INIS)

    Alexander, J.

    1982-01-01

    Vaccinating CBA mice with radioattenuated Leishmania major amastigotes but not with radioattenuated L. mexicana amastigotes rendered them highly resistant to subsequent infection with L. m. mexicana. Unvaccinated CBA mice were highly susceptible to infection with L. m. mexicana producing rapidly growing non-ulcerating cutaneous lesions. Two manifestations of resistance were induced in vaccinated animals depending on the timing of the challenge infection: no lesions appeared at the site of subcutaneous challenge in animals vaccinated four or more weeks previously, while lesions grew rapidly but ulcerated and healed in animals vaccinated less than 3 weeks beforehand. L. major amastigotes were found to be markedly more resistant to γ irradiation than L. m.mexicana amastigotes both as measured by their ability to infect susceptible strains of mice and to transform and multiply as promastigotes in NNN medium. (author)

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  1. LACK, a RACK1 ortholog, facilitates cytochrome c oxidase subunit expression to promote Leishmania major fitness.

    Science.gov (United States)

    Cardenas, Daviel; Carter, Pamela M; Nation, Catherine S; Pizarro, Juan C; Guidry, Jessie; Aiyar, Ashok; Kelly, Ben L

    2015-04-01

    Leishmania are kinetoplastid parasites that cause the sandfly-transmitted disease leishmaniasis. To maintain fitness throughout their infectious life cycle, Leishmania must undergo rapid metabolic adaptations to the dramatically distinct environments encountered during transition between sandfly and vertebrate hosts. We performed proteomic and immunoblot analyses of attenuated L. major strains deficient for LACK, the Leishmania ortholog of the mammalian receptor for activated c kinase (RACK1), that is important for parasite thermotolerance and virulence. This approach identified cytochrome c oxidase (LmCOX) subunit IV as a LACK-dependent fitness protein. Consistent with decreased levels of LmCOX subunit IV at mammalian temperature, and in amastigotes, LmCOX activity and mitochondrial function were also impaired in LACK-deficient L. major under these conditions. Importantly, overexpression of LmCOX subunit IV in LACK-deficient L. major restored thermotolerance and macrophage infectivity. Interestingly, overexpression of LmCOX subunit IV enhanced LmCOX subunit VI expression at mammalian temperature. Collectively, our data suggest LACK promotes Leishmania adaptation to the mammalian host environment by sustaining LmCOX subunit IV expression and hence energy metabolism in response to stress stimuli such as heat. These findings extend the repertoire of RACK1 protein utility to include a role in mitochondrial function. © 2015 John Wiley & Sons Ltd.

  2. 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 mitoch......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...

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Expression of hsa Let-7a MicroRNA of Macrophages Infected by Leishmania Major

    Directory of Open Access Journals (Sweden)

    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.

  5. Characterization of a Monoclonal Antibody Specific for the Parasite Surface Antigen-2 of Leishmania major

    OpenAIRE

    "AR Khabiri; F Bagheri; SR Naddaf; M Assmar; A Hosseini Taghavi"

    2004-01-01

    The Leishmania major Parasite surface Antigen-2 (PSA-2) is a family of glycoinositol phospholipids anchored glycoprotoins expressed in both promastigotes and amastigotes. Promastigote PSA-2 comprises three polypeptides with approximate molecular weight of 96, 80 and 50 kDa. Amastigote express a distinct but closely PSA-2 polypeptide with molecular weight of 50 kDa. In this study fusion of SP2/0 myeloma cells with immunized mice spleenocytes infected with promastigotes of L. major intraperiton...

  6. First Detection of Leishmania major DNA in Sergentomyia (Spelaeomyia) darlingi from Cutaneous Leishmaniasis Foci in Mali

    Science.gov (United States)

    Berdjane-Brouk, Zohra; Koné, Abdoulaye K.; Djimdé, Abdoulaye A.; Charrel, Rémi N.; Ravel, Christophe; Delaunay, Pascal; del Giudice, Pascal; Diarra, Adama Z.; Doumbo, Siala; Goita, Siaka; Thera, Mahamadou A.; Depaquit, Jérôme; Marty, Pierre; Doumbo, Ogobara K.; Izri, Arezki

    2012-01-01

    Background Leishmania major complex is the main causative agent of zoonotic cutaneous leishmaniasis (ZCL) in the Old World. Phlebotomus papatasi and Phlebotomus duboscqi are recognized vectors of L. major complex in Northern and Southern Sahara, respectively. In Mali, ZCL due to L. major is an emerging public health problem, with several cases reported from different parts of the country. The main objective of the present study was to identify the vectors of Leishmania major in the Bandiagara area, in Mali. Methodology/Principal Findings An entomological survey was carried out in the ZCL foci of Bandiagara area. Sandflies were collected using CDC miniature light traps and sticky papers. In the field, live female Phlebotomine sandflies were identified and examined for the presence of promastigotes. The remaining sandflies were identified morphologically and tested for Leishmania by PCR in the ITS2 gene. The source of blood meal of the engorged females was determined using the cyt-b sequence. Out of the 3,259 collected sandflies, 1,324 were identified morphologically, and consisted of 20 species, of which four belonged to the genus Phlebotomus and 16 to the genus Sergentomyia. Leishmania major DNA was detected by PCR in 7 of the 446 females (1.6%), specifically 2 out of 115 Phlebotomus duboscqi specimens, and 5 from 198 Sergentomyia darlingi specimens. Human DNA was detected in one blood-fed female S. darlingi positive for L. major DNA. Conclusion Our data suggest the possible involvement of P. duboscqi and potentially S. darlingi in the transmission of ZCL in Mali. PMID:22276095

  7. First detection of Leishmania major DNA in Sergentomyia (Spelaeomyia darlingi from cutaneous leishmaniasis foci in Mali.

    Directory of Open Access Journals (Sweden)

    Zohra Berdjane-Brouk

    Full Text Available BACKGROUND: Leishmania major complex is the main causative agent of zoonotic cutaneous leishmaniasis (ZCL in the Old World. Phlebotomus papatasi and Phlebotomus duboscqi are recognized vectors of L. major complex in Northern and Southern Sahara, respectively. In Mali, ZCL due to L. major is an emerging public health problem, with several cases reported from different parts of the country. The main objective of the present study was to identify the vectors of Leishmania major in the Bandiagara area, in Mali. METHODOLOGY/PRINCIPAL FINDINGS: An entomological survey was carried out in the ZCL foci of Bandiagara area. Sandflies were collected using CDC miniature light traps and sticky papers. In the field, live female Phlebotomine sandflies were identified and examined for the presence of promastigotes. The remaining sandflies were identified morphologically and tested for Leishmania by PCR in the ITS2 gene. The source of blood meal of the engorged females was determined using the cyt-b sequence. Out of the 3,259 collected sandflies, 1,324 were identified morphologically, and consisted of 20 species, of which four belonged to the genus Phlebotomus and 16 to the genus Sergentomyia. Leishmania major DNA was detected by PCR in 7 of the 446 females (1.6%, specifically 2 out of 115 Phlebotomus duboscqi specimens, and 5 from 198 Sergentomyia darlingi specimens. Human DNA was detected in one blood-fed female S. darlingi positive for L. major DNA. CONCLUSION: Our data suggest the possible involvement of P. duboscqi and potentially S. darlingi in the transmission of ZCL in Mali.

  8. In vivo antileishmanial action of Ir-(COD-pentamidine tetraphenylborate on Leishmania donovani and Leishmania major mouse models

    Directory of Open Access Journals (Sweden)

    Loiseau P.M.

    2000-06-01

    Full Text Available lr-(COD-pentamidine tetraphenylborate which has previously been studied on promastigote forms of Leishmania, was investigated for its antileishmanial properties compared with pentamidine used as reference compound. In vitro, the iridium complex had the same IC50 value on intracellular forms of Leishmania as pentamidine (15 μM. In vivo, the compound could not be injected intravenously due to the DMSO excipient so that the treatments were performed intraperitoneally or subcutaneously. On the L. donovani LV9 /Balb/C mouse model, the iridium complex was not toxic after intraperitoneal treatment at 232 mg/kg/day x 5 or 147 μmoles/ kg/day x 5, whereas all the mice died within five days when treated at the same dose with pentamidine isethionate. However, only 23 % of parasite suppression was observed with the iridium complex. On a L. major MON 74/Balb/C mouse model, susceptible to intravenously administered pentamidine at 6.7 μmoles/ kg/day x 5 (54 % of parasite suppression, the iridium complex exhibited 32 % of parasite suppression after a treatment at 76 μmoles/ kg/day x 5 administered subcutaneously. This slight activity is of interest since pentamidine isethionate is not active under these conditions. Transmission electron microscopy of amastigotes from infected and treated mice show aggregation of ribosomal material, distension of the nuclear membrane and kDNA depolymerization. The mechanism of action therefore involves several targets: membranes, ribosomes and kDNA. According to our results, the Iridium complex is a suitable candidate to be encapsulated in drug carriers such as liposomes or nanoparticles.

  9. Dichotomy of the human T cell response to Leishmania antigens. I. Th1-like response to Leishmania major promastigote antigens in individuals recovered from cutaneous leishmaniasis

    DEFF Research Database (Denmark)

    Kemp, M; Hey, A S; Kurtzhals, J A

    1994-01-01

    The T cell response to antigens from Leishmania major promastigotes was investigated in peripheral blood mononuclear cells from Sudanese individuals with a history of cutaneous leishmaniasis (CL), Sudanese individuals with positive DTH reaction in the leishmanin skin test but with no history of s...... or subclinical L. major infection is an IFN-gamma-producing Th1-like response....

  10. Evaluation of the anti-Leishmania major activity of Satureja bakhtiarica essential oil in vitro.

    Science.gov (United States)

    Mohammadpour, Ghasem; Marzony, Eisa Tahmasbpour; Farahmand, Mahin

    2012-01-01

    Leishmaniasis is a painless chronic skin disease that is caused by the protozoan parasite Leishmania. Due to the importance of this disease and the side effects of chemical drugs, use of drugs of plant origin to treat Leishmaniasis is very important. In the present study, the chemical composition and the anti-Leishmania major activity of the essential oils obtained from Satureja bakhtiarica were evaluated in vitro. The oils were extracted using a Clevenger apparatus and then the chemical composition was analyzed by GC-MS. Promastigotes of L. major were cultured in both N.N.N and RPMI1640 media. GC-MS analysis showed 13 compounds, in which the major components were the phenolic (37.4%) compounds, thymol (22.6%) and p-cymene (19.3%). The essential oil of S. bakhtiarica showed higher activity against L. major than the standard anti-Leishmania drug, glucantime,. Perhaps because of the high concentration of phenolic compounds in the essential oil, all the parasites were killed after 24 hours. The essential oil from S. bakhtiarica is a potential plant drug against leishmaniasis. Further studies are necessary to evaluate this oil in animal models (in vivo) for future drug applications.

  11. Distinct genetic control of parasite elimination, dissemination, and disease after Leishmania major infection

    Czech Academy of Sciences Publication Activity Database

    Kurey, Irina; Kobets, Tetyana; Havelková, Helena; Slapničková, Martina; Quan, L.; Trtková, Kateřina; Grekov, Igor; Svobodová, M.; Stassen, A. P. M.; Hutson, A.; Demant, P.; Lipoldová, Marie

    2009-01-01

    Roč. 61, č. 9 (2009), s. 619-633 ISSN 0093-7711 R&D Projects: GA ČR GA310/06/1745; GA MŠk(CZ) LC06009 Grant - others:EC(XE) 05-1000004-7761 Institutional research plan: CEZ:AV0Z50520514 Keywords : Leishmania major * Parasite elimination * QTL mapping Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.988, year: 2009

  12. In vitro antileishmanial activity of methanolic and aqueous extracts of Eucalyptus camaldulensis against Leishmania major

    OpenAIRE

    Nosratabadi, Seyyed Jafar; Sharifi, Iraj; Sharififar, Fariba; Bamorovat, Mehdi; Daneshvar, Hamid; Mirzaie, Mohammad

    2013-01-01

    Pentavalent antimony compounds are expensive, toxic and drug resistance is prevalent, whereas the plant extract derivatives are safe. In the present study, the effect of methanolic and aqueous extracts of Eucalyptus camaldulensis on the promastigotes of Leishmania major was evaluated. The methanolic and aqueous extracts of E. camaldulensis leaves were prepared. The compounds were dried and powdered. Serial dilutions of the extracts and control drugs in phosphate buffer solution were prepared....

  13. Effect of BMAP-28 antimicrobial peptides on Leishmania major promastigote and amastigote growth

    DEFF Research Database (Denmark)

    Lynn, Miriam A.; Kindrachuk, Jason; Marr, Alexandra K.

    2011-01-01

    of the cathelicidin family of HDPs have demonstrated significant antimicrobial activities against various parasites including Leishmania. The cathelicidin bovine myeloid antimicrobial peptide 28 (BMAP-28) has broad antimicrobial activities and confers protection in animal models of bacterial infection or sepsis. We...... tested the effectiveness of the use of BMAP-28 and two of its isomers the D-amino acid form (D-BMAP-28) and the retro-inverso form (RI-BMAP-28), as anti-leishmanial agents against the promastigote and amastigote intracellular Leishmania major lifecycle stages. Methodology/Principal Findings: An MTS...... with early osmotic cell lysis caused by the antimicrobial peptides. Furthermore, BMAP-28 and its isomers demonstrated anti-leishmanial activities against intracellular amastigotes within a macrophage infection model. Conclusions/Significance: Interestingly, D-BMAP-28 appears to be the most potent...

  14. Dichotomy of the human T cell response to Leishmania antigens. I. Th1-like response to Leishmania major promastigote antigens in individuals recovered from cutaneous leishmaniasis

    DEFF Research Database (Denmark)

    Kemp, M; Hey, A S; Kurtzhals, J A

    1994-01-01

    The T cell response to antigens from Leishmania major promastigotes was investigated in peripheral blood mononuclear cells from Sudanese individuals with a history of cutaneous leishmaniasis (CL), Sudanese individuals with positive DTH reaction in the leishmanin skin test but with no history...

  15. Leishmania major UDP-sugar pyrophosphorylase salvages galactose for glycoconjugate biosynthesis.

    Science.gov (United States)

    Damerow, Sebastian; Hoppe, Carolin; Bandini, Giulia; Zarnovican, Patricia; Buettner, Falk F R; Ferguson, Michael A J; Routier, Françoise H

    2015-10-01

    Leishmaniases are a set of tropical and sub-tropical diseases caused by protozoan parasites of the genus Leishmania whose severity ranges from self-healing cutaneous lesions to fatal visceral infections. Leishmania parasites synthesise a wide array of cell surface and secreted glycoconjugates that play important roles in infection. These glycoconjugates are particularly abundant in the promastigote form and known to be essential for establishment of infection in the insect midgut and effective transmission to the mammalian host. Since they are rich in galactose, their biosynthesis requires an ample supply of UDP-galactose. This nucleotide-sugar arises from epimerisation of UDP-glucose but also from an uncharacterised galactose salvage pathway. In this study, we evaluated the role of the newly characterised UDP-sugar pyrophosphorylase (USP) of Leishmania major in UDP-galactose biosynthesis. Upon deletion of the USP encoding gene, L. major lost the ability to synthesise UDP-galactose from galactose-1-phosphate but its ability to convert glucose-1-phosphate into UDP-glucose was fully maintained. Thus USP plays a role in UDP-galactose activation but does not significantly contribute to the de novo synthesis of UDP-glucose. Accordingly, USP was shown to be dispensable for growth and glycoconjugate biosynthesis under standard growth conditions. However, in a mutant seriously impaired in the de novo synthesis of UDP-galactose (due to deficiency of the UDP-glucose pyrophosphorylase) addition of extracellular galactose increased biosynthesis of the cell surface lipophosphoglycan. Thus under restrictive conditions, such as those encountered by Leishmania in its natural habitat, galactose salvage by USP may play a substantial role in biosynthesis of the UDP-galactose pool. We hypothesise that USP recycles galactose from the blood meal within the midgut of the insect for synthesis of the promastigote glycocalyx and thereby contributes to successful vector infection. Copyright

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

    Science.gov (United States)

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

    2001-01-01

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

  17. Coadministration of the Three Antigenic Leishmania infantum Poly (A Binding Proteins as a DNA Vaccine Induces Protection against Leishmania major Infection in BALB/c Mice.

    Directory of Open Access Journals (Sweden)

    Manuel Soto

    2015-05-01

    Full Text Available Highly conserved intracellular proteins from Leishmania have been described as antigens in natural and experimental infected mammals. The present study aimed to evaluate the antigenicity and prophylactic properties of the Leishmania infantum Poly (A binding proteins (LiPABPs.Three different members of the LiPABP family have been described. Recombinant tools based on these proteins were constructed: recombinant proteins and DNA vaccines. The three recombinant proteins were employed for coating ELISA plates. Sera from human and canine patients of visceral leishmaniasis and human patients of mucosal leishmaniasis recognized the three LiPABPs. In addition, the protective efficacy of a DNA vaccine based on the combination of the three Leishmania PABPs has been tested in a model of progressive murine leishmaniasis: BALB/c mice infected with Leishmania major. The induction of a Th1-like response against the LiPABP family by genetic vaccination was able to down-regulate the IL-10 predominant responses elicited by parasite LiPABPs after infection in this murine model. This modulation resulted in a partial protection against L. major infection. LiPABP vaccinated mice showed a reduction on the pathology that was accompanied by a decrease in parasite burdens, in antibody titers against Leishmania antigens and in the IL-4 and IL-10 parasite-specific mediated responses in comparison to control mice groups immunized with saline or with the non-recombinant plasmid.The results presented here demonstrate for the first time the prophylactic properties of a new family of Leishmania antigenic intracellular proteins, the LiPABPs. The redirection of the immune response elicited against the LiPABP family (from IL-10 towards IFN-γ mediated responses by genetic vaccination was able to induce a partial protection against the development of the disease in a highly susceptible murine model of leishmaniasis.

  18. Detection of amastigote-like forms in the valve of Phlebotomus papatasi infected with Leishmania major

    Directory of Open Access Journals (Sweden)

    Nestor Añez

    2003-06-01

    Full Text Available A massive and homogeneous amount of amastigote-like forms was detected in the stomodeal valve (SV and the thoracic mid-gut (TMG of Leishmania major-infected Phlebotomus papatasi, which received a second blood meal 13 to 21 days post-infection on healthy anaesthetized hamsters. After re-feeding, the infected sand flies were dissected out to examine the morphology of the parasite in SV, TMG and the abdominal mid-gut (AMG. Different promastigote forms were seen in the infected flies. Among these included typical promastigotes (nectomonads and haptomonads, paramastigotes, metacyclic promastigotes and, in some samples, the here-reported amastigote-like forms. The Leishmania amastigote-like forms were detected in the SV of sand flies with 14, 18 and 21 days of infection as well as in the TMG at 13 and 18 days post-infection. However, the amastigote-like forms were not detected in the AMG. Factors such as the acidic pH predominating the TMG and the SV, as well as the temperature of the ingested blood, among others, are suggested as contributing to the transformation of the typical promastigotes into the amastigote-like forms. The significance of this finding is discussed and the possible biological advantage for transmission of Leishmania is considered.

  19. Efficacy of Permethrin Treated Bed Nets Against Leishmania major Infected Sand Flies.

    Science.gov (United States)

    Rowland, Tobin; Davidson, Silas A; Kobylinski, Kevin; Menses, Claudio; Rowton, Edgar

    2015-01-01

    Insecticide treated nets (ITNs) are a potential tool to help control sand flies and prevent Leishmaniasis. However, little is currently known about the response of Leishmania infected sand flies to ITNs. In this study, Phlebotomus duboscqi sand flies were infected with the parasite Leishmania major. Infected and noninfected sand flies were then evaluated against permethrin treated and untreated bed nets in a laboratory assay that required sand flies to pass through suspended netting material to feed on a mouse serving as an attractive host. The number of sand flies passing through the nets and blood feeding was recorded. There was not a significant difference in the ability of infected or noninfected sand flies to move through treated or untreated nets. Fewer sand flies entered the permethrin treated nets compared to the untreated nets, indicating that permethrin creates an effective barrier. The results show that in addition to reducing the nuisance bites of noninfected sand flies, ITNs also protect against Leishmania infected sand flies and therefore can play in key role in reducing the rates of Leishmaniasis. This study is important to the Department of Defense as it continues to develop and field new bed nets to protect service members.

  20. CD8+ T cells Are Preferentially Activated during Primary Low Dose Leishmania major Infection but Are Completely Dispensable during Secondary Anti-Leishmania Immunity

    OpenAIRE

    Okwor, Ifeoma B.; Jia, Ping; Mou, Zhirong; Onyilagha, Chukwunonso; Uzonna, Jude E.

    2014-01-01

    We previously showed that CD8+ T cells are required for optimal primary immunity to low dose Leishmania major infection. However, it is not known whether immunity induced by low dose infection is durable and whether CD8+ T cells contribute to secondary immunity following recovery from low dose infection. Here, we compared primary and secondary immunity to low and high dose L. major infections and assessed the influence of infectious dose on the quality and magnitude of secondary anti-Leishman...

  1. Spatio-temporal Genetic Structuring of Leishmania major in Tunisia by Microsatellite Analysis.

    Science.gov (United States)

    Harrabi, Myriam; Bettaieb, Jihène; Ghawar, Wissem; Toumi, Amine; Zaâtour, Amor; Yazidi, Rihab; Chaâbane, Sana; Chalghaf, Bilel; Hide, Mallorie; Bañuls, Anne-Laure; Ben Salah, Afif

    2015-01-01

    In Tunisia, cases of zoonotic cutaneous leishmaniasis caused by Leishmania major are increasing and spreading from the south-west to new areas in the center. To improve the current knowledge on L. major evolution and population dynamics, we performed multi-locus microsatellite typing of human isolates from Tunisian governorates where the disease is endemic (Gafsa, Kairouan and Sidi Bouzid governorates) and collected during two periods: 1991-1992 and 2008-2012. Analysis (F-statistics and Bayesian model-based approach) of the genotyping results of isolates collected in Sidi Bouzid in 1991-1992 and 2008-2012 shows that, over two decades, in the same area, Leishmania parasites evolved by generating genetically differentiated populations. The genetic patterns of 2008-2012 isolates from the three governorates indicate that L. major populations did not spread gradually from the south to the center of Tunisia, according to a geographical gradient, suggesting that human activities might be the source of the disease expansion. The genotype analysis also suggests previous (Bayesian model-based approach) and current (F-statistics) flows of genotypes between governorates and districts. Human activities as well as reservoir dynamics and the effects of environmental changes could explain how the disease progresses. This study provides new insights into the evolution and spread of L. major in Tunisia that might improve our understanding of the parasite flow between geographically and temporally distinct populations.

  2. Different genetic control of cutaneous and visceral disease after Leishmania major infection in mice

    Czech Academy of Sciences Publication Activity Database

    Vladimirov, Vladimir; Badalová, Jana; Svobodová, M.; Havelková, Helena; Hart, A. A. M.; Blažková, Hana; Demant, P.; Lipoldová, Marie

    2003-01-01

    Roč. 71, č. 4 (2003), s. 2041-2046 ISSN 0019-9567 R&D Projects: GA ČR GA310/00/0760; GA ČR GA310/03/1381; GA MŠk OK 394 Grant - others:Howard Hughes Medical Institute(US) HHMI55000323; EC(XE) ERBI-C15-CT98-0317; EC(XE) BIO-4-CT98-0445 Institutional research plan: CEZ:AV0Z5052915 Keywords : genetic control * Leishmania major Subject RIV: EC - Immunology Impact factor: 3.875, year: 2003

  3. Immunotherapeutic effects of chitin in comparison with chitosan against Leishmania major infection.

    Science.gov (United States)

    Hoseini, Mostafa Haji Molla; Moradi, Maryam; Alimohammadian, Mohammad Hossein; Shahgoli, Vahid Khaze; Darabi, Hayedeh; Rostami, Ali

    2016-04-01

    Chitin and chitosan microparticles (MPs) are important immune system stimulators. The aim of this study was to evaluate the protective effects of these compounds in comparison with each other against Leishmania infection in BALB/c mice infected with Leishmania major (L. major). Female BALB/c mice were injected subcutaneously with 2×10(5) promastigotes. Chitin and/or chitosan MPs (chitinous MPs on the proliferation and production of cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) were determined. The mean sizes of lesions were significantly smaller in chitin (0.6±0.12 mm) and chitosan treated groups (1.2±0.8 mm) than in the control group (6.2±1.7 mm) (Pchitin and chitosan MPs treatment, respectively). We found that chitinous MPs induced cell proliferation and that chitin but not chitosan increased TNF-α and IL-10 production. Chitin appears that it has more effect than chitosan against leishmaniasis. The current study revealed that chitinous MPs had significant activity against L. major and could be considered as new therapeutic modality in leishmaniasis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  4. Expression of Leishmania major LmSTI1 in Yeast Pichia Pastoris

    Directory of Open Access Journals (Sweden)

    Mehdi Shokri

    2017-01-01

    Full Text Available Background: Leishmania major LmSTI1 is a conserved protein among different species of leishmania, and expressed in both amastigote and promastigote forms of L. major life cycle. It has previously been expressed in bacterial systems.Materials and Methods: To express LmSTI1 in the methylotrophic yeast         Pichia pastoris (P. pastoris, the shuttle vector pPICZA containing gene lmsti1 was constructed under the control of the AOX1 promoter. The recombinant vector was electro-transformed into P. pastoris, and induced by 0.5% methanol in the buffered medium. The expression of the LmSTI1 protein was visualized in the total soluble protein of P. pastoris by 12% SDS-PAGE, and further confirmed by Western blotting with L.major-infected mouse sera and HRP-conjugated goat anti-mouse IgG as the first and secondary antibodies, respectively.Results: The expression level was 0.2% of total soluble proteins.Conclusion: It might be possible to use this formulation as a whole yeast candidate vaccine against cutaneous leishmanization.

  5. Immunization with Leishmania major exogenous antigens protects susceptible BALB/c mice against challenge infection with L. major.

    Science.gov (United States)

    Tonui, Willy K; Mejia, J Santiago; Hochberg, Lisa; Mbow, M Lamine; Ryan, Jeffrey R; Chan, Adeline S T; Martin, Samuel K; Titus, Richard G

    2004-10-01

    The potential of Leishmania major culture-derived soluble exogenous antigens (SEAgs) to induce a protective response in susceptible BALB/c mice challenged with L. major promastigotes was investigated. Groups of BALB/c mice were immunized with L. major SEAgs alone, L. major SEAgs coadministered with either alum (aluminum hydroxide gel) or recombinant murine interleukin-12 (rmIL-12), L. major SEAgs coadministered with both alum and rmIL-12, and L. major SEAgs coadministered with Montanide ISA 720. Importantly and surprisingly, the greatest and most consistent protection against challenge with L. major was seen in mice immunized with L. major SEAgs alone, in the absence of any adjuvant. Mice immunized with L. major SEAgs had significantly smaller lesions that at times contained more than 100-fold fewer parasites. When lymphoid cells from L. major SEAg-immunized mice were stimulated with leishmanial antigen in vitro, they proliferated and secreted a mixed profile of type 1 and type 2 cytokines. Finally, analyses with Western blot analyses and antibodies against three surface-expressed and secreted molecules of L. major (lipophosphoglycan, gp46/M2/PSA-2, and gp63) revealed that two of these molecules are present in L. major SEAgs, lipophosphoglycan and the molecules that associate with it and gp46/M2/PSA-2.

  6. The Transcriptome of Leishmania major Developmental Stages in Their Natural Sand Fly Vector.

    Science.gov (United States)

    Inbar, Ehud; Hughitt, V Keith; Dillon, Laura A L; Ghosh, Kashinath; El-Sayed, Najib M; Sacks, David L

    2017-04-04

    The life cycle of the Leishmania parasite in the sand fly vector involves differentiation into several distinctive forms, each thought to represent an adaptation to specific microenvironments in the midgut of the fly. Based on transcriptome sequencing (RNA-Seq) results, we describe the first high-resolution analysis of the transcriptome dynamics of four distinct stages of Leishmania major as they develop in a natural vector, Phlebotomus duboscqi The early transformation from tissue amastigotes to procyclic promastigotes in the blood-fed midgut was accompanied by the greatest number of differentially expressed genes, including the downregulation of amastins, and upregulation of multiple cell surface proteins, sugar and amino acid transporters, and genes related to glucose metabolism and cell cycle progression. The global changes accompanying post-blood meal differentiation of procyclic promastigotes to the nectomonad and metacyclic stages were less extensive, though each displayed a unique signature. The transcriptome of nectomonads, which has not been studied previously, revealed changes consistent with cell cycle arrest and the upregulation of genes associated with starvation and stress, including autophagic pathways of protein recycling. Maturation to the infective, metacyclic stage was accompanied by changes suggesting preadaptation to the intracellular environment of the mammalian host, demonstrated by the amastigote-like profiles of surface proteins and metabolism-related genes. Finally, a direct comparison between sand fly-derived and culture-derived metacyclics revealed a reassuring similarity between the two forms, with the in vivo forms distinguished mainly by a stronger upregulation of transcripts associated with nutrient stress. IMPORTANCE The life cycle of Leishmania parasites in the sand fly vector includes their growth and development as morphologically distinct forms of extracellular promastigotes found within the different microenvironments of the

  7. The Role of Vitamin D and Vitamin D Receptor in Immunity to Leishmania major Infection

    Directory of Open Access Journals (Sweden)

    James P. Whitcomb

    2012-01-01

    Full Text Available Vitamin D signaling modulates a variety of immune responses. Here, we assessed the role of vitamin D in immunity to experimental leishmaniasis infection in vitamin D receptor-deficient mice (VDRKO. We observed that VDRKO mice on a genetically resistant background have decreased Leishmania major-induced lesion development compared to wild-type (WT mice; additionally, parasite loads in infected dermis were significantly lower at the height of infection. Enzymatic depletion of the active form of vitamin D mimics the ablation of VDR resulting in an increased resistance to L. major. Conversely, VDRKO or vitamin D-deficient mice on the susceptible Th2-biased background had no change in susceptibility. These studies indicate vitamin D deficiency, either through the ablation of VDR or elimination of its ligand, 1,25D3, leads to an increase resistance to L. major infection but only in a host that is predisposed for Th-1 immune responses.

  8. Crystallization and preliminary X-ray analysis of Leishmania major glyoxalase I

    Energy Technology Data Exchange (ETDEWEB)

    Ariza, Antonio; Vickers, Tim J.; Greig, Neil; Fairlamb, Alan H.; Bond, Charles S., E-mail: c.s.bond@dundee.ac.uk [Division of Biological Chemistry and Molecular Microbiology, Wellcome Trust Biocentre, School of Life Sciences, University of Dundee, Dundee DD1 5EH,Scotland (United Kingdom)

    2005-08-01

    The detoxification enzyme glyoxalase I from L. major has been crystallized. Preliminary molecular-replacement calculations indicate the presence of three glyoxalase I dimers in the asymmetric unit. Glyoxalase I (GLO1) is a putative drug target for trypanosomatids, which are pathogenic protozoa that include the causative agents of leishmaniasis. Significant sequence and functional differences between Leishmania major and human GLO1 suggest that it may make a suitable template for rational inhibitor design. L. major GLO1 was crystallized in two forms: the first is extremely disordered and does not diffract, while the second, an orthorhombic form, produces diffraction to 2.0 Å. Molecular-replacement calculations indicate that there are three GLO1 dimers in the asymmetric unit, which take up a helical arrangement with their molecular dyads arranged approximately perpendicular to the c axis. Further analysis of these data are under way.

  9. CD8+ T cells are preferentially activated during primary low dose leishmania major infection but are completely dispensable during secondary anti-Leishmania immunity.

    Science.gov (United States)

    Okwor, Ifeoma B; Jia, Ping; Mou, Zhirong; Onyilagha, Chukwunonso; Uzonna, Jude E

    2014-01-01

    We previously showed that CD8+ T cells are required for optimal primary immunity to low dose Leishmania major infection. However, it is not known whether immunity induced by low dose infection is durable and whether CD8+ T cells contribute to secondary immunity following recovery from low dose infection. Here, we compared primary and secondary immunity to low and high dose L. major infections and assessed the influence of infectious dose on the quality and magnitude of secondary anti-Leishmania immunity. In addition, we investigated the contribution of CD8+ T cells in secondary anti-Leishmania immunity following recovery from low and high dose infections. We found that the early immune response to low and high dose infections were strikingly different: while low dose infection preferentially induced proliferation and effector cytokine production by CD8+ T cells, high dose infection predominantly induced proliferation and cytokine production by CD4+ T cells. This differential activation of CD4+ and CD8+ T cells by high and low dose infections respectively, was imprinted during in vitro and in vivo recall responses in healed mice. Both low and high dose-infected mice displayed strong infection-induced immunity and were protected against secondary L. major challenge. While depletion of CD4+ cells in mice that healed low and high dose infections abolished resistance to secondary challenge, depletion of CD8+ cells had no effect. Collectively, our results show that although CD8+ T cells are preferentially activated and may contribute to optimal primary anti-Leishmania immunity following low dose infection, they are completely dispensable during secondary immunity.

  10. Leishmania major surface protease Gp63 interferes with the function of human monocytes and neutrophils in vitro

    DEFF Research Database (Denmark)

    Sørensen, A L; Hey, A S; Kharazmi, A

    1994-01-01

    In the present study the effect of Leishmania major surface protease Gp63 on the chemotaxis and oxidative burst response of human peripheral blood monocytes and neutrophils was investigated. It was shown that prior incubation of cells with Gp63 inhibited chemotaxis of neutrophils but not monocytes...... by heat inactivation of the protease at 70 degrees C for 15 min. Neither neutrophil nor monocyte chemiluminescence was inhibited by Gp63 when cells were stimulated with PMA. Our data suggest that the major surface protease Gp63 might play an important role in the initial stages of Leishmania...

  11. Dendritic cells in Leishmania major infections: mechanisms of parasite uptake, cell activation and evidence for physiological relevance.

    Science.gov (United States)

    Kautz-Neu, Kordula; Schwonberg, Kirsten; Fischer, Michael R; Schermann, Anja I; von Stebut, Esther

    2012-11-01

    Leishmaniasis is one of the most important infectious diseases worldwide; a vaccine is still not available. Infected dendritic cells (DC) are critical for the initiation of protective Th1 immunity against Leishmania major. Phagocytosis of L. major by DC leads to cell activation, IL-12 release and (cross-) presentation of Leishmania antigens by DC. Here, we review the role of Fcγ receptor- and B cell-mediated processes for parasite internalization by DC. In addition, the early events after parasite inoculation that consist of mast cell activation, parasite uptake by skin-resident macrophages (MΦ), followed by neutrophil and monocyte immigration and DC activation are described. All these events contribute significantly to antigen processing in infected DC and influence resulting T cell priming in vivo. A detailed understanding of the role of DC for the development of efficient anti-Leishmania immunity will aid the development of potent anti-parasite drugs and/or vaccines.

  12. Murine immune response induced by Leishmania major during the implantation of paraffin tablets.

    Science.gov (United States)

    Reis, Maria Letícia Costa; Ferreira, Vanessa Martins; Zhang, Xia; Gonçalves, Ricardo; Vieira, Leda Quércia; Tafuri, Washington Luiz; Mosser, David M; Tafuri, Wagner Luiz

    2010-11-01

    We carried out a model of chronic inflammation using a subcutaneous paraffin tablet in mice experimentally infected with Leishmania major. It was previously reported that the parasite load following paraffin implantation occurred at a peak of 21 days in both BALB/c and C57BL/6 mice. At the present study, we have investigated what cytokines and chemokines are directly related to the parasite load in C57BL/6 mice. All mice were divided in four groups: mice implanted with paraffin tablets; mice experimentally infected with L. major; mice implanted with paraffin tablets and experimentally infected with L. major; and mice submitted only to the surgery were used for the Real-Time Polymerase Chain Reaction (RT-PCR) controls. Fragments of skin tissue and the tissue surrounding the paraffin tablets (inflammatory capsule) were collected for histopathology and RT-PCR studies. By 21 days, a diffuse chronic inflammatory reaction was mainly observed in the deep dermis where macrophages parasitized with Leishmania amastigotes were also found. RT-PCR analysis has shown that BALB/c mice showed strong IL-4 and IL-10 mRNA expression than controls with very little expression of IFN-γ. In contrast, both IFN-γ and IL-10 mRNA was found in higher levels in C57BL/6 animals. Moreover, in C57BL/6 mice the expression of chemokines mRNA of CCL3/MIP-1α was more highly expressed than CCL2/MCP-1. We conclude that the Th1 immune response C57BL/6 did not change to a Th2 response, even though C57BL/6 animals presented higher parasitism than BALB/c mice 21 days after infection and paraffin implantation.

  13. 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.

  14. Description of a novel eukaryotic deoxyuridine 5'-triphosphate nucleotidohydrolase in Leishmania major

    DEFF Research Database (Denmark)

    Camacho, A; Arrebola, R; Pena Diaz, Javier

    1997-01-01

    A Leishmania major full-length cDNA encoding a functional dUTP nucleotidohydrolase (dUTPase; EC 3.6.1.23) was isolated from a cDNA expression library by genetic complementation of dUTPase deficiency in Escherichia coli. The cDNA contained an open reading frame that encoded a protein of 269 amino...... complemented dUTPase deficiency in Escherichia coli. The gene is of single copy; Northern blots indicated a transcript of the same size as the cDNA isolated in the screening procedure. The enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. The availability...

  15. MicroRNA expression profile in human macrophages in response to Leishmania major infection.

    Directory of Open Access Journals (Sweden)

    Julien Lemaire

    Full Text Available Leishmania (L. are intracellular protozoan parasites able to survive and replicate in the hostile phagolysosomal environment of infected macrophages. They cause leishmaniasis, a heterogeneous group of worldwide-distributed affections, representing a paradigm of neglected diseases that are mainly embedded in impoverished populations. To establish successful infection and ensure their own survival, Leishmania have developed sophisticated strategies to subvert the host macrophage responses. Despite a wealth of gained crucial information, these strategies still remain poorly understood. MicroRNAs (miRNAs, an evolutionarily conserved class of endogenous 22-nucleotide non-coding RNAs, are described to participate in the regulation of almost every cellular process investigated so far. They regulate the expression of target genes both at the levels of mRNA stability and translation; changes in their expression have a profound effect on their target transcripts.We report in this study a comprehensive analysis of miRNA expression profiles in L. major-infected human primary macrophages of three healthy donors assessed at different time-points post-infection (three to 24 h. We show that expression of 64 out of 365 analyzed miRNAs was consistently deregulated upon infection with the same trends in all donors. Among these, several are known to be induced by TLR-dependent responses. GO enrichment analysis of experimentally validated miRNA-targeted genes revealed that several pathways and molecular functions were disturbed upon parasite infection. Finally, following parasite infection, miR-210 abundance was enhanced in HIF-1α-dependent manner, though it did not contribute to inhibiting anti-apoptotic pathways through pro-apoptotic caspase-3 regulation.Our data suggest that alteration in miRNA levels likely plays an important role in regulating macrophage functions following L. major infection. These results could contribute to better understanding of the

  16. Separation and mapping of multiple genes that control IgE level in Leishmania major infected mice

    Czech Academy of Sciences Publication Activity Database

    Bádalová, J.; Svobodová, M.; Havelková, Helena; Vladimirov, Vladimir; Vojtíšková, Jarmila; Engová, J.; Pilčík, Tomáš; Volf, P.; Demant, P.; Lipoldová, Marie

    2002-01-01

    Roč. 2002, č. 3 (2002), s. 187-195 ISSN 1466-4879 Institutional research plan: CEZ:AV0Z5052915 Keywords : IgE * quantitative trait * Leishmania major, Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.060, year: 2002

  17. In vitro and in vivo susceptibility of Leishmania major to some medicinal plants

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    Fatemeh Maleki

    2017-01-01

    Conclusions: Our findings indicate that the tested plant extracts are effective against Leishmania major both during in vitro and in vivo experiments, but further researches are required to recommend a potential plant extract as an alternative drug.

  18. Variation in clinical presentation and genotype of causative Leishmania major strain in cutaneous leishmaniasis in north and south Afghanistan

    NARCIS (Netherlands)

    van Thiel, Pieter-Paul A. M.; van Gool, Tom; Faber, William R.; Leenstra, Tjalling; Kager, Piet A.; Bart, Aldert

    2011-01-01

    A different clinical picture and therapeutic response were observed when data from Leishmania major-infected Dutch military personnel stationed in southern (N = 8) and northern (N = 169) Afghanistan were analyzed. Clinical presentation of cutaneous leishmaniasis in personnel in the south was milder

  19. Characterization of the local and systemic immune responses in patients with cutaneous leishmaniasis due to Leishmania major

    DEFF Research Database (Denmark)

    Gaafar, A; Veress, B; Permin, H

    1999-01-01

    In this study skin biopsies and peripheral blood samples were obtained from patients with cutaneous leishmaniasis caused by Leishmania major. Samples were obtained at diagnosis and during healing when the lesions had regressed to half the original size. At diagnosis most of the cells expressed HLA...

  20. Characterization of the local and systemic immune responses in patients with cutaneous leishmaniasis due to Leishmania major

    DEFF Research Database (Denmark)

    Gaafar, A; Veress, B; Permin, H

    1999-01-01

    In this study skin biopsies and peripheral blood samples were obtained from patients with cutaneous leishmaniasis caused by Leishmania major. Samples were obtained at diagnosis and during healing when the lesions had regressed to half the original size. At diagnosis most of the cells expressed HL...

  1. Evaluation of recombinant Leishmania polyprotein plus glucopyranosyl lipid A stable emulsion vaccines against sand fly-transmitted Leishmania major in C57BL/6 mice.

    Science.gov (United States)

    Peters, Nathan C; Bertholet, Sylvie; Lawyer, Phillip G; Charmoy, Melanie; Romano, Audrey; Ribeiro-Gomes, Flavia L; Stamper, Lisa W; Sacks, David L

    2012-11-15

    Numerous experimental Leishmania vaccines have been developed to prevent the visceral and cutaneous forms of Leishmaniasis, which occur after exposure to the bite of an infected sand fly, yet only one is under evaluation in humans. KSAC and L110f, recombinant Leishmania polyproteins delivered in a stable emulsion (SE) with the TLR4 agonists monophosphoryl lipid A or glucopyranosyl lipid A (GLA) have shown protection in animal models. KSAC+GLA-SE protected against cutaneous disease following sand fly transmission of Leishmania major in susceptible BALB/c mice. Similar polyprotein adjuvant combinations are the vaccine candidates most likely to see clinical evaluation. We assessed immunity generated by KSAC or L110f vaccination with GLA-SE following challenge with L. major by needle or infected sand fly bite in resistant C57BL/6 mice. Polyprotein-vaccinated mice had a 60-fold increase in CD4(+)IFN-γ(+) T cell numbers versus control animals at 2 wk post-needle inoculation of L. major, and this correlated with a 100-fold reduction in parasite load. Immunity did not, however, reach levels observed in mice with a healed primary infection. Following challenge by infected sand fly bite, polyprotein-vaccinated animals had comparable parasite loads, greater numbers of neutrophils at the challenge site, and reduced CD4(+)IFN-γ(+)/IL-17(+) ratios versus nonvaccinated controls. In contrast, healed animals had significantly reduced parasite loads and higher CD4(+)IFN-γ(+)/IL-17(+) ratios. These observations demonstrate that vaccine-induced protection against needle challenge does not necessarily translate to protection following challenge by infected sand fly bite.

  2. Cloning and Expression of TRYP6 Gene from Leishmania major (MRHO/IR/75/ER

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    G Eslami

    2008-06-01

    Full Text Available Background: Leishmania, needs to detoxify the macrophage derived potent peroxides (H2O2. Tryparedoxin path­way contains tryparedoxin peroxidase (TSA or TRYP. The aim of the study was to detect the full-length gene se­quence and its encoded protein of the LmTRYP6 gene (EU251502, and comparison the gene sequence with LmTRYP6 (LmjF15.1140, another previously reported member of this gene family.Methods: L.major (MRHO/IR/75/ER promastigotes were cultured, DNA and RNA were extracted and the inter­ested gene was amplified using PCR and RT-PCR methods.  PCR/ RT-PCR fragments were purified and cloned first in pTZ57R/T and then in pET15b expression vector. The expressed protein was verified using western blot method. Char­acterization of the expressed protein was performed bioinformatically.Results: Molecular evaluation revealed that the cloned LmTRYP6 gene (EU251502 encoded a predicted 184 amino acid long protein with a theoretical isoelectric point of 6.1101. Alignment showed a number of changes in amino acid composition including the replacement of highly conserved Trp177 by Cys in LmTRYP6 (ABX26130.Conclusion: So far no study has been done on this group, i.e.  TRYP6 gene, from tryparedoxin peroxidase family. The low homology with LmTRYP6 (LmjF15.1140 and vast array of differences observed in the gene under study (LmTRYP6; EU251502 could open new windows in the field of anti-Leishmania combat. Based on its important role in the viability and successful establishment of the parasite in the host organism it looks to be very good candi­date for vaccine development and any other sort of novel drug development.

  3. The Transcriptome of Leishmania major Developmental Stages in Their Natural Sand Fly Vector

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    Ehud Inbar

    2017-04-01

    Full Text Available The life cycle of the Leishmania parasite in the sand fly vector involves differentiation into several distinctive forms, each thought to represent an adaptation to specific microenvironments in the midgut of the fly. Based on transcriptome sequencing (RNA-Seq results, we describe the first high-resolution analysis of the transcriptome dynamics of four distinct stages of Leishmania major as they develop in a natural vector, Phlebotomus duboscqi. The early transformation from tissue amastigotes to procyclic promastigotes in the blood-fed midgut was accompanied by the greatest number of differentially expressed genes, including the downregulation of amastins, and upregulation of multiple cell surface proteins, sugar and amino acid transporters, and genes related to glucose metabolism and cell cycle progression. The global changes accompanying post-blood meal differentiation of procyclic promastigotes to the nectomonad and metacyclic stages were less extensive, though each displayed a unique signature. The transcriptome of nectomonads, which has not been studied previously, revealed changes consistent with cell cycle arrest and the upregulation of genes associated with starvation and stress, including autophagic pathways of protein recycling. Maturation to the infective, metacyclic stage was accompanied by changes suggesting preadaptation to the intracellular environment of the mammalian host, demonstrated by the amastigote-like profiles of surface proteins and metabolism-related genes. Finally, a direct comparison between sand fly-derived and culture-derived metacyclics revealed a reassuring similarity between the two forms, with the in vivo forms distinguished mainly by a stronger upregulation of transcripts associated with nutrient stress.

  4. First molecular detection of Leishmania major within naturally infected Phlebotomus salehi from a zoonotic cutaneous leishmaniasis focus in southern Iran.

    Science.gov (United States)

    Azizi, K; Fakoorziba, M R; Jalali, M; Moemenbellah-Fard, M D

    2012-03-01

    Human cutaneous leishmaniasis (CL) is a major notifiable public health problem in many parts of Iran. It is often caused by the zooflagellate parasite Leishmania major which is mainly transmitted by the bites of female phlebotomine sandflies belonging to the genus Phlebotomus (Diptera: Psychodidae). The annual incidence of CL in Fars province, southern Iran, was about 108-144 in 2007. The leishmanial infections of wild sandflies that may act as vectors were thus investigated at an endemic focus in this province. In all 330 female Phlebotomus sandflies were screened for the detection of Leishmania-specific kinetoplast DNA (kDNA) by polymerase chain reaction (PCR) methods. A two stage nested PCR protocol was used to establish the identity of Leishmania major species in naturally infected sandflies. The L. major kDNA was detected in 18 (5.5%) individual sandflies which belonged to four different Phlebotomus species (Phlebotomus papatasi, Phlebotomus salehi, Phlebotomus sergenti and P. major group). For the first time, one naturally infected P. salehi specimen contained the kDNA of the protozoan parasite, L. major, with a main band of 560 base pairs identified using the nested PCR method. It seems most likely therefore that P. salehi is potentially a rare sylvatic vector of L. major parasites in parts of this province. This is the first combined morphological and molecular studies of P. salehi in Iran.

  5. The major surface glycoprotein (gp63) from Leishmania major and Leishmania donovani cleaves CD4 molecules on human T cells

    DEFF Research Database (Denmark)

    Hey, A S; Theander, T G; Hviid, L

    1994-01-01

    Abs to human T cells, whereas the binding of one Ab, OKT4, was not inhibited. Heat inactivation of the protease before the incubation with cells abolished the effect on binding of anti-CD4 Abs. Cells incubated for 2 h with the protease and subsequently washed free of the protease showed a gradual re...... in interfering with the induction of the immune response and thus disease progression in Leishmania infections....

  6. Protective vaccination with promastigote surface antigen 2 from Leishmania major is mediated by a TH1 type of immune response.

    OpenAIRE

    Handman, E; Symons, F M; Baldwin, T M; Curtis, J M; Scheerlinck, J P

    1995-01-01

    Leishmania major promastigote surface antigen-2 complex (PSA-2) comprises a family of three similar but distinct polypeptides. The three PSA-2 polypeptides were purified from cultured promastigotes by a combination of detergent phase separation and monoclonal antibody affinity chromatography. Intraperitoneal vaccination of C3H/He mice with PSA-2 with Corynebacterium parvum as an adjuvant resulted in complete protection from lesion development after challenge infection with virulent L. major. ...

  7. Vaccination with the divergent portion of the protein histone H2B of Leishmania protects susceptible BALB/c mice against a virulent challenge with Leishmania major.

    Science.gov (United States)

    Chenik, M; Louzir, H; Ksontini, H; Dilou, A; Abdmouleh, I; Dellagi, K

    2006-03-24

    To identify approaches for vaccination against leishmaniasis, we analyzed the protective effect of different constructions using recombinant peptides from the protein Leishmania (L.) major histone H2B. H2B sequence displays two distinct regions: an amino-terminal region divergent from mammalian H2B (27% identity) and a carboxy-terminal region highly conserved with mammalian H2B (55% identity). We tested the ability of the entire H2B protein, its divergent or conserved regions to provide protection against virulent L. major challenge. While the recombinant H2B protein adjuved with CpG induces potent cellular and antibody responses when injected to BALB/c mice, only the divergent amino-terminal region of H2B is able to confer potent protection against a virulent challenge. These findings indicate that different portions of the same parasite protein may express contrasting protective effects likely through the induction of different effector mechanisms. Due to its potent protective properties in the BALB/c mouse model, the amino-terminal region of Leishmania H2B could constitute a good vaccine candidate.

  8. Immunization against Leishmania major infection using LACK- and IL-12-expressing Lactococcus lactis induces delay in footpad swelling.

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    Felix Hugentobler

    Full Text Available BACKGROUND: Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines. METHODOLOGY/PRINCIPAL FINDINGS: We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional T(H1 CD4(+ and CD8(+ T cells and a systemic LACK-specific T(H1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific T(H1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective T(H1 response. CONCLUSIONS/SIGNIFICANCE: This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania.

  9. Immunization against Leishmania major Infection Using LACK- and IL-12-Expressing Lactococcus lactis Induces Delay in Footpad Swelling

    Science.gov (United States)

    Hugentobler, Felix; Yam, Karen K.; Gillard, Joshua; Mahbuba, Raya; Olivier, Martin; Cousineau, Benoit

    2012-01-01

    Background Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines. Methodology/Principal findings We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional TH1 CD4+ and CD8+ T cells and a systemic LACK-specific TH1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific TH1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective TH1 response. Conclusions/Significance This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania. PMID:22348031

  10. Crystallization and preliminary X-ray diffraction analysis of Leishmania major dihydroorotate dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Cordeiro, Artur T.; Feliciano, Patricia R.; Nonato, M. Cristina, E-mail: cristy@fcfrp.usp.br [Laboratório de Cristalografia de Proteínas, Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto-USP, Ribeirão Preto, SP 14040-903 (Brazil)

    2006-10-01

    Dihydroorotate dehydrogenase from L. major has been crystallized by the vapour-diffusion technique using lithium sulfate as the precipitant agent. A complete data set from a native crystal has been collected to 2.0 Å resolution using an in-house rotating-anode generator. Dihydroorotate dehydrogenases (DHODHs) are flavin-containing enzymes that catalyze the oxidation of l-dihydroorotate to orotate, the fourth step in the de novo pyrimidine nucleotide synthesis pathway. In this study, DHODH from Leishmania major has been crystallized by the vapour-diffusion technique using lithium sulfate as the precipitating agent. The crystals belong to space group P6{sub 1}, with unit-cell parameters a = 143.7, c = 69.8 Å. X-ray diffraction data were collected to 2.0 Å resolution using an in-house rotating-anode generator. Analysis of the solvent content and the self-rotation function indicate the presence of two molecules in the asymmetric unit. The structure has been solved by the molecular-replacement technique.

  11. In vitro antileishmanial activity of methanolic and aqueous extracts of Eucalyptus camaldulensis against Leishmania major.

    Science.gov (United States)

    Nosratabadi, Seyyed Jafar; Sharifi, Iraj; Sharififar, Fariba; Bamorovat, Mehdi; Daneshvar, Hamid; Mirzaie, Mohammad

    2015-03-01

    Pentavalent antimony compounds are expensive, toxic and drug resistance is prevalent, whereas the plant extract derivatives are safe. In the present study, the effect of methanolic and aqueous extracts of Eucalyptus camaldulensis on the promastigotes of Leishmania major was evaluated. The methanolic and aqueous extracts of E. camaldulensis leaves were prepared. The compounds were dried and powdered. Serial dilutions of the extracts and control drugs in phosphate buffer solution were prepared. The stationary phase promastigotes of L. major were incubated to the methanolic and aqueous extractions in vitro. Tartar emetic was used as the positive control drug. After 72 h of incubation the activity of the extracts was measured, using MTT method. The IC50 values (50 % inhibitory concentration) were 586.2 ± 47.6 and 1,108.6 ± 51.9 μg/ml for methanolic and aqueous extracts, respectively, whereas it was 32.5 ± 6.8 μg/ml for tartar emetic. The results indicated that the methanolic extract was more effective than aqueous extract, although there was no significant difference. The extracts were less effective as compared to the control drug. Further investigation is required to evaluate these extracts on clinical stage in macrophage-amastigote model.

  12. The PSA-2 glycoprotein complex of Leishmania major is a glycosylphosphatidylinositol-linked promastigote surface antigen.

    Science.gov (United States)

    Murray, P J; Spithill, T W; Handman, E

    1989-12-15

    Polyclonal rabbit antiserum to the Triton X-114 phase material of Leishmania major, which comprises the surface and internal integral membrane proteins of the parasite, was used to screen a lambda gt11 genomic expression library. A recombinant clone producing a Mr 123,000 beta-galactosidase fusion protein was isolated. Antibodies affinity-purified on this fusion protein recognized a complex of three surface-oriented proteins of promastigotes of L. major of Mr 94,000, 90,000, and 80,000 that we have termed the promastigote surface Ag 2 (PSA-2) complex. The DNA sequence of the insert in this clone predicted the 3' end of an open reading frame encoding a hydrophobic C-terminus. The inferred C-terminal sequence was suggestive of a glycosylphosphatidyl-inositol membrane anchoring mechanism. Phosphatidylinositol-specific phospholipase C treatment of the native PSA-2 proteins caused a shift in their electrophoretic mobility with an apparent reduction in the molecular weight of the PSA-2 complex. After phospholipase C treatment these proteins also displayed the cryptic cross-reacting determinant recognized by antibodies to the Trypanosoma brucei variant surface Ag. Moreover, PSA-2, which previously partitioned in the detergent phase after Triton X-114 phase separation, became water-soluble after phospholipase C treatment. Immunoprecipitation of the PSA-2 proteins with sera directed to lectin-binding proteins indicated that these polypeptides may be differentially glycosylated. Finally, these PSA-2 proteins were recognized by sera from some patients with cutaneous leishmaniasis.

  13. Control of cutaneous leishmaniasis caused by Leishmania major in south-eastern Morocco.

    Science.gov (United States)

    Bennis, Issam; De Brouwere, Vincent; Ameur, Btissam; El Idrissi Laamrani, Abderrahmane; Chichaoui, Smaine; Hamid, Sahibi; Boelaert, Marleen

    2015-10-01

    The incidence of cutaneous leishmaniasis (CL) caused by Leishmania major has increased in Morocco over the last decade, prompting the Ministry of Health to take intersectoral response measures including vector and reservoir control. The aim of this article was to describe the CL outbreak response measures taken in the province of Errachidia, where the reservoir of L. major, a sand rat (Meriones shawi), was targeted using strychnine-poisoned wheat baits from 2010 to 2012. We analysed routine surveillance data and other information using the data of the CL control programme. We present data on the evolution and the extension of CL in this province as well as the epidemiological profile of the disease. Between 2004 and 2013, 7099 cases of CL were recorded in Errachidia Province, gradually affecting all districts. Our results demonstrate that more women were affected than men and that all age groups were represented. Errachidia Province was the epicentre of the recent CL outbreak in Morocco. A notable decline in incidence rates was observed after 2011. The outbreak control measures may have contributed to this decline, as well as climatic trends or progressing herd immunity. © 2015 John Wiley & Sons Ltd.

  14. Mapping of a Leishmania major gene/locus that confers pentamidine resistance by deletion and insertion of transposable element

    Directory of Open Access Journals (Sweden)

    Coelho Adriano C.

    2004-01-01

    Full Text Available Pentamidine (PEN is an alternative compound to treat antimony-resistant leishmaniasis patients, which cellular target remains unclear. One approach to the identification of prospective targets is to identify genes able to mediate PEN resistance following overexpression. Starting from a genomic library of transfected parasites bearing a multicopy episomal cosmid vector containing wild-type Leishmania major DNA, we isolated one locus capable to render PEN resistance to wild type cells after DNA transfection. In order to map this Leishmania locus, cosmid insert was deleted by two successive sets of partial digestion with restriction enzymes, followed by transfection into wild type cells, overexpression, induction and functional tests in the presence of PEN. To determine the Leishmania gene related to PEN resistance, nucleotide sequencing experiments were done through insertion of the transposon Mariner element of Drosophila melanogaster (mosK into the deleted insert to work as primer island. Using general molecular techniques, we described here this method that permits a quickly identification of a functional gene facilitating nucleotide sequence experiments from large DNA fragments. Followed experiments revealed the presence of a P-Glycoprotein gene in this locus which role in Leishmania metabolism has now been analyzed.

  15. Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major.

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    Juliana Ide Aoki

    2016-09-01

    Full Text Available Tubercidin (TUB is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this drug, we employed in this study an overexpression/selection approach using TUB for the identification of potential targets, as well as, drug resistance genes in L. major. Although, TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis.After transfection of a cosmid genomic library into L. major Friedlin (LmjF parasites and application of the overexpression/selection method, we identified two cosmids (cosTUB1 and cosTU2 containing two different loci capable of conferring significant levels of TUB resistance. In the cosTUB1 contained a gene encoding NUPM1-like protein, which has been previously described as associated with TUB resistance in L. amazonensis. In the cosTUB2 we identified and characterized a gene encoding a 63 kDa protein that we denoted as tubercidin-resistance protein (TRP. Functional analysis revealed that the transfectants were less susceptible to TUB than LmjF parasites or those transfected with the control vector. In addition, the trp mRNA and protein levels in cosTUB2 transfectants were higher than LmjF. TRP immunolocalization revealed that it was co-localized to the endoplasmic reticulum (ER, a cellular compartment with many functions. In silico predictions indicated that TRP contains only a hypothetical transmembrane domain. Thus, it is likely that TRP is a lumen protein involved in multidrug efflux transport that may be involved in the purine metabolic pathway.This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how TRP mediates TUB resistance and whether purine

  16. Antigenic profile of heat-killed versus thimerosal-treated Leishmania major using sodium dodecyl sulfate-polyacrylamide gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Reza Arjmand

    2015-01-01

    Full Text Available Background: Leishmania is a parasitic protozoan of trypanosomatidae family which causes a wide spectrum of diseases ranging from self-healing cutaneous lesions to deadly visceral forms. In endemic areas, field trials of different preparations of Leishmania total antigen were tested as leishmaniasis vaccine. Two preparations of killed Leishmania major were produced In Iran, which were heat-killed vaccine called autoclaved L. major (ALM and thimerosal-treated freeze-thawed vaccine called killed L. major (KLM. In this study, the protein content of both ALM and KLM were compared with that of freshly harvested intact L. major promastigotes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE. Materials and Methods: L. major (MRHO/IR/75/ER from pre-infected Balb/c mice was isolated with modified Novy-MacNeal-Nicolle (NNN medium and then subcultured in liquid RPMI 1640 medium supplemented with fetal calf serum (FCS 20% for mass production. Two preparations of KLM and ALM were produced by Razi Vaccine and Serum Research Institute, Iran, under WHO/TDR supervision. Electrophoresis was performed by SDS-PAGE method and the gel was stained by Coomassie brilliant blue dye. The resultant unit bands were compared using standard molecular proteins. Results: Electrophoresis of the two preparations produced many bands from 10 kDa to 100 kDa. KLM bands were much like those of freshly harvested intact L. major. Conclusion: It is concluded that although there are similar bands in the three forms of Leishmania antigens, there are some variations which might be considered for identification and purification of protective immunogens in a total crude antigen, and detection of their stability is essential for the production and marketing of a putative vaccine.

  17. In Vitro Infectivity of Leishmania major Isolated from Patients with Different Clinical Forms of Cutaneous Leishmaniasis and Its Association with Parasite Zymodems

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    B Sarkari

    2009-07-01

    Full Text Available "nBackground: The aim of this study was to characterize the Leishmania parasites isolated from cuta­neous leishmaniasis (CL patients in Fars Province in Iran and to compare the potential infectivity of the isolates in macrophage cell line. Moreover, attempt was made to find out the association between parasite infectivity and their zymodems. "nMethods: Twenty samples were taken from the skin lesion of CL patients. The samples were cultured in biphasic media followed by mass cultivation in RPMI medium. Each isolate was tested for the ac­tivity of the 5 enzymes including glucose phosphate isomerase (GPI, malate dehydrogenase (MDH, nucleoside hydrolase 1& 2 (NH1 & NH2, and phosphoglucomutase (PGM.   The enzymatic profiles of the isolates were compared with WHO reference strains. Specific PCR (primers: LIN17 & LIN R4 and RAPD-PCR were used as complementary methods for characterization of the isolates. "nResults:  Isoenzyme electrophoresis showed that all of the isolates were L. major. PCR with LIN17 and LIN R4 and RAPD-PCR with AB-07 primers further determined the isolates as L. major. Results of macrophage infectivity experiment, using J774 cell line, showed that the most virulent isolates were related to Z1 with 63% macrophage infectivity rate. A well correlation was found between the infec­tivity rate of the isolates and type of ulcer. Those isolates with high infectivity rate were involved in more severe, ulcerative or erythmatose lesions in CL patients. "nConclusion: The most invasive isolates might be a good candidate for immunological studies and for vaccine development.

  18. B-1 cells modulate the murine macrophage response to Leishmania major infection.

    Science.gov (United States)

    Arcanjo, Angelica F; Nunes, Marise P; Silva-Junior, Elias B; Leandro, Monique; da Rocha, Juliana Dutra Barbosa; Morrot, Alexandre; Decote-Ricardo, Debora; Freire-de-Lima, Celio Geraldo

    2017-05-26

    To investigate the modulatory effect of B-1 cells on murine peritoneal macrophages infected with Leishmania major ( L. major ) in vitro . Peritoneal macrophages obtained from BALB/c and BALB/c XID mice were infected with L. major and cultured in the presence or absence of B-1 cells obtained from wild-type BALB/c mice. Intracellular amastigotes were counted, and interleukin-10 (IL-10) production was quantified in the cellular supernatants using an enzyme-linked immunosorbent assay. The levels of the lipid mediator prostaglandin E2 (PGE 2 ) were determined using a PGE 2 enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI), and the number of lipid bodies was quantified in the cytoplasm of infected macrophages in the presence and absence of B-1 cells. Culturing the cells with selective PGE 2 -neutralizing drugs inhibited PGE 2 production and confirmed the role of this lipid mediator in IL-10 production. In contrast, we demonstrated that B-1 cells derived from IL-10 KO mice did not favor the intracellular growth of L. major . We report that B-1 cells promote the growth of L. major amastigotes inside peritoneal murine macrophages. We demonstrated that the modulatory effect was independent of physical contact between the cells, suggesting that soluble factor(s) were released into the cultures. We demonstrated in our co-culture system that B-1 cells trigger IL-10 production by L. major -infected macrophages. Furthermore, the increased secretion of IL-10 was attributed to the presence of the lipid mediator PGE 2 in supernatants of L. major -infected macrophages. The presence of B-1 cells also favors the production of lipid bodies by infected macrophages. In contrast, we failed to obtain the same effect on parasite replication inside L. major -infected macrophages when the B-1 cells were isolated from IL-10 knockout mice. Our results show that elevated levels of PGE 2 and IL-10 produced by B-1 cells increase L. major growth, as indicated by the number of parasites in cell

  19. Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major.

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    Rafael Ricci-Azevedo

    2016-04-01

    Full Text Available ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1 immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS, form neutrophil extracellular traps (NETs and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF and interleukin-1beta (IL-1β release; otherwise, transforming growth factor-beta (TGF-β production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be

  20. Conditions Influencing the Efficacy of Vaccination with Live Organisms against Leishmania major Infection

    Science.gov (United States)

    Tabbara, Khaled S.; Peters, Nathan C.; Afrin, Farhat; Mendez, Susana; Bertholet, Sylvie; Belkaid, Yasmine; Sacks, David L.

    2005-01-01

    Numerous experimental vaccines have been developed with the goal of generating long-term cell-mediated immunity to the obligate intracellular parasite Leishmania major, yet inoculation with live, wild-type L. major remains the only successful vaccine in humans. We examined the expression of immunity at the site of secondary, low-dose challenge in the ear dermis to determine the kinetics of parasite clearance and the early events associated with the protection conferred by vaccination with live L. major organisms in C57BL/6 mice. Particular attention was given to the route of vaccination. We observed that the rapidity, strength, and durability of the memory response following subcutaneous vaccination with live parasites in the footpad are even greater than previously appreciated. Antigen-specific gamma interferon (IFN-γ)-producing T cells infiltrate the secondary site by 1.5 weeks, and viable parasites are cleared as early as 2.5 weeks following rechallenge, followed by a rapid drop in IFN-γ+ CD4+ cell numbers in the site. In comparison, intradermal vaccination with live parasites in the ear generates immunity that is delayed in effector cell recruitment to the rechallenge site and in the clearance of parasites from the site. This compromised immunity was associated with a rapid recruitment of interleukin-10 (IL-10)-producing CD4+ T cells to the rechallenge site. Treatment with anti-IL-10-receptor or anti-CD25 antibody enhanced early parasite clearance in ear-vaccinated mice, indicating that chronic infection in the skin generates a population of regulatory cells capable of influencing the level of resistance to reinfection. A delicate balance of effector and regulatory T cells may be required to optimize the potency and durability of vaccines against Leishmaniasis and other intracellular pathogens. PMID:16040984

  1. Comparative genomics of Tunisian Leishmania major isolates causing human cutaneous leishmaniasis with contrasting clinical severity.

    Science.gov (United States)

    Ghouila, Amel; Guerfali, Fatma Z; Atri, Chiraz; Bali, Aymen; Attia, Hanene; Sghaier, Rabiaa M; Mkannez, Ghada; Dickens, Nicholas J; Laouini, Dhafer

    2017-06-01

    Zoonotic cutaneous leishmaniasis caused by Leishmania (L.) major parasites affects urban and suburban areas in the center and south of Tunisia where the disease is endemo-epidemic. Several cases were reported in human patients for which infection due to L. major induced lesions with a broad range of severity. However, very little is known about the mechanisms underlying this diversity. Our hypothesis is that parasite genomic variability could, in addition to the host immunological background, contribute to the intra-species clinical variability observed in patients and explain the lesion size differences observed in the experimental model. Based on several epidemiological, in vivo and in vitro experiments, we focused on two clinical isolates showing contrasted severity in patients and BALB/c experimental mice model. We used DNA-seq as a high-throughput technology to facilitate the identification of genetic variants with discriminating potential between both isolates. Our results demonstrate that various levels of heterogeneity could be found between both L. major isolates in terms of chromosome or gene copy number variation (CNV), and that the intra-species divergence could surprisingly be related to single nucleotide polymorphisms (SNPs) and Insertion/Deletion (InDels) events. Interestingly, we particularly focused here on genes affected by both types of variants and correlated them with the observed gene CNV. Whether these differences are sufficient to explain the severity in patients is obviously still open to debate, but we do believe that additional layers of -omic information is needed to complement the genomic screen in order to draw a more complete map of severity determinants. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Effect of distal His mutation on the peroxynitrite reactivity of Leishmania major peroxidase.

    Science.gov (United States)

    Saha, Rina; Bose, Moumita; Santara, Sumit Sen; Roy, Jayasree; Yadav, Rajesh K; Adak, Subrata

    2013-10-01

    The conserved distal histidine in peroxidases has been considered to play a major role as a general acid-base catalyst for heterolytic cleavage of an OO bond in H2O2. However, heme peroxidases react with peroxynitrite to form transient intermediates but the role of the distal histidine in this reaction is still unknown. In order to investigate catalytic roles of the histidine at the distal cavity, two Leishmania major peroxidase (LmP) mutants (H68E, H68V) were prepared. The rate of transition from ferric H68V to Compound ES by H2O2 is decreased by approximately five orders of magnitude relative to wild type, which is consistent with electron donor oxidation data where the H68V is ~1000 fold less active than wild type. In the reaction with peroxynitrite, the formation rate of intermediates in the mutants is not significantly lower than that for the wild type, indicating that the His68 has no major role in homolytic cleavage of an OO bond in peroxynitrite. EPR spectroscopic data suggest that the transient intermediates formed by the reaction of LmP with H2O2 exhibits an intense and stable signal similar to CCP Compound ES whereas in case of the reaction with peroxynitrite, this signal disappears, indicating that the transient intermediate is Compound II. Rapid kinetics data suggest that the distal His68 mutants display higher decay rates of Compound II than wild type. Thus, His68 mutations minimize Compound II formation (inactive species in peroxynitrite scavenging cycles) by increasing decay rates during the steady state and results in higher peroxynitrite degrading activity. © 2013.

  3. Resolution of an Infection with Leishmania braziliensis Confers Complete Protection to a Subsequent Challenge with Leishmania major in BALB/c Mice

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    Lima Hermenio C

    1999-01-01

    Full Text Available Both Leishmania major and L. braziliensis induce cutaneous leishmaniasis in BALB/c mice. Whereas BALB/c mice die of infection with L. major, they cure an infection with L. braziliensis. We report here that after curing an infection with L. braziliensis, BALB/c mice are resistant to challenge with L. major. When challenged with L. major, L. braziliensis pre-treated BALB/c mice mounted a delayed-type hypersensitivity response to L. major and produced high amounts of interferon-g (IFN-g but low amounts of interleukin-4. The IFN-g produced by the L. braziliensis pre-infected mice was involved in the protection seen against L. major challenge since treating the mice with a neutralizing anti-IFN-g abrogated the protection. This suggests that cross-reactive antigen epitopes exist between L. braziliensis and L. major and that pre-infection with L. braziliensis primes BALB/c mice to epitopes on L. major that can elicit a protective Th1 response to the parasite.

  4. 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.

  5. Investigation and Comparison of Leishmania major Promastigote and Amastigote Protein Content by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    S. Soleimanifard

    2013-04-01

    Full Text Available ntroduction & Objective: Leishmania is a protozoan of the trypanosomatidae family. This pro-tozoan has two stages in its life cycle, promastigote form in sand flies and amastigote form in macrophage of mammalian hosts. The purpose of this study was identification and compari-son of proteins of Leishmania amastigote and promastigote stages. Materials & Methods: The present study is a cross sectional study of two forms of Leishmania major. To culture promastigotes , L.major (MRHO/IR/75/ER from previously infected Balb/c mice was transferred to modified N.N.N medium with overlay of liquid BHI and then transferred to RPMI-1640 at 26oc ± 1 for mass production. After isolation and growth, pro-mastigotes were transferred to liquid cell culture medium RPMI-1640 with pH 5.5 and incu-bated at 5% CO2 at 37oc for 72 hours until promastigote to amastigote transformation. Elec-trophoresis was performed with SDS-PAGE method to find and compare the molecular weight of the antigens of two stages. Results: The molecular weights of the bands observed in both forms were as follows: 19, 36, 50, 63, 65, 80, 90, 94, 96, 110- 130 KDa. The proteins in the surface of only promastigote were 22, 28 and 46 KDa and special proteins in the surface of amastigote were 12 and 32 KDa. Conclusion : According to this study Leishmania parasite has stage specific proteins. Various studies have shown that axenic amastigotes and tissue amastigotes are similar in their protein content. Therefore, based on stage specific proteins ,effective drugs and vaccines can be de-signed against leishmaniasis. (Sci J Hamadan Univ Med Sci 2013; 20 (1:1-8

  6. Sheep blood-LB agar base medium (SLM) as a simple and suitable medium for the cultivation of Leishmania major promastigotes.

    Science.gov (United States)

    Nasiri, Vahid

    2013-11-01

    Several methods of cultivation of Leishmania parasites are available, and among them, biphasic media like Nicolle-Novy-MacNeal medium are considered to be superior for the initial isolation of the parasites and their in vivo infectivity. In the present work, the efficacy of sheep blood-LB agar base medium (SLM) was evaluated for the cultivation of Leishmania major promastigotes. The SLM supports the proliferation of the parasites and can be used for routine isolation and cultivation of Leishmania parasites with acceptable in vivo infectivity for research purposes.

  7. Cinnamic Acid Bornyl Ester Derivatives from Valeriana wallichii Exhibit Antileishmanial In Vivo Activity in Leishmania major-Infected BALB/c Mice.

    Science.gov (United States)

    Masic, Anita; Valencia Hernandez, Ana Maria; Hazra, Sudipta; Glaser, Jan; Holzgrabe, Ulrike; Hazra, Banasri; Schurigt, Uta

    2015-01-01

    Human leishmaniasis covers a broad spectrum of clinical manifestations ranging from self-healing cutaneous leishmaniasis to severe and lethal visceral leishmaniasis caused among other species by Leishmania major or Leishmania donovani, respectively. Some drug candidates are in clinical trials to substitute current therapies, which are facing emerging drug-resistance accompanied with serious side effects. Here, two cinnamic acid bornyl ester derivatives (1 and 2) were assessed for their antileishmanial activity. Good selectivity and antileishmanial activity of bornyl 3-phenylpropanoate (2) in vitro prompted the antileishmanial assessment in vivo. For this purpose, BALB/c mice were infected with Leishmania major promastigotes and treated with three doses of 50 mg/kg/day of compound 2. The treatment prevented the characteristic swelling at the site of infection and correlated with reduced parasite burden. Transmitted light microscopy and transmission electron microscopy of Leishmania major promastigotes revealed that compounds 1 and 2 induce mitochondrial swelling. Subsequent studies on Leishmania major promastigotes showed the loss of mitochondrial transmembrane potential (ΔΨm) as a putative mode of action. As the cinnamic acid bornyl ester derivatives 1 and 2 had exhibited antileishmanial activity in vitro, and compound 2 in Leishmania major-infected BALB/c mice in vivo, they can be regarded as possible lead structures for the development of new antileishmanial therapeutic approaches.

  8. Diverse modes of binding in structures of Leishmania majorN-myristoyltransferase with selective inhibitors

    Directory of Open Access Journals (Sweden)

    James A. Brannigan

    2014-07-01

    Full Text Available The leishmaniases are a spectrum of global diseases of poverty associated with immune dysfunction and are the cause of high morbidity. Despite the long history of these diseases, no effective vaccine is available and the currently used drugs are variously compromised by moderate efficacy, complex side effects and the emergence of resistance. It is therefore widely accepted that new therapies are needed. N-Myristoyltransferase (NMT has been validated pre-clinically as a target for the treatment of fungal and parasitic infections. In a previously reported high-throughput screening program, a number of hit compounds with activity against NMT from Leishmania donovani have been identified. Here, high-resolution crystal structures of representative compounds from four hit series in ternary complexes with myristoyl-CoA and NMT from the closely related L. major are reported. The structures reveal that the inhibitors associate with the peptide-binding groove at a site adjacent to the bound myristoyl-CoA and the catalytic α-carboxylate of Leu421. Each inhibitor makes extensive apolar contacts as well as a small number of polar contacts with the protein. Remarkably, the compounds exploit different features of the peptide-binding groove and collectively occupy a substantial volume of this pocket, suggesting that there is potential for the design of chimaeric inhibitors with significantly enhanced binding. Despite the high conservation of the active sites of the parasite and human NMTs, the inhibitors act selectively over the host enzyme. The role of conformational flexibility in the side chain of Tyr217 in conferring selectivity is discussed.

  9. Lower galactosylation levels of the Lipophosphoglycan from Leishmania (Leishmania) major-like strains affect interaction with Phlebotomus papatasi and Lutzomyia longipalpis.

    Science.gov (United States)

    Guimarães, Agna Cristina; Nogueira, Paula Monalisa; Silva, Soraia de Oliveira; Sadlova, Jovana; Pruzinova, Katerina; Hlavacova, Jana; Melo, Maria Norma; Soares, Rodrigo Pedro

    2018-01-01

    BACKGROUND Leishmania major is an Old World species causing cutaneous leishmaniasis and is transmitted by Phlebotomus papatasi and Phlebotomus duboscqi. In Brazil, two isolates from patients who never left the country were characterised as L. major-like (BH49 and BH121). Using molecular techniques, these isolates were indistinguishable from the L. major reference strain (FV1). OBJECTIVES We evaluated the lipophosphoglycans (LPGs) of the strains and their behaviour in Old and New World sand fly vectors. METHODS LPGs were purified, and repeat units were qualitatively evaluated by immunoblotting. Experimental in vivo infection with L. major-like strains was performed in Lutzomyia longipalpis (New World, permissive vector) and Ph. papatasi (Old World, restrictive or specific vector). FINDINGS The LPGs of both strains were devoid of arabinosylated side chains, whereas the LPG of strain BH49 was more galactosylated than that of strain BH121. All strains with different levels of galactosylation in their LPGs were able to infect both vectors, exhibiting colonisation of the stomodeal valve and metacyclogenesis. The BH121 strain (less galactosylated) exhibited lower infection intensity compared to BH49 and FV1 in both vectors. MAIN CONCLUSIONS Intraspecific variation in the LPG of L. major-like strains occur, and the different galactosylation levels affected interactions with the invertebrate host.

  10. The in vitro antileishmanial activity of essential oil from Aloysia gratissima and guaiol, its major sesquiterpene against Leishmania amazonensis.

    Science.gov (United States)

    Garcia, Maria Carolina Freitas; Soares, Deivid Costa; Santana, Raissa Couto; Saraiva, Elvira Maria; Siani, Antonio Carlos; Ramos, Mônica Freiman S; Danelli, Maria das Graças Miranda; Souto-Padron, Thaïs Cristina; Pinto-da-Silva, Lucia H

    2018-01-21

    Leishmaniases is a tropical disease caused by protozoa of the genus Leishmania for which the current treatment is expensive, besides increasing reports of parasite resistance. This study investigated the anti-Leishmania amazonensis activity of the essential oil from Aloysia gratissima (AgEO) and guaiol, the major sesquiterpene constituent in the oil. Our results showed that AgEO killed promastigotes and intracellular amastigotes at an IC50 of 25 and 0·16 µg mL-1, respectively, while guaiol killed amastigotes at an IC50 of 0·01 µg mL-1. Both AgEO and guaiol were safe for macrophages up to 100 µg mL-1, as evaluated by the dehydrogenase activity, membrane integrity and phagocytic capacity. AgEO and guaiol did not induce nitrite oxide (NO) in resting macrophages and inhibited the production of NO in lipopolysaccharide-stimulated macrophages. The ultrastructural analysis suggested that AgEO and guaiol act directly on parasites, affecting promastigotes kinetoplast, mitochondrial matrix and plasma membrane. Together, these results pointed out that AgEO and guaiol could be promising candidates to develop anti-Leishmania drugs.

  11. Antigen-presenting cells in human cutaneous leishmaniasis due to Leishmania major

    DEFF Research Database (Denmark)

    ElHassan, A M; Gaafar, A; Theander, T G

    1995-01-01

    keratinocytes and endothelial cells also showed these characteristics, they may also act as APC. By examining tissue samples from skin lesions and draining lymph nodes it was possible to follow the probable route of trafficking of various inflammatory cells between the skin lesion and lymph nodes. Leishmania...

  12. Differential Impact of LPG-and PG-Deficient Leishmania major Mutants on the Immune Response of Human Dendritic Cells.

    Directory of Open Access Journals (Sweden)

    Michelle A Favila

    2015-12-01

    Full Text Available Leishmania major infection induces robust interleukin-12 (IL12 production in human dendritic cells (hDC, ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of Leishmania parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG and other phosphoglycan-containing molecules (PGs, making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS responsible for IL12 induction.Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating L. major Friedlin V1 mutants defective in LPG alone, (FV1 lpg1-, or generally deficient for all PGs, (FV1 lpg2-. Infection with metacyclic, infective stage, L. major or purified LPG induced high levels of IL12B subunit gene transcripts in hDCs, which was abrogated with FV1 lpg1- infections. In contrast, hDC infections with FV1 lpg2- displayed increased IL12B expression, suggesting other PG-related/LPG2 dependent molecules may act to dampen the immune response. Global transcriptional profiling comparing WT, FV1 lpg1-, FV1 lpg2- infections revealed that FV1 lpg1- mutants entered hDCs in a silent fashion as indicated by repression of gene expression. Transcription factor binding site analysis suggests that LPG recognition by hDCs induces IL-12 in a signaling cascade resulting in Nuclear Factor κ B (NFκB and Interferon Regulatory Factor (IRF mediated transcription.These data suggest that L. major LPG is a major PAMP recognized by hDC to induce IL12-mediated protective immunity and that there is a complex interplay between PG-baring Leishmania surface glycoconjugates that result in modulation of host cellular IL12.

  13. Th1-like human T-cell clones recognizing Leishmania gp63 inhibit Leishmania major in human macrophages

    DEFF Research Database (Denmark)

    Kemp, M; Hey, A S; Bendtzen, K

    1994-01-01

    resembling Th1 cells. Autologous mononuclear cells and Epstein-Barr virus-transformed B cell lines were equally efficient in presenting the antigen to the T cells. The gp63 reactive T cells induced resistance to infection in cultured human macrophages by L. major. The data confirm that human CD4+ T cells...

  14. The molecular karyotype of Leishmania major and mapping of alpha and beta tubulin gene families to multiple unlinked chromosomal loci.

    OpenAIRE

    Spithill, T W; Samaras, N

    1985-01-01

    The arrangement of tubulin genes in the genome of the protozoan parasite Leishmania major was studied by genomic Southern blot analysis and mapping of genes to chromosomes fractionated by pulsed field gradient gel (PFG) electrophoresis. alpha-tubulin genes exist as a tandem array of 2.4 kb PstI fragments. beta-tubulin genes are found as a tandem array of 3.9 kb AvaI or PvuI fragments, but additional genes are also found on other genomic DNA fragments. Chromosome-sized DNA molecules released f...

  15. Sense and antisense transcripts in the histone H1 (HIS-1) locus of Leishmania major.

    Science.gov (United States)

    Belli, Sabina I; Monnerat, Séverine; Schaff, Cédric; Masina, Slavica; Noll, Tanja; Myler, Peter J; Stuart, Kenneth; Fasel, Nicolas

    2003-08-01

    Histone H1 in the parasitic protozoan Leishmania is a developmentally regulated protein encoded by two genes, HIS-1.1 and HIS-1.2. These genes are separated by approximately 20 kb of sequence and are located on the same DNA strand of chromosome 27. When Northern blots of parasite RNA were probed with HIS-1 strand-specific riboprobes, we detected sense and antisense transcripts that were polyadenylated and developmentally regulated. When the HIS-1.2 coding region was replaced with the coding region of the neomycin phosphotransferase gene, antisense transcription of this gene was unaffected, indicating that the regulatory elements controlling antisense transcription were located outside of the HIS-1.2 gene, and that transcription in Leishmania can occur from both DNA strands even in the presence of transcription of a selectable marker in the complementary strand. A search for other antisense transcripts within the HIS-1 locus identified an additional transcript (SC-1) within the intervening HIS-1 sequence, downstream of adenine and thymine-rich sequences. These results show that gene expression in Leishmania is not only regulated polycistronically from the sense strand of genomic DNA, but that the complementary strand of DNA also contains sequences that could drive expression of open reading frames from the antisense strand of DNA. These findings suggest that the parasite has evolved in such a way as to maximise the transcription of its genome, a mechanism that might be important for it to maintain virulence.

  16. Immunization against leishmaniasis by PLGA nanospheres loaded with an experimental autoclaved Leishmania major (ALM) and Quillaja saponins.

    Science.gov (United States)

    Tafaghodi, M; Eskandari, M; Kharazizadeh, M; Khamesipour, A; Jaafari, M R

    2010-12-01

    Immune responses against the Leishmania antigens are not sufficient to protect against a leishmania challenge. Therefore these antigens need to be potentiated by various adjuvants and delivery systems. In this study, Poly (d,l-lactide-co-glycolide (PLGA) nanospheres as antigen delivery system and Quillaja saponins (QS) as immunoadjuvant have been used to enhance the immune response against autoclaved Leishmania major (ALM). PLGA nanospheres were prepared by a double-emulsion (W/O/W) technique. Particulate characteristics were studied by scanning electron microscopy and particle size analysis. Mean diameter for nanospheres loaded with ALM+QS was 294 ± 106 nm. BALB/c mice were immunized three times in 3-weeks intervals using ALM plus QS loaded nanospheres [(ALM+QS)PLGA], ALM encapsulated with PLGA nanospheres [(ALM)PLGA], (ALM)PLGA + QS, ALM + QS, ALM alone or PBS. The intensity of infection induced by L. major challenge was assessed by measuring size of footpad swelling. The strongest protection, showed by significantly (P < 0.05) smaller footpad, were observed in mice immunized with (ALM)PLGA. The (ALM+QS)PLGA group showed the least protection and highest swelling, while the (ALM)PLGA+QS, ALM+QS and ALM showed an intermediate protection with no significant difference. The mice immunized with ALM and ALM+QS showed the highest IgG2a/IgG1 ratio (P < 0.01), followed by (ALM)PLGA+QS. The highest IFN-γ and lowest IL-4 production was seen in (ALM)PLGA+QS, ALM+QS groups. The highest parasite burden was observed in (ALM)PLGA+QS and (ALM+QS)PLGA groups. It is concluded that PLGA nanospheres as a vaccine delivery system could increase the protective immune responses, but QS adjuvant has a reverse effect on protective immune responses and the least protective responses were seen in the presence of this adjuvant.

  17. DNA vaccination with a plasmid encoding LACK-TSA fusion against Leishmania major infection in BALB/c mice.

    Science.gov (United States)

    Maspi, N; Ghaffarifar, F; Sharifi, Z; Dalimi, A; Khademi, S Z

    2017-12-01

    Vaccination would be the most important strategy for the prevention and elimination of leishmaniasis. The aim of the present study was to compare the immune responses induced following DNA vaccination with LACK (Leishmania analogue of the receptor kinase C), TSA (Thiol-specific-antioxidant) genes alone or LACK-TSA fusion against cutaneous leishmaniasis (CL). Cellular and humoral immune responses were evaluated before and after challenge with Leishmania major (L. major). In addition, the mean lesion size was also measured from 3th week post-infection. All immunized mice showed a partial immunity characterized by higher interferon (IFN)-γ and Immunoglobulin G (IgG2a) levels compared to control groups (pLACK-TSA fusion. Mean lesion sizes reduced significantly in all immunized mice compared with control groups at 7th week post-infection (pLACK-TSA and TSA groups than LACK group after challenge (pLACK and TSA antigens against CL. Furthermore, this study demonstrated that a bivalent vaccine can induce stronger immune responses and protection against infectious challenge with L. major.

  18. Leishmaniasis in Turkey: first clinical isolation of Leishmania major from 18 autochthonous cases of cutaneous leishmaniasis in four geographical regions.

    Science.gov (United States)

    Özbilgin, Ahmet; Çulha, Gülnaz; Uzun, Soner; Harman, Mehmet; Topal, Suhan Günaştı; Okudan, Fulya; Zeyrek, Fadile; Gündüz, Cumhur; Östan, İpek; Karakuş, Mehmet; Töz, Seray; Kurt, Özgür; Akyar, Işın; Erat, Ayşegül; Güngör, Dilek; Kayabaşı, Çağla; Çavuş, İbrahim; Bastien, Patrick; Pratlong, Francine; Kocagöz, Tanıl; Özbel, Yusuf

    2016-06-01

    To report isolation of Leishmania major strains obtained from 18 Turkish autochthonous cutaneous leishmaniasis (CL) patients infected with L. major between 2011 and 2014. Initial diagnosis relied on microscopy and culture in enriched medium, prepared by adding specific amounts of liver extract, protein and lipid sources to NNN medium. Promastigotes were then transferred to RPMI medium including 10% of foetal calf serum for mass culture. Species-specific real-time PCR targeting ITS1 region of Leishmania spp. was performed using both lesion aspiration samples and cultured promastigotes. Two of 18 isolates were identified by isoenzyme analysis in the Leishmaniasis Reference Center in Montpellier, France. Each isolate was inoculated into the footpads of six mice to observe the pathogenicity of L. major. Developing lesions were observed, and the thickening of footpads was measured weekly. Melting curve analyses of 18 isolates showed a peak concordant with L. major, and two of them were confirmed by isoenzyme analyses as L. major zymodeme MON103. In the mouse model, acute lesions seen on day 21 were accepted as an indication of heavy infection. Severe impairments were observed on all mouse footpads over 3 weeks, which even progressed to extremity amputation. Cutaneous leishmaniasis-causing L. major was recently identified in Adana province in southern Turkey, with PCR. Our study shows that such CL cases are not limited to Adana but currently present from western to Southeastern Anatolia, and along the Mediterranean coast. The role of small mammals, the main reservoirs of L. major in Anatolia, needs to be elucidated, as do the underlying factors that cause severe clinical manifestations in L. major infections in Turkey, contrary to the infections in neighbouring countries. © 2016 John Wiley & Sons Ltd.

  19. Leishmania mexicana can utilize amino acids as major carbon sources in macrophages but not in animal models.

    Science.gov (United States)

    Saunders, Eleanor C; Naderer, Thomas; Chambers, Jenny; Landfear, Scott M; McConville, Malcolm J

    2018-04-01

    Leishmania parasites target macrophages in their mammalian hosts and proliferate within the mature phagolysosome compartment of these cells. Intracellular amastigote stages are dependent on sugars as a major carbon source in vivo, but retain the capacity to utilize other carbon sources. To investigate whether amastigotes can switch to using other carbon sources, we have screened for suppressor strains of the L. mexicana Δlmxgt1-3 mutant which lacks the major glucose transporters LmxGT1-3. We identified a novel suppressor line (Δlmxgt1-3 s2 ) that has restored growth in rich culture medium and virulence in ex vivo infected macrophages, but failed to induce lesions in mice. Δlmxgt1-3 s2 amastigotes had lower rates of glucose utilization than the parental line and primarily catabolized non-essential amino acids. The increased mitochondrial metabolism of this line was associated with elevated levels of intracellular reactive oxygen species, as well as increased sensitivity to inhibitors of the tricarboxylic acid (TCA) cycle, including nitric oxide. These results suggest that hardwired sugar addiction of Leishmania amastigotes contributes to the intrinsic resistance of this stage to macrophage microbicidal processes in vivo, and that these stages have limited capacity to switch to using other carbon sources. © 2018 John Wiley & Sons Ltd.

  20. KSAC, a defined Leishmania antigen, plus adjuvant protects against the virulence of L. major transmitted by its natural vector Phlebotomus duboscqi.

    Directory of Open Access Journals (Sweden)

    Regis Gomes

    Full Text Available Recombinant KSAC and L110f are promising Leishmania vaccine candidates. Both antigens formulated in stable emulsions (SE with the natural TLR4 agonist MPL® and L110f with the synthetic TLR4 agonist GLA in SE protected BALB/c mice against L. major infection following needle challenge. Considering the virulence of vector-transmitted Leishmania infections, we vaccinated BALB/c mice with either KSAC+GLA-SE or L110f+GLA-SE to assess protection against L. major transmitted via its vector Phlebotomus duboscqi.Mice receiving the KSAC or L110f vaccines were challenged by needle or L. major-infected sand flies. Weekly disease progression and terminal parasite loads were determined. Immunological responses to KSAC, L110f, or soluble Leishmania antigen (SLA were assessed throughout vaccination, three and twelve weeks after immunization, and one week post-challenge.Following sand fly challenge, KSAC-vaccinated mice were protected while L110f-vaccinated animals showed partial protection. Protection correlated with the ability of SLA to induce IFN-γ-producing CD4(+CD62L(lowCCR7(low effector memory T cells pre- and post-sand fly challenge.This study demonstrates the protective efficacy of KSAC+GLA-SE against sand fly challenge; the importance of vector-transmitted challenge in evaluating vaccine candidates against Leishmania infection; and the necessity of a rapid potent Th1 response against Leishmania to attain true protection.

  1. Infection parameters in the sand fly vector that predict transmission of Leishmania major.

    Science.gov (United States)

    Stamper, Lisa W; Patrick, Rachel L; Fay, Michael P; Lawyer, Phillip G; Elnaiem, Dia-Eldin A; Secundino, Nagila; Debrabant, Alain; Sacks, David L; Peters, Nathan C

    2011-08-01

    To identify parameters of Leishmania infection within a population of infected sand flies that reliably predict subsequent transmission to the mammalian host, we sampled groups of infected flies and compared infection intensity and degree of metacyclogenesis with the frequency of transmission. The percentage of parasites within the midgut that were metacyclic promastigotes had the highest correlation with the frequency of transmission. Meta-analysis of multiple transmission experiments allowed us to establish a percent-metacyclic "cutoff" value that predicted transmission competence. Sand fly infections initiated with variable doses of parasites resulted in correspondingly altered percentages of metacyclic promastigotes, resulting in altered transmission frequency and disease severity. Lastly, alteration of sand fly oviposition status and environmental conditions at the time of transmission also influenced transmission frequency. These observations have implications for transmission of Leishmania by the sand fly vector in both the laboratory and in nature, including how the number of organisms acquired by the sand fly from an infection reservoir may influence the clinical outcome of infection following transmission by bite.

  2. Effect of IL-22 on DNA vaccine encoding LACK gene of Leishmania major in BALB/c mice.

    Science.gov (United States)

    Hezarjaribi, Hajar Ziaee; Ghaffarifar, Fatemeh; Dalimi, Abdolhosein; Sharifi, Zohreh; Jorjani, Ogholniaz

    2013-07-01

    In the present study, the effect of IL-22 together with the plasmid encoding LACK (Leishmania homolog of receptors for activated C-kinase) gene of Leishmania major on the trend of leishmaniasis in BALB/c mice was evaluated. Evaluation of the cellular and humoral immunity was performed by measurement of IL-4 and IFN-γ, culture of splenocytes and MTT assay, and measurement of total IgG, IgG1, and IgG2a in the control and immunized groups. Clinical evaluations were also carried out by measurement of the lesion size, survival rate, and body weight of mice. Comparison of the mean size of lesions in the LACK and LACK+IL-22 groups demonstrated that the mean size of lesions of the two groups was significantly different from week four (pLACK gene), and pcLACK+IL-22 groups were 20%, 40%, 60%, and 80%, respectively. According to the results of IFN-γ, IL-4, total IgG, IgG1, and IgG2a measurement and the MTT assay, IL-22 obviously caused an increase in IFN-γ production and a decrease in IL-4 production before and after the challenge (p<0.05). The results showed the effectiveness of IL-22 in DNA vaccine. It showed that IL-22 brought about Th1 cytokine responses and high survival rate of mice. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. In vitro effect of essential oils and isolated mono- and sesquiterpenes on Leishmania major and Trypanosoma brucei.

    Science.gov (United States)

    Mikus, J; Harkenthal, M; Steverding, D; Reichling, J

    2000-05-01

    The effect of different essential oils as well as of isolated mono- and sesquiterpenes on the viability of bloodstream forms of Trypanosoma brucei, promastigotes of Leishmania major and human HL-60 cells was evaluated using the Almar Blue assay. Of the 12 essential oils and 8 terpenes investigated, only three essential oils, Melissa officinalis (balmmint) oil, Thymus vulgaris (thyme) oil, and Melaleuca alternifolia (tea tree) oil were about 50-fold and 80-fold more toxic to bloodstream forms of T. brucei than to HL-60 cells, respectively. Terpinen-4-ol, the main compound of the Australian tea tree oil, was even 1000-fold more toxic to trypanosomes than to the human cells. On the other hand, none of the essential oils and terpenes tested were more toxic to promastigotes of L. major than to HL-60 cells.

  4. Sporotrichoid cutaneous leishmaniasis due to Leishmania major of different zymodemes in the Sudan and Saudi Arabia: a comparative study

    DEFF Research Database (Denmark)

    Gaafar, A; Fadl, A; el Kadaro, A Y

    1994-01-01

    Sporotrichoid cutaneous leishmaniasis is due to dissemination of amastigotes via the lymphatics to the subcutaneous tissues. A comparison was made between the potential to disseminate by this route of 2 parasites of different zymodemes in Sudan and Saudi Arabia. In Sudan cutaneous leishmaniasis...... is caused by Leishmania major zymodeme LON-1, and in Saudi Arabia by L. major LON-4. Sporotrichoid leishmaniasis was significantly more common in Sudan, occurring in 23% of patients compared with 10% in Saudi Arabia. Lymph node involvement was slightly more prevalent in the Sudan. Clinical and pathological...... differences between subcutaneous nodules, particularly when they ulcerate, and multiple primary cutaneous lesions are described and treatment of localized and sporotrichoid leishmaniasis is discussed. The pathological features of the primary lesions in the Sudan and Saudi Arabia were similar....

  5. Sporotrichoid cutaneous leishmaniasis due to Leishmania major of different zymodemes in the Sudan and Saudi Arabia: a comparative study

    DEFF Research Database (Denmark)

    Gaafar, A; Fadl, A; el Kadaro, A Y

    1994-01-01

    Sporotrichoid cutaneous leishmaniasis is due to dissemination of amastigotes via the lymphatics to the subcutaneous tissues. A comparison was made between the potential to disseminate by this route of 2 parasites of different zymodemes in Sudan and Saudi Arabia. In Sudan cutaneous leishmaniasis...... differences between subcutaneous nodules, particularly when they ulcerate, and multiple primary cutaneous lesions are described and treatment of localized and sporotrichoid leishmaniasis is discussed. The pathological features of the primary lesions in the Sudan and Saudi Arabia were similar....... is caused by Leishmania major zymodeme LON-1, and in Saudi Arabia by L. major LON-4. Sporotrichoid leishmaniasis was significantly more common in Sudan, occurring in 23% of patients compared with 10% in Saudi Arabia. Lymph node involvement was slightly more prevalent in the Sudan. Clinical and pathological...

  6. Transitory or long-lasting immunity to Leishmania major infection: the result of immunogenicity and multicomponent properties of histone DNA vaccines.

    Science.gov (United States)

    Carrión, Javier; Folgueira, Cristina; Alonso, Carlos

    2008-02-26

    The present studies were designed to analyze the immunization against cutaneous leishmaniosis with plasmids encoding Leishmania histones either individually or genetically linked in tandem, or with cocktails encoding the four nucleosomal histones (H2A, H2B, H3 and H4). Genetic immunization of BALB/c mice with the individual histones only resulted in a delay in lesion development, whereas the immunization with any one of the plasmids encoding a pair of histones provided stronger, though still partial protection against Leishmania major infection compared to the combination of the four histones. These results provide direct evidence that all four nucleosomal histones of Leishmania are necessary to maintain complete protection against L. major reinfection.

  7. Cathepsin B in antigen-presenting cells controls mediators of the Th1 immune response during Leishmania major infection.

    Directory of Open Access Journals (Sweden)

    Iris J Gonzalez-Leal

    2014-09-01

    Full Text Available Resistance and susceptibility to Leishmania major infection in the murine model is determined by the capacity of the host to mount either a protective Th1 response or a Th2 response associated with disease progression. Previous reports involving the use of cysteine cathepsin inhibitors indicated that cathepsins B (Ctsb and L (Ctsl play important roles in Th1/Th2 polarization during L. major infection in both susceptible and resistant mouse strains. Although it was hypothesized that these effects are a consequence of differential patterns of antigen processing, the mechanisms underlying these differences were not further investigated. Given the pivotal roles that dendritic cells and macrophages play during Leishmania infection, we generated bone-marrow derived dendritic cells (BMDC and macrophages (BMM from Ctsb-/- and Ctsl-/- mice, and studied the effects of Ctsb and Ctsl deficiency on the survival of L. major in infected cells. Furthermore, the signals used by dendritic cells to instruct Th cell polarization were addressed: the expression of MHC class II and co-stimulatory molecules, and cytokine production. We found that Ctsb-/- BMDC express higher levels of MHC class II molecules than wild-type (WT and Ctsl-/- BMDC, while there were no significant differences in the expression of co-stimulatory molecules between cathepsin-deficient and WT cells. Moreover, both BMDC and BMM from Ctsb-/- mice significantly up-regulated the levels of interleukin 12 (IL-12 expression, a key Th1-inducing cytokine. These findings indicate that Ctsb-/- BMDC display more pro-Th1 properties than their WT and Ctsl-/- counterparts, and therefore suggest that Ctsb down-regulates the Th1 response to L. major. Moreover, they propose a novel role for Ctsb as a regulator of cytokine expression.

  8. The mating competence of geographically diverse Leishmania major strains in their natural and unnatural sand fly vectors.

    Science.gov (United States)

    Inbar, Ehud; Akopyants, Natalia S; Charmoy, Melanie; Romano, Audrey; Lawyer, Phillip; Elnaiem, Dia-Eldin A; Kauffmann, Florence; Barhoumi, Mourad; Grigg, Michael; Owens, Katherine; Fay, Michael; Dobson, Deborah E; Shaik, Jahangheer; Beverley, Stephen M; Sacks, David

    2013-01-01

    Invertebrate stages of Leishmania are capable of genetic exchange during their extracellular growth and development in the sand fly vector. Here we explore two variables: the ability of diverse L. major strains from across its natural range to undergo mating in pairwise tests; and the timing of the appearance of hybrids and their developmental stage associations within both natural (Phlebotomus duboscqi) and unnatural (Lutzomyia longipalpis) sand fly vectors. Following co-infection of flies with parental lines bearing independent drug markers, doubly-drug resistant hybrid progeny were selected, from which 96 clonal lines were analyzed for DNA content and genotyped for parent alleles at 4-6 unlinked nuclear loci as well as the maxicircle DNA. As seen previously, the majority of hybrids showed '2n' DNA contents, but with a significant number of '3n' and one '4n' offspring. In the natural vector, 97% of the nuclear loci showed both parental alleles; however, 3% (4/150) showed only one parental allele. In the unnatural vector, the frequency of uniparental inheritance rose to 10% (27/275). We attribute this to loss of heterozygosity after mating, most likely arising from aneuploidy which is both common and temporally variable in Leishmania. As seen previously, only uniparental inheritance of maxicircle kDNA was observed. Hybrids were recovered at similar efficiencies in all pairwise crosses tested, suggesting that L. major lacks detectable 'mating types' that limit free genetic exchange. In the natural vector, comparisons of the timing of hybrid formation with the presence of developmental stages suggest nectomonads as the most likely sexually competent stage, with hybrids emerging well before the first appearance of metacyclic promastigotes. These studies provide an important perspective on the prevalence of genetic exchange in natural populations of L. major and a guide for experimental studies to understand the biology of mating.

  9. Protective vaccination with promastigote surface antigen 2 from Leishmania major is mediated by a TH1 type of immune response.

    Science.gov (United States)

    Handman, E; Symons, F M; Baldwin, T M; Curtis, J M; Scheerlinck, J P

    1995-11-01

    Leishmania major promastigote surface antigen-2 complex (PSA-2) comprises a family of three similar but distinct polypeptides. The three PSA-2 polypeptides were purified from cultured promastigotes by a combination of detergent phase separation and monoclonal antibody affinity chromatography. Intraperitoneal vaccination of C3H/He mice with PSA-2 with Corynebacterium parvum as an adjuvant resulted in complete protection from lesion development after challenge infection with virulent L. major. Significant protection was also obtained in the genetically susceptible BALB/cH-2k and BALB/c mice. One of the PSA-2 genes was cloned and expressed in both Escherichia coli and Leishmania mexicana promastigotes. Vaccination with the recombinant PSA-2 purified from E. coli did not confer protection, in contrast to the L. mexicana-derived recombinant PSA-2, which provided excellent protection. CD4+ T cells isolated from the spleens of vaccinated mice produced large amounts of gamma interferon but no detectable interleukin 4 upon stimulation with PSA-2 in vitro. Limiting dilution analysis showed a marked increase in the precursor frequency of PSA-2-specific gamma interferon-secreting CD4+ T cells. No substantial change in precursor frequency was observed for interleukin 4-secreting T cells in the vaccinated mice. A CD4+ PSA-2 specific T-cell line generated from splenocytes of a vaccinated mouse produces a cytokine pattern consistent with a TH1 phenotype. Intravenous injection of this line into naive mice reduced significantly the parasite burden upon challenge infection. Taken together, the data suggest that vaccination with PSA-2 induces a TH1 type of immune response which protects mice from L. major infection. Moreover, a single recombinant PSA-2 polypeptide derived from a genomic clone can also vaccinate, provided that the structural form of the antigen is near native.

  10. 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 develo...

  11. Crystallization and preliminary X-ray diffraction analysis of selenophosphate synthetases from Trypanosoma brucei and Leishmania major.

    Science.gov (United States)

    Faim, Lívia Maria; Rosa e Silva, Ivan; Bertacine Dias, Marcio Vinicius; D'Muniz Pereira, Humberto; Brandao-Neto, José; Alves da Silva, Marco Túlio; Thiemann, Otavio Henrique

    2013-08-01

    Selenophosphate synthetase (SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the activation of selenide with adenosine 5'-triphosphate (ATP) to generate selenophosphate, the essential selenium donor for selenocysteine synthesis. Recombinant full-length Leishmania major SPS (LmSPS2) was recalcitrant to crystallization. Therefore, a limited proteolysis technique was used and a stable N-terminal truncated construct (ΔN-LmSPS2) yielded suitable crystals. The Trypanosoma brucei SPS orthologue (TbSPS2) was crystallized by the microbatch method using paraffin oil. X-ray diffraction data were collected to resolutions of 1.9 Å for ΔN-LmSPS2 and 3.4 Å for TbSPS2.

  12. Immunization against leishmaniasis by PLGA nanospheres encapsulated with autoclaved Leishmania major (ALM) and CpG-ODN.

    Science.gov (United States)

    Tafaghodi, Mohsen; Khamesipour, Ali; Jaafari, Mahmoud R

    2011-05-01

    Various adjuvants and delivery systems have been evaluated for increasing the protective immune responses against leishmaniasis and mostly have been shown not to be effective enough. In this study, poly(D,L-lactide-co-glycolide) (PLGA) nanospheres as an antigen delivery system and CpG-ODN as an immunoadjuvant have been used for the first time to enhance the immune response against autoclaved Leishmania major (ALM). PLGA nanospheres were prepared by a double-emulsion (W/O/W) technique. Particulate characteristics were studied by scanning electron microscopy and particle size analysis. Mean diameter of ALM + CpG-ODN-loaded nanospheres was 300 ± 128 nm. BALB/c mice were immunized three times in 3-week intervals using ALM plus CpG-ODN-loaded nanospheres [(ALM + CpG-ODN)(PLGA)], ALM encapsulated PLGA nanospheres [(ALM)(PLGA)], (ALM)(PLGA) + CpG, ALM + CpG, ALM alone, or phosphate buffer solution (PBS). The intensity of infection induced by L. major challenge was assessed by measuring size of footpad swelling. The strongest protection, showed by significantly (P<0.05) smaller footpad, was observed in mice immunized with (ALM + CpG-ODN)(PLGA). The (ALM)(PLGA), (ALM)(PLGA) + CpG, and ALM + CpG were also showed a significantly (P<0.05) smaller footpad swelling compared to the groups received either PBS or ALM alone. The mice immunized with (ALM + CpG-ODN)(PLGA), (ALM)(PLGA) + CpG, and ALM + CpG showed the highest IgG2a/IgG1 ratio, interferon-γ production, and lowest interleukin-4 production compared to the other groups. It is concluded that when both PLGA nanospheres and CpG-ODN adjuvants were used simultaneously, it induce stronger immune response and enhance protection rate against Leishmania infection.

  13. Monoterpenic aldehydes as potential anti-Leishmania agents: activity of Cymbopogon citratus and citral on L. infantum, L. tropica and L. major.

    Science.gov (United States)

    Machado, M; Pires, P; Dinis, A M; Santos-Rosa, M; Alves, V; Salgueiro, L; Cavaleiro, C; Sousa, M C

    2012-03-01

    In order to contribute for the search of new drugs for leishmaniasis, we study the susceptibility of Leishmania infantum, Leishmania tropica and Leishmania major to Cymbopogon citratus essential oil and major compounds, mrycene and citral. C. citratus and citral were the most active inhibiting L. infantum, L. tropica and L. major growth at IC(50) concentrations ranging from 25 to 52 μg/ml and from 34 to 42 μg/ml, respectively. L. infantum promastigotes exposed to essential oil and citral underwent considerable ultrastructural alterations, namely mitochondrial and kinetoplast swelling, autophagosomal structures, disruption of nuclear membrane and nuclear chromatin condensation. C. citratus essential oil and citral promoted the leishmanicidal effect by triggering a programmed cell death. In fact, the leishmanicidal activity was mediated via apoptosis as evidenced by externalization of phosphatidylserine, loss of mitochondrial membrane potential, and cell-cycle arrest at the G(0)/G(1) phase. Taken together, ours findings lead us to propose that citral was responsible for anti-Leishmania activity of the C. citratus and both may represent a valuable source for therapeutic control of leishmaniasis. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. Crystallization and preliminary crystallographic characterization of LmACR2, an arsenate/antimonate reductase from Leishmania major

    Energy Technology Data Exchange (ETDEWEB)

    Bisacchi, Davide [Bioinformatics and Structural Proteomics, IST-National Cancer Research Institute, Genova (Italy); Zhou, Yao; Rosen, Barry P.; Mukhopadhyay, Rita [Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan (United States); Bordo, Domenico, E-mail: domenico.bordo@istge.it [Bioinformatics and Structural Proteomics, IST-National Cancer Research Institute, Genova (Italy)

    2006-10-01

    LmACR2 from L. major is the first rhodanese-like enzyme directly involved in the reduction of arsenate and antimonate to be crystallized. Diffraction data have been collected to 1.99 Å resolution using synchrotron X-rays. Arsenic is present in the biosphere owing either to the presence of pesticides and herbicides used in agricultural and industrial activities or to leaching from geological formations. The health effects of prolonged exposure to arsenic can be devastating and may lead to various forms of cancer. Antimony(V), which is chemically very similar to arsenic, is used instead in the treatment of leishmaniasis, an infection caused by the protozoan parasite Leishmania sp.; the reduction of pentavalent antimony contained in the drug Pentostam to the active trivalent form arises from the presence in the Leishmania genome of a gene, LmACR2, coding for the protein LmACR2 (14.5 kDa, 127 amino acids) that displays weak but significant sequence similarity to the catalytic domain of Cdc25 phosphatase and to rhodanese enzymes. For structural characterization, LmACR2 was overexpressed, purified to homogeneity and crystallized in a trigonal space group (P321 or P3{sub 1}21/P3{sub 2}21). The protein crystallized in two distinct trigonal crystal forms, with unit-cell parameters a = b = 111.0, c = 86.1 Å and a = b = 111.0, c = 175.6 Å, respectively. At a synchrotron beamline, the diffraction pattern extended to a resolution limit of 1.99 Å.

  15. The Contribution of the Fas/FasL Apoptotic Pathway in Ulcer Formation during Leishmania major-Induced Cutaneous Leishmaniasis

    Science.gov (United States)

    Eidsmo, Liv; Nylen, Susanne; Khamesipour, Ali; Hedblad, Mari-Anne; Chiodi, Francesca; Akuffo, Hannah

    2005-01-01

    Cutaneous leishmaniasis (CL), caused by the intracellular protozoan Leishmania major, is characterized by lesion formation and ulceration at the site of infection. The mechanism of ulcer formation during CL is not fully understood. The expression of Fas and FasL and the levels of apoptosis in skin biopsies and in restimulated blood mononuclear cells from patients with 1 to 7 months of L. major-induced CL were analyzed using immunohistochemistry and fluorescence-activated cell sorting analysis. The levels of soluble Fas and FasL were also analyzed by enzyme-linked immunosorbent assay. A substantial number of apoptotic keratinocytes were observed mainly in the superficial epidermis of morphologically active and healing CL skin samples. Fas expression was increased on epidermis in active CL, whereas Fas expression was similar in healing and healthy epidermis. FasL-expressing macrophages and T cells were found in subepidermal infiltrate, mainly in active disease. When CL peripheral blood mononuclear cells were restimulated with L. major, Fas was up-regulated on effector T cells, and high levels of sFasL were secreted. Supernatants from restimulated cultures induced apoptosis in human keratinocytes (HaCaT), possibly through Fas/FasL interactions. Our results indicate that FasL-expressing effector T cells and macrophages may act to induce apoptosis and ulcer formation in Fas-expressing keratinocytes during L. major infection. PMID:15793290

  16. Dectin-1 Positive Dendritic Cells Expand after Infection with Leishmania major Parasites and Represent Promising Targets for Vaccine Development

    Directory of Open Access Journals (Sweden)

    Nicole Zimara

    2018-02-01

    Full Text Available Resistant mouse strains mount a protective T cell-mediated immune response upon infection with Leishmania (L. parasites. Healing correlates with a T helper (Th cell-type 1 response characterized by a pronounced IFN-γ production, while susceptibility is associated with an IL-4-dependent Th2-type response. It has been shown that dermal dendritic cells are crucial for inducing protective Th1-mediated immunity. Additionally, there is growing evidence that C-type lectin receptor (CLR-mediated signaling is involved in directing adaptive immunity against pathogens. However, little is known about the function of the CLR Dectin-1 in modulating Th1- or Th2-type immune responses by DC subsets in leishmaniasis. We characterized the expression of Dectin-1 on CD11c+ DCs in peripheral blood, at the site of infection, and skin-draining lymph nodes of L. major-infected C57BL/6 and BALB/c mice and in peripheral blood of patients suffering from cutaneous leishmaniasis (CL. Both mouse strains responded with an expansion of Dectin-1+ DCs within the analyzed tissues. In accordance with the experimental model, Dectin-1+ DCs expanded as well in the peripheral blood of CL patients. To study the role of Dectin-1+ DCs in adaptive immunity against L. major, we analyzed the T cell stimulating potential of bone marrow-derived dendritic cells (BMDCs in the presence of the Dectin-1 agonist Curdlan. These experiments revealed that Curdlan induces the maturation of BMDCs and the expansion of Leishmania-specific CD4+ T cells. Based on these findings, we evaluated the impact of Curdlan/Dectin-1 interactions in experimental leishmaniasis and were able to demonstrate that the presence of Curdlan at the site of infection modulates the course of disease in BALB/c mice: wild-type BALB/c mice treated intradermally with Curdlan developed a protective immune response against L. major whereas Dectin-1−/− BALB/c mice still developed the fatal course of disease after Curdlan treatment

  17. Antigen Requirements for Efficient Priming of CD8+ T Cells by Leishmania major-Infected Dendritic Cells

    Science.gov (United States)

    Bertholet, Sylvie; Debrabant, Alain; Afrin, Farhat; Caler, Elisabeth; Mendez, Susana; Tabbara, Khaled S.; Belkaid, Yasmine; Sacks, David L.

    2005-01-01

    CD4+ and CD8+ T-cell responses have been shown to be critical for the development and maintenance of acquired resistance to infections with the protozoan parasite Leishmania major. Monitoring the development of immunodominant or clonally restricted T-cell subsets in response to infection has been difficult, however, due to the paucity of known epitopes. We have analyzed the potential of L. major transgenic parasites, expressing the model antigen ovalbumin (OVA), to be presented by antigen-presenting cells to OVA-specific OT-II CD4+ or OT-I CD8+ T cells. Truncated OVA was expressed in L. major as part of a secreted or nonsecreted chimeric protein with L. donovani 3′ nucleotidase (NT-OVA). Dendritic cells (DC) but not macrophages infected with L. major that secreted NT-OVA could prime OT-I T cells to proliferate and release gamma interferon. A diminished T-cell response was observed when DC were infected with parasites expressing nonsecreted NT-OVA or with heat-killed parasites. Inoculation of mice with transgenic parasites elicited the proliferation of adoptively transferred OT-I T cells and their recruitment to the site of infection in the skin. Together, these results demonstrate the possibility of targeting heterologous antigens to specific cellular compartments in L. major and suggest that proteins secreted or released by L. major in infected DC are a major source of peptides for the generation of parasite-specific CD8+ T cells. The ability of L. major transgenic parasites to activate OT-I CD8+ T cells in vivo will permit the analysis of parasite-driven T-cell expansion, differentiation, and recruitment at the clonal level. PMID:16177338

  18. The Leishmania promastigote surface antigen-2 (PSA-2) is specifically recognised by Th1 cells in humans with naturally acquired immunity to L. major

    DEFF Research Database (Denmark)

    Kemp, M; Handman, E; Kemp, K

    1998-01-01

    The promastigote surface antigen-2 (PSA-2) is a Leishmania parasite antigen, which can induce Th1-mediated protection against murine leishmaniasis when used as a vaccine. To evaluate PSA-2 as a human vaccine candidate the specific T-cell response to PSA-2 was characterised in individuals immune...... to cutaneous leishmaniasis. Peripheral blood mononuclear cells from Sudanese individuals with a past history of self-healing cutaneous leishmaniasis proliferated vigorously in response to PSA-2 isolated from Leishmania major, whereas the antigen did not activate cells from presumably unexposed Danes....... Peripheral blood mononuclear cells from individuals with previous L. major infection had varying proliferative responses to PSA-2 derived from L. donovani promastigotes. Peripheral blood mononuclear cells activated by PSA-2 from L. major produced high amounts of interferon-gamma and tumour necrosis factor...

  19. An α-Gal-containing neoglycoprotein-based vaccine partially protects against murine cutaneous leishmaniasis caused by Leishmania major.

    Science.gov (United States)

    Iniguez, Eva; Schocker, Nathaniel S; Subramaniam, Krishanthi; Portillo, Susana; Montoya, Alba L; Al-Salem, Waleed S; Torres, Caresse L; Rodriguez, Felipe; Moreira, Otacilio C; Acosta-Serrano, Alvaro; Michael, Katja; Almeida, Igor C; Maldonado, Rosa A

    2017-10-01

    Protozoan parasites from the genus Leishmania cause broad clinical manifestations known as leishmaniases, which affect millions of people worldwide. Cutaneous leishmaniasis (CL), caused by L. major, is one the most common forms of the disease in the Old World. There is no preventive or therapeutic human vaccine available for L. major CL, and existing drug treatments are expensive, have toxic side effects, and resistant parasite strains have been reported. Hence, further therapeutic interventions against the disease are necessary. Terminal, non-reducing, and linear α-galactopyranosyl (α-Gal) epitopes are abundantly found on the plasma membrane glycolipids of L. major known as glycoinositolphospholipids. The absence of these α-Gal epitopes in human cells makes these glycans highly immunogenic and thus potential targets for vaccine development against CL. Here, we evaluated three neoglycoproteins (NGPs), containing synthetic α-Gal epitopes covalently attached to bovine serum albumin (BSA), as vaccine candidates against L. major, using α1,3-galactosyltransferase-knockout (α1,3GalT-KO) mice. These transgenic mice, similarly to humans, do not express nonreducing, linear α-Gal epitopes in their cells and are, therefore, capable of producing high levels of anti-α-Gal antibodies. We observed that Galα(1,6)Galβ-BSA (NGP5B), but not Galα(1,4)Galβ-BSA (NGP12B) or Galα(1,3)Galα-BSA (NGP17B), was able to significantly reduce the size of footpad lesions by 96% in comparison to control groups. Furthermore, we observed a robust humoral and cellular immune response with production of high levels of protective lytic anti-α-Gal antibodies and induction of Th1 cytokines. We propose that NGP5B is an attractive candidate for the study of potential synthetic α-Gal-neoglycoprotein-based vaccines against L. major infection.

  20. An α-Gal-containing neoglycoprotein-based vaccine partially protects against murine cutaneous leishmaniasis caused by Leishmania major.

    Directory of Open Access Journals (Sweden)

    Eva Iniguez

    2017-10-01

    Full Text Available Protozoan parasites from the genus Leishmania cause broad clinical manifestations known as leishmaniases, which affect millions of people worldwide. Cutaneous leishmaniasis (CL, caused by L. major, is one the most common forms of the disease in the Old World. There is no preventive or therapeutic human vaccine available for L. major CL, and existing drug treatments are expensive, have toxic side effects, and resistant parasite strains have been reported. Hence, further therapeutic interventions against the disease are necessary. Terminal, non-reducing, and linear α-galactopyranosyl (α-Gal epitopes are abundantly found on the plasma membrane glycolipids of L. major known as glycoinositolphospholipids. The absence of these α-Gal epitopes in human cells makes these glycans highly immunogenic and thus potential targets for vaccine development against CL.Here, we evaluated three neoglycoproteins (NGPs, containing synthetic α-Gal epitopes covalently attached to bovine serum albumin (BSA, as vaccine candidates against L. major, using α1,3-galactosyltransferase-knockout (α1,3GalT-KO mice. These transgenic mice, similarly to humans, do not express nonreducing, linear α-Gal epitopes in their cells and are, therefore, capable of producing high levels of anti-α-Gal antibodies. We observed that Galα(1,6Galβ-BSA (NGP5B, but not Galα(1,4Galβ-BSA (NGP12B or Galα(1,3Galα-BSA (NGP17B, was able to significantly reduce the size of footpad lesions by 96% in comparison to control groups. Furthermore, we observed a robust humoral and cellular immune response with production of high levels of protective lytic anti-α-Gal antibodies and induction of Th1 cytokines.We propose that NGP5B is an attractive candidate for the study of potential synthetic α-Gal-neoglycoprotein-based vaccines against L. major infection.

  1. Comparison on the performance of Leishmania major-like and Leishmania braziliensis braziliensis as antigen for new world leishmaniasis IgG-immunofluorescence test Comparação do desempenho de antígenos de Leishmania major-like e Leishmania braziliensis braziliensis no teste de imunofluorescência-IgG para leishmaniose mucocutânea

    Directory of Open Access Journals (Sweden)

    Maria Carolina Soares Guimarães

    1991-12-01

    Full Text Available The performance of an antigen of L. major-like promastigotes for the serological diagnosis of mucocutaneous leishmaniasis in the IgG-immunofluorescent test was compared to that of an antigen of L. braziliensis braziliensis. Each antigen was used to test two hundred and twenty-four sera of etiologies such as mucocutaneous leishmaniasis, deep mycoses, toxoplasmosis, malaria, Chagas' disease, visceral leishmaniasis, anti-nuclear factor, schistosomaiasis, rheumatoid factor and normal controls. Agreement between responses to each antigen was high: 77.2% of leishmaniases sera agreed on a positive or a negative result to both antigens and 91.1 % of control sera. Cross reactivity was restricted to Chagas' disease sera, visceral leishmaniasis, anti-nuclear factor and paracoccidiodomycosis. The quantitative response of leishmaniasis and Chagas' disease sera to both antigens was evaluated by a linear regression; although the y-intercept and the slope were different for each antigen, neither was better than the other in the disclosure of anti-Leishmania antibodies. In the case of Chagas' disease sera the L. major-like antigen was better than L. b. braziliensis' to disclose cross-reacting antibodies.O desempenho de um antígeno de promastigotas de L. major-like para o diagnóstico sorológico de leishmaniose mucocutânea pelo teste de imunofluorescência-IgG foi comparado com o desempenho de um antígeno de L. braziliensis braziliensis. Cada antígeno foi usado para testar 224 soros de etiologias como leishmaniose mucocutânea, micoses profundas, toxoplasmose, malária, doença de Chagas, leishmaniose visceral, fator anti-núcleo, esquistossomose mansônica, fator reumatóide e controles normais. A concordância entre as respostas a cada antígeno foi grande: 77,2% dos soros de leishmaniose mostraram resultado positivo para ambos os antígenos ou negativo, assim como 91,1% dos soros controle negativos. Reações cruzadas ficaram restritas à doença de

  2. Diversity among Leishmania isolates from the Sudan: isoenzyme homogeneity of L. donovani versus heterogeneity of L. major

    DEFF Research Database (Denmark)

    Ibrahim, M E; Evans, D A; Theander, T G

    1995-01-01

    Leishmania isolates from patients in the Sudan suffering from either visceral or cutaneous leishmaniasis were characterized using a battery of 12 enzymes. Aspartate aminotransferase separated the L. donovani isolates into 2 distinct zymodemes, but the overall results showed no significant...

  3. Ox40L-Ox40 pathway plays distinct roles in regulating Th2 responses but does not determine outcome of cutaneous leishmaniasis caused by Leishmania mexicana and Leishmania major.

    Science.gov (United States)

    Tuladhar, Rashmi; Oghumu, Steve; Dong, Ran; Peterson, Allison; Sharpe, Arlene H; Satoskar, Abhay R

    2015-01-01

    Ox40 ligand (Ox40L)-Ox40 pathway has been shown to enhance Th2 responses and play a role in pathogenesis of cutaneous leishmaniasis (CL) caused by Leishmania major. Using Ox40l(-/-) BALB/c mice we analyzed the role of this pathway in determining the outcome to CL caused by L. mexicana and compared to L. major. Contrary to our expectations, Ox40l(-/-) mice were highly susceptible to both L. major (LV39) and L. mexicana (M379) and developed large non-healing lesions containing parasites comparable to Ox40l(+/+) BALB/c mice. Interestingly, upon in vitro stimulation with Leishmania antigen (LmAg), the lymph node cells from L. major infected Ox40l(-/-) mice produced significantly less IL-4 and IL-10 compared to Ox40l(+/+) mice. L. mexicana infected Ox40l(-/-) and Ox40l(+/+) mice did not show any difference in the production of IL-4 and IL-10. No difference was noted in the amount of Th1 cytokines IFN-ү and IL-12 produced by Ox40l(-/-) and Ox40l(+/+) mice infected with either parasite. These results indicate that the Ox40L-Ox40 pathway promotes Th2 bias only in L. major infection but not L. mexicana infection and this pathway is not critical for susceptibility to CL. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Structural and thermodynamic basis of the inhibition of Leishmania major farnesyl diphosphate synthase by nitrogen-containing bisphosphonates

    Energy Technology Data Exchange (ETDEWEB)

    Aripirala, Srinivas [Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States); Gonzalez-Pacanowska, Dolores [López-Neyra Institute of Parasitology and Biomedicine, 18001 Granada (Spain); Oldfield, Eric [University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States); Kaiser, Marcel [University of Basel, Petersplatz 1, CH-4003 Basel (Switzerland); Amzel, L. Mario, E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Gabelli, Sandra B., E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States)

    2014-03-01

    Structural insights into L. major farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, are described. Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneous leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca{sup 2+} ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg{sup 2+} ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.

  5. Detection of Leishmania major DNA within wild caught Phlebotomus papatasi and species composition of sand flies in endemic focus of cutaneous leishmaniasis, in western Iran.

    Science.gov (United States)

    Vahabi, A; Rassi, Y; Oshaghi, M A; Sayyadi, M; Rafizadeh, S

    2016-03-01

    Cutaneous leishmaniasis is one of the most important public health problem in many developing countries. The present study was conducted to determine the vector(s), the parasite and the species composition of sand flies in the Dehloran County during May-November 2012. Sand flies were collected by sticky traps and mounted in Puri's medium for species identification. Polymerase chain reaction (PCR) techniques of kDNA, ITS1-rDNA, followed by restriction fragment length polymorphism (RFLP) were used for identification of DNA of Leishmania parasites in infected sand flies. A total of 82443 specimens comprising 15 species of sand flies (5 Phlebotomus and 10 Sergentomyia) were collected and identified. The species of Phlebotomus papatasi was dominant in outdoor and indoor resting places. Among the 280 specimens of female P. papatasi tested by PCR of kDNA, ITS1-rDNA genes of the parasite followed by RFLP, only 5 of them (1.8 %) were positive to Leishmania major parasites. This is the first molecular detection of leishmania infection of P. papatasi to L. major in this region. The results indicated that, P. papatasi was only species found infected by L. major and the principal vector of disease agent to human.

  6. First microscopical and molecular-based characterization of Leishmania major within naturally infected Phlebotomus salehi (Diptera; Psychodidae) in Fars province, southern Iran

    Science.gov (United States)

    DAVAMI, M H; MOTAZEDIAN, M H; KALANTARI, M; ASGARI, Q; BADZOHRE, A; MOHAMMADPOUR, I

    2011-01-01

    Zoonotoc cutaneous leishmaniasis is endemic in several parts of Iran. Jahrom district is one of the most important endemic foci of leishmaniasis located in Fars province, southern Iran. To identify the vectors of leishmaniasis in this area, a total of 349 sandflies were collected during May to August 2009. They were caught from outdoors in five regions of Jahrom district including villages of Mousavieh, Ghotb-Abad, Heydar-Abad, Fath-Abad and Jahrom County. Eleven species of Phlebotomine (three Phlebotomus spp. and eight Sergentomyia spp.) were detected. To determine the sandflies naturally infected by Leishmania spp., 122 female sandflies were dissected and evaluated microscopically using Giemsa-stained slides. Natural infection of 2 out of 38 (5.26%) P. papatasi and 1 out of 8 (12.5%) P. salehi to Leishmania major was confirmed in the region. Sequencing and nested polymerase chain reaction-based detection of Leishmania were carried out to confirm the microscopic findings. Five (13.16%) P. papatasi and two (25%) P. salehi were positive in nested polymerase chain reaction assay. All positive samples were shown 72–76% similarity with L. major Friedlin. On the basis of our knowledge, this is the first molecular detection of L. major within naturally infected P. salehi in this region in southern Iran. PMID:22185942

  7. Deletion of IL-4Ralpha on CD4 T cells renders BALB/c mice resistant to Leishmania major infection.

    Directory of Open Access Journals (Sweden)

    Magdalena Radwanska

    2007-05-01

    Full Text Available Effector responses induced by polarized CD4+ T helper 2 (Th2 cells drive nonhealing responses in BALB/c mice infected with Leishmania major. Th2 cytokines IL-4 and IL-13 are known susceptibility factors for L. major infection in BALB/c mice and induce their biological functions through a common receptor, the IL-4 receptor alpha chain (IL-4Ralpha. IL-4Ralpha-deficient BALB/c mice, however, remain susceptible to L. major infection, indicating that IL-4/IL-13 may induce protective responses. Therefore, the roles of polarized Th2 CD4+ T cells and IL-4/IL-13 responsiveness of non-CD4+ T cells in inducing non-healer or healer responses have yet to be elucidated. CD4+ T cell-specific IL-4Ralpha (Lck(creIL-4Ralpha(-/lox deficient BALB/c mice were generated and characterized to elucidate the importance of IL-4Ralpha signaling during cutaneous leishmaniasis in the absence of IL-4-responsive CD4+ T cells. Efficient deletion was confirmed by loss of IL-4Ralpha expression on CD4+ T cells and impaired IL-4-induced CD4+ T cell proliferation and Th2 differentiation. CD8+, gammadelta+, and NK-T cells expressed residual IL-4Ralpha, and representative non-T cell populations maintained IL-4/IL-13 responsiveness. In contrast to IL-4Ralpha(-/lox BALB/c mice, which developed ulcerating lesions following infection with L. major, Lck(creIL-4Ralpha(-/lox mice were resistant and showed protection to rechallenge, similar to healer C57BL/6 mice. Resistance to L. major in Lck(creIL-4Ralpha(-/lox mice correlated with reduced numbers of IL-10-secreting cells and early IL-12p35 mRNA induction, leading to increased delayed type hypersensitivity responses, interferon-gamma production, and elevated ratios of inducible nitric oxide synthase mRNA/parasite, similar to C57BL/6 mice. These data demonstrate that abrogation of IL-4 signaling in CD4+ T cells is required to transform non-healer BALB/c mice to a healer phenotype. Furthermore, a beneficial role for IL-4Ralpha signaling in L

  8. Antileishmanial activity of Ferula assa-foetida oleo gum resin against Leishmania major: An in vitro study

    Directory of Open Access Journals (Sweden)

    Ali Fatahi Bafghi

    2014-01-01

    Full Text Available Background: In Ayurveda, asafetida is introduced as a valuable remedy for flatulence, hysteria, nervous disorders, whooping cough, pneumonia and bronchitis in children and also considered as an aphrodisiac agent. Presently, Leishmaniasis is common in most countries of the world and is a serious health problem in the world. Some plant medicines and natural products have a new candidate for treatment of leishmaniasis. Objective: This study was designed to evaluate Ferula assa-foetida oleo gum resin (asafetida on mortality and morbidity Leishmania major in vitro. Materials and Methods: Mostigotes were isolated from mice spleens and then transformed to promastigotes in Novy-Nicolle-Mac Neal (NNN medium supplemented with penicillin (100 U/ml, streptomycin (100 μg/ml and 20% heat-inactivated fetal calf serum (FCS at 25°C. A fixed initial density of the parasites was transferred to screw-capped vials containing 5 ml of RPMI 1640 media to which different concentrations of 2.5, 5, 10 and 20 μg asafetida were added and each concentration was done in triplicates. Each run also included control. The mortality of parasitoids was measured by the slide and the enzyme-linked immunosorbent assay (ELISA methods. Results: After 72 h, asafetida inhibited growth of parasites in all doses in stationary and logarithmic phases. The ELISA measurement suggested that the viability of parasites significantly decreased after 48h (P < 0.05. Conclusion: The results show that asafetida could prevent from growth and viability of parasites and this oleo gum resin can be useful for treatment of leishmaniasis.

  9. H-11-linked gene has a parallel effect on Leishmania major and L. donovani infections in mice

    Energy Technology Data Exchange (ETDEWEB)

    Blackwell, J.M.; Hale, C.; Roberts, M.B.; Ulczak, O.M.; Liew, F.Y.; Howard, J.G.

    1985-01-01

    The courses of visceral infection following intravenous injection of Leishmania donovani amastigotes, or lesion growth following subcutaneous injection of L. major promastigotes, were examined in B10.129(10M) (H-2b, H-11b) mice and compared with disease profiles observed in congenic C57BL/10ScSn(= B10) (H-2b, H-11a) and B10.D2/n (H-2d, H-11a) mice, and in BALB/mice. Possession of alternative alleles at H-11 and closely linked loci transformed the normal curing/healing phenotype of B10 mice into a characteristically different noncuring/nonhealing phenotype affecting both visceral and subcutaneous infections in B10.129(10M) mice. In reciprocal radiation bone marrow chimeras made between the congenic B10 and B10.129(10M) strains, both cure and noncure phenotypes were transferable with the donor hematopoietic system. Although it was possible to demonstrate transfer of suppression with T-enriched spleen cells from day 61 L. donovani-infected B10.129(10M) donor mice into 550 rad syngeneic recipients, the pretreatment of mice with sublethal irradiation did not, as in the earlier studies of Scl-controlled L. major nonhealing or H-2-controlled L. donovani noncure phenotypes, have a clear or consistent prophylactic effect. Together with the progressive disease profile observed even for L. donovani at low parasite doses this suggests that, despite their ability to develop initial delayed-type hypersensitivity reactions to parasite antigen early in L. major infection, B10.129(10M) mice possess some inherent defect in ability to mount a cell-mediated response effective at the level of macrophage neishmanial activity in vivo even when suppressor T cells are not generated. Elucidation of this characteristically different noncuring/nonhealing phenotye may provide important insight into common events involved in the development of the cell-mediated immune response to both visceral and subcutaneous forms of leishmaniasis.

  10. First detection of Leishmania major DNA in Sergentomyia (Sintonius) clydei (Sinton, 1928, Psychodidae: Phlebotominae), from an outbreak area of cutaneous leishmaniasis in Tunisia.

    Science.gov (United States)

    Ayari, Chiraz; Ben Othman, Souad; Chemkhi, Jomaa; Tabbabi, Ahmed; Fisa, Roser; Ben Salah, Afif; BenAbderrazak, Souha

    2016-04-01

    In recent years there has been growing interest in Sergentomyia species. Their role in the spread of mammalian leishmaniasis appears repeatedly in the literature and the possibility of its implication in Leishmania transmission to humans remains controversial. Sergentomyia (Sintonius) clydei is one of several cryptic species sharing therefore common morphologic criteria with others species of the subgenera Sintonius. Little is known about this specie in Tunisia. We sampled and identified different specimens including four specimens of S. clydei collected from Sidi Bouzid and Kairouan areas (center of Tunisia) using morphological tools. Male Sergentomyia clydei and Sergentomyia christophersi are known to share several morphological characters and can be mistaken for. Consequently we took advantage of 5 male S. christophersi available in our collection (Tataouin, South of Tunisia). In our study morphological tools were completed by molecular study of cytochrome b gene to identify S. clydei. For the detection of Leishmania spp. that might infect our specimens, Leishmania DNA was analyzed by amplification of kinetoplast minicircle DNA using real-time PCR and nested-PCR. Obtained result was confirmed by restriction analysis of the amplified ribosomal internal transcribed spacer 1 (ITS1). We provide in our study, the first molecular identification of S. clydei, in Tunisia. Our Neighbor Joining tree based on mitochondrial cytochrome b gene shows two different clusters. The first includes the Tunisians S. clydei and other specimens from Africa, Middle East and the Arabic peninsula, and the second cluster containing the specimens from Seychelle. Unexpectedly, we also demonstrate the infection of one anthropophilic female S. clydei by Leishmania major DNA. This finding shows that more attention should be paid when identifying parasites by molecular tools within sandfly vector. Copyright © 2015. Published by Elsevier B.V.

  11. Exploring the midgut transcriptome of Phlebotomus papatasi: comparative analysis of expression profiles of sugar-fed, blood-fed and Leishmania-major-infected sandflies.

    Science.gov (United States)

    Ramalho-Ortigão, Marcelo; Jochim, Ryan C; Anderson, Jennifer M; Lawyer, Phillip G; Pham, Van-My; Kamhawi, Shaden; Valenzuela, Jesus G

    2007-08-30

    In sandflies, the blood meal is responsible for the induction of several physiologic processes that culminate in egg development and maturation. During blood feeding, infected sandflies are also able to transmit the parasite Leishmania to a suitable host. Many blood-induced molecules play significant roles during Leishmania development in the sandfly midgut, including parasite killing within the endoperitrophic space. In this work, we randomly sequenced transcripts from three distinct high quality full-length female Phlebotomus papatasi midgut-specific cDNA libraries from sugar-fed, blood-fed and Leishmania major-infected sandflies. Furthermore, we compared the transcript expression profiles from the three different cDNA libraries by customized bioinformatics analysis and validated these findings by semi-quantitative PCR and real-time PCR. Transcriptome analysis of 4010 cDNA clones resulted in the identification of the most abundant P. papatasi midgut-specific transcripts. The identified molecules included those with putative roles in digestion and peritrophic matrix formation, among others. Moreover, we identified sandfly midgut transcripts that are expressed only after a blood meal, such as microvilli associated-like protein (PpMVP1, PpMVP2 and PpMVP3), a peritrophin (PpPer1), trypsin 4 (PpTryp4), chymotrypsin PpChym2, and two unknown proteins. Of interest, many of these overabundant transcripts such as PpChym2, PpMVP1, PpMVP2, PpPer1 and PpPer2 were of lower abundance when the sandfly was given a blood meal in the presence of L. major. This tissue-specific transcriptome analysis provides a comprehensive look at the repertoire of transcripts present in the midgut of the sandfly P. papatasi. Furthermore, the customized bioinformatic analysis allowed us to compare and identify the overall transcript abundance from sugar-fed, blood-fed and Leishmania-infected sandflies. The suggested upregulation of specific transcripts in a blood-fed cDNA library were validated by

  12. Exploring the midgut transcriptome of Phlebotomus papatasi: comparative analysis of expression profiles of sugar-fed, blood-fed and Leishmania major-infected sandflies

    Science.gov (United States)

    Ramalho-Ortigão, Marcelo; Jochim, Ryan C; Anderson, Jennifer M; Lawyer, Phillip G; Pham, Van-My; Kamhawi, Shaden; Valenzuela, Jesus G

    2007-01-01

    Background In sandflies, the blood meal is responsible for the induction of several physiologic processes that culminate in egg development and maturation. During blood feeding, infected sandflies are also able to transmit the parasite Leishmania to a suitable host. Many blood-induced molecules play significant roles during Leishmania development in the sandfly midgut, including parasite killing within the endoperitrophic space. In this work, we randomly sequenced transcripts from three distinct high quality full-length female Phlebotomus papatasi midgut-specific cDNA libraries from sugar-fed, blood-fed and Leishmania major-infected sandflies. Furthermore, we compared the transcript expression profiles from the three different cDNA libraries by customized bioinformatics analysis and validated these findings by semi-quantitative PCR and real-time PCR. Results Transcriptome analysis of 4010 cDNA clones resulted in the identification of the most abundant P. papatasi midgut-specific transcripts. The identified molecules included those with putative roles in digestion and peritrophic matrix formation, among others. Moreover, we identified sandfly midgut transcripts that are expressed only after a blood meal, such as microvilli associated-like protein (PpMVP1, PpMVP2 and PpMVP3), a peritrophin (PpPer1), trypsin 4 (PpTryp4), chymotrypsin PpChym2, and two unknown proteins. Of interest, many of these overabundant transcripts such as PpChym2, PpMVP1, PpMVP2, PpPer1 and PpPer2 were of lower abundance when the sandfly was given a blood meal in the presence of L. major. Conclusion This tissue-specific transcriptome analysis provides a comprehensive look at the repertoire of transcripts present in the midgut of the sandfly P. papatasi. Furthermore, the customized bioinformatic analysis allowed us to compare and identify the overall transcript abundance from sugar-fed, blood-fed and Leishmania-infected sandflies. The suggested upregulation of specific transcripts in a blood-fed c

  13. Exploring the midgut transcriptome of Phlebotomus papatasi: comparative analysis of expression profiles of sugar-fed, blood-fed and Leishmania major-infected sandflies

    Directory of Open Access Journals (Sweden)

    Pham Van-My

    2007-08-01

    Full Text Available Abstract Background In sandflies, the blood meal is responsible for the induction of several physiologic processes that culminate in egg development and maturation. During blood feeding, infected sandflies are also able to transmit the parasite Leishmania to a suitable host. Many blood-induced molecules play significant roles during Leishmania development in the sandfly midgut, including parasite killing within the endoperitrophic space. In this work, we randomly sequenced transcripts from three distinct high quality full-length female Phlebotomus papatasi midgut-specific cDNA libraries from sugar-fed, blood-fed and Leishmania major-infected sandflies. Furthermore, we compared the transcript expression profiles from the three different cDNA libraries by customized bioinformatics analysis and validated these findings by semi-quantitative PCR and real-time PCR. Results Transcriptome analysis of 4010 cDNA clones resulted in the identification of the most abundant P. papatasi midgut-specific transcripts. The identified molecules included those with putative roles in digestion and peritrophic matrix formation, among others. Moreover, we identified sandfly midgut transcripts that are expressed only after a blood meal, such as microvilli associated-like protein (PpMVP1, PpMVP2 and PpMVP3, a peritrophin (PpPer1, trypsin 4 (PpTryp4, chymotrypsin PpChym2, and two unknown proteins. Of interest, many of these overabundant transcripts such as PpChym2, PpMVP1, PpMVP2, PpPer1 and PpPer2 were of lower abundance when the sandfly was given a blood meal in the presence of L. major. Conclusion This tissue-specific transcriptome analysis provides a comprehensive look at the repertoire of transcripts present in the midgut of the sandfly P. papatasi. Furthermore, the customized bioinformatic analysis allowed us to compare and identify the overall transcript abundance from sugar-fed, blood-fed and Leishmania-infected sandflies. The suggested upregulation of specific

  14. Oral immunization using live Lactococcus lactis co-expressing LACK and IL-12 protects BALB/c mice against Leishmania major infection.

    Science.gov (United States)

    Hugentobler, Felix; Di Roberto, Raphaël B; Gillard, Joshua; Cousineau, Benoit

    2012-08-24

    Leishmaniasis is a parasitic disease affecting over 12 million individuals worldwide. Current treatments are laborious, expensive, cause severe side effects, and emerging drug resistance has been reported. While vaccination is the most cost-effective means to control infectious diseases there is no human vaccine currently available against Leishmania infections. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used for the expression and delivery of biologically active molecules, such as antigens and cytokines, in mice and humans. In this study, we report the generation of L. lactis(alr-) strains solely expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall or co-expressing mouse IL-12. We show that oral immunization using live L. lactis, secreting both LACK and IL-12 was the only regimen that partially protected BALB/c mice against subsequent Leishmania major challenge. This highlights the importance of temporal and physical proximity of the delivered antigen and adjuvant for optimal immune priming by oral immunization since co-administration of L. lactis strains independently expressing secLACK and secIL-12 did not induce protective immunity. Protected animals displayed a delay in footpad swelling, which correlated with a significant reduction of parasite burden. Immunization with the L. lactis strain secreting both LACK and IL-12 induced an antigen-specific mucosal immune response and a LACK-specific T(H)1 immune response in splenocytes and mesenteric lymph node cells. Further, protection in immunized animals correlated with a strong Leishmania-specific T(H)1 immune response post-challenge, detectable in splenocytes and lymph node cells draining the site of infection. This report demonstrates the use of L. lactis as an oral live vaccine against L. major infection in susceptible BALB/c mice. The

  15. The Leishmania promastigote surface antigen-2 (PSA-2) is specifically recognised by Th1 cells in humans with naturally acquired immunity to L. major.

    Science.gov (United States)

    Kemp, M; Handman, E; Kemp, K; Ismail, A; Mustafa, M D; Kordofani, A Y; Bendtzen, K; Kharazmi, A; Theander, T G

    1998-03-01

    The promastigote surface antigen-2 (PSA-2) is a Leishmania parasite antigen, which can induce Th1-mediated protection against murine leishmaniasis when used as a vaccine. To evaluate PSA-2 as a human vaccine candidate the specific T-cell response to PSA-2 was characterised in individuals immune to cutaneous leishmaniasis. Peripheral blood mononuclear cells from Sudanese individuals with a past history of self-healing cutaneous leishmaniasis proliferated vigorously in response to PSA-2 isolated from Leishmania major, whereas the antigen did not activate cells from presumably unexposed Danes. Peripheral blood mononuclear cells from individuals with previous L. major infection had varying proliferative responses to PSA-2 derived from L. donovani promastigotes. Peripheral blood mononuclear cells activated by PSA-2 from L. major produced high amounts of interferon-gamma and tumour necrosis factor-beta, and little interleukin-4, thereby showing a Th1 cytokine pattern. Parallel cultures showed clear Th1 and Th2 response patterns to purified protein derivative of tuberculin or tetanus toxoid, respectively. Flow cytometric analysis revealed that PSA-2 induced blastogenesis in the CD3 positive population and that these cells were the major source of interferon-gamma. The results show that Th1-like cells recognising PSA-2 are expanded during infection by L. major and that they maintain their Th1-like cytokine profile upon reactivation in vitro. Since immunity to cutaneous leishmaniasis is mediated by antigen-specific Th1-like cells, PSA-2 might be considered a vaccine candidate for human leishmaniasis.

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

    Science.gov (United States)

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

    2015-02-03

    Syzygium cumini (L.) Skeels (Myrtaceae), commonly known as "jambolão" in Brazil is widely used in folk medicine against leishmaniasis, inflammation, chronic diarrhea, and ulcers. It is one of the most commonly used plants for the treatment of diabetes worldwide. In previous studies, Syzygium cumini was shown to possess antihyperlipidemic and anti-allergic properties, and to exhibit good performance as an antimicrobial agent against bacteria, fungi, and protozoa parasites of the genus Leishmania and Trypanosoma. This study was aimed at evaluating the effects of S. cumini essential oil (ScEO) and its major component α-pinene on Leishmania (Leishmania) amazonensis, as well as their cytotoxicity and possible mechanisms of action. To evaluate the anti-proliferative effect on Leishmania, effects on promastigote and axenic amastigote forms were assessed using tetrazolium salt (MTT) assay. The intramacrophagic amastigotes were exposed to ScEO and α-pinene to determine the survival index. To gain insight into the mechanism of action involved in the effect on the samples, we evaluated the modulation of macrophage activation state by observing structural (phagocytic and lysosomal activities) and cellular (nitric oxide increase) changes. To assess the safety profile of ScEO and α-pinene, murine macrophages and human red blood cells were treated with ScEO and α-pinene and the selectivity index was calculated for each treatment. α-Pinene was effective against Leishmania amazonensis promastigote forms, with a half-maximal inhibitory concentration (IC50) value of 19.7µg/mL. α-Pinene was more active (IC50 values of 16.1 and 15.6µg/mL against axenic and intracellular amastigotes, respectively) than ScEO (IC50 values of 43.9 and 38.1µg/mL against axenic and intracellular amastigotes, respectively). Our results showed that the anti-Leishmania effects were mediated by immunomodulatory activity, as evidenced by the observed increases in both phagocytic and lysosomal activity

  17. Characterization of the Early Inflammatory Infiltrate at the Feeding Site of Infected Sand Flies in Mice Protected from Vector-Transmitted Leishmania major by Exposure to Uninfected Bites

    Science.gov (United States)

    Teixeira, Clarissa; Gomes, Regis; Oliveira, Fabiano; Meneses, Claudio; Gilmore, Dana C.; Elnaiem, Dia-Eldin A.; Valenzuela, Jesus G.; Kamhawi, Shaden

    2014-01-01

    Background Mice exposed to sand fly saliva are protected against vector-transmitted Leishmania major. Although protection has been related to IFN- γ producing T cells, the early inflammatory response orchestrating this outcome has not been defined. Methodology/Principal findings Mice exposed to uninfected P. duboscqi bites and naïve mice were challenged with L. major-infected flies to characterize their early immune response at the bite site. Mostly, chemokine and cytokine transcript expression post-infected bites was amplified in exposed compared to naïve mice. In exposed mice, induced chemokines were mostly involved in leukocyte recruitment and T cell and NK cell activation; IL-4 was expressed at 6 h followed by IFN-γ and iNOS2 as well as IL-5 and IL-10 expression. In naïve animals, the transcript expression following Leishmania-infected sand fly bites was suppressed. Expression profiles translated to an earlier and significantly larger recruitment of leukocytes including neutrophils, macrophages, Gr+ monocytes, NK cells and CD4+ T cells to the bite site of exposed compared to naïve mice post-infected bites. Additionally, up to 48 hours post-infected bites the number of IFN-γ-producing CD4+T cells and NK cells arriving at the bite site was significantly higher in exposed compared to naïve mice. Thereafter, NK cells become cytolytic and persist at the bite site up to a week post-bite. Conclusion/Significance The quiet environment induced by a Leishmania-infected sand fly bite in naïve mice was significantly altered in animals previously exposed to saliva of uninfected flies. We propose that the enhanced recruitment of Gr+ monocytes, NK cells and CD4 Th1 cells observed at the bite site of exposed mice creates an inhospitable environment that counters the establishment of L. major infection. PMID:24762408

  18. DDA/TDB liposomes containing soluble Leishmania major antigens induced a mixed Th1/Th2 immune response in BALB/c mice

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    Mansure Hojatizade

    2017-04-01

    Full Text Available Objective(s: Leishmaniasis is a complex parasitic disease that represents a major public health problem. Despite numerous attempts over the past decades, yet there is no effective vaccine against human leishmaniasis probably due to the lack of suitable adjuvants. In this study, a first generation liposomal-based Leishmania vaccine was developed using soluble Leishmania major antigens (SLA and á, Ü-trehalose6, 6'-dibehenat (TDB as an immunostimulatory adjuvant. In this liposome structure, the cationic lipid Dimethyldioctadecylammonium (DDA provides intrinsic adjuvant activity and cholesterol was added as a membrane stabilizer. Liposomes containing SLA were prepared.Materials and Methods: BALB/c mice were subcutaneously (sc immunized with Lip (DDA/TDB/CHOL-SLA+, Lip (DDA/TDB-SLA+, Lip (DDA-SLA+, Lip (DDA/CHOL-SLA+, SLA or Tris-HCl buffer. Immunization was done every two weeks for three weeks. The immunized mice were then challenged sc in the left footpad with 1×106 stationary phase L. major promastigotes (50 ìl, at 2 weeks after last booster injection.Results: mice immunized with any of the liposomal formulations containing SLA (Lip-SLA+, substantially increased footpad swelling and parasite loads of foot and spleen with no significant difference compared to Tris-HCl buffer or SLA alone. Lip-SLA+ formulations induced a mixed Th1/Th2 immune response characterized by IFN-ã and IL-4 production as well as high levels of IgG1 anti-Leishmania antibody. Conclusion: immunization with liposomes containing DDA and/or TDB in combination with SLA induces a mixed Th1/Th2 immune response and is not an appropriate strategy for preferential induction of a Th1 response and protection against leishmaniasis.

  19. In Vitro Anti-leishmanial Effects of Hydroalcoholic Extracts From Six Iranian Medicinal Herbs on Leishmania major (MRHO/IR/75/ER Promastigotes

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    Feiz Haddad

    2016-02-01

    Full Text Available Background Cutaneous leishmaniasis is endemic in 11 Iranian provinces, and almost 80% of all cases are the rural cutaneous form caused by Leishmania major. The main important drug used to treat leishmaniasis is a five-capacity antimony compound that has hinders disease recurrence, drug resistance, complications, and long treatment duration. Objectives It seems necessary to search for new, natural medicine to replace the use of drugs, particularly herbal compounds with no side effects. Materials and Methods Different dilutions of extracts were prepared with 1 mg/mL of the extracts in 96-well plates. Cultivated 106 Leishmania major promastigotes in culture flasks containing RPMI1640 medium and 10% of brain heart infusion (BHI were counted using neobar lam and poured into each well. Following 72 hours incubation, MTT solution was added to each well, and then absorbance was documented with an enzyme-linked immunosorbent assay (ELISA reader at 570 nm. Results IC50 of Allium hirtifolium (87 μg/mL and Ziziphus spina-christi (112 μg/mL extracts showed higher efficiency in inhibiting the growth of promastigotes over 72 hours. Conclusions Allium hirtifolium and Z. spina-christi extracts showed the most efficient activity in the inhibition of promastigote growth, but this was not total. The remaining plant extracts indicated ineffective or very weak efficiency on the Leishmania promastigotes. There is a possibility of achieving better results by changing the extraction method; determining materials affecting promastigotes and extracting these materials exactly; or converting extracts to other medicinal forms, such as ointment or gel.

  20. LB broth-lyophilized Rabbit serum (LLR) as a new and suitable culture medium for cultivation of promastigotes ofLeishmania major.

    Science.gov (United States)

    Nasiri, Vahid; Dalimi, Abdolhossein; Ghaffarifar, Fatemeh

    2017-03-01

    Fetal calf serum is the major part and the most expensive ingredient of the Leishmania culture media. Here, the efficacy of the LB broth-lyophilized Rabbit serum medium (LLR) was evaluated in cultivation of Leishmania major . Conventional Luria-Bertani (LB) broth medium was prepared and autoclaved for 15 min at 121 °C and then lyophilized Rabbit serum was added at the 1, 2.5, 5 and 10 % final concentrations. The efficacy of medium was evaluated by assessing the growth ability and replication pattern of the promastigotes of L. major . According to our finding, the LLR medium with 5-10 % lyophilized Rabbit serum supported the growth of the parasites and can be used for cultivation of Leishmanian parasites with acceptable In vivo infectivity for research purpose. The ability of the parasites to survive and proliferating in the presence of lyophilized Rabbit serum indicating that this serum is a good nutritional source. This study opens a new way to make low-cost medium that could be used in cultivation of Leishmanian parasites.

  1. Genomic cartography and proposal of nomenclature for the repeated, interspersed elements of the Leishmania major SIDER2 family and identification of SIDER2-containing transcripts.

    Science.gov (United States)

    Requena, Jose M; Rastrojo, Alberto; Garde, Esther; López, Manuel C; Thomas, M Carmen; Aguado, Begoña

    2017-03-01

    The genomes of most eukaryotic organisms contain a large number of transposable elements that are able to move from one genomic site to another either by transferring of DNA mobile elements (transposons) or transpose via reverse transcription of an RNA intermediate (retroposons). An exception to this rule is found in protists of the subgenus Leishmania, in which active retroposons degenerated after a flourishing era, leaving only retroposon remains; these have been classified into two families: SIDER1 and SIDER2. In this work, we have re-examined the elements belonging to the family SIDER2 present in the genome of Leishmania major with the aim of providing a nomenclature that will facilitate a future reference to particular elements. According to sequence conservation, the 1100 SIDER2 elements have been grouped into subfamilies, and the inferred taxonomic relationships have also been incorporated into the nomenclature. Additionally, we are providing detailed data regarding the genomic distribution of these elements and their association with specific transcripts, based on the recently established transcriptome for L. major. Thus, the presented data can help to study and better understand the roles played by these degenerated retroposons in both regulation of gene expression and genome plasticity. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Habitats of the sandfly vectors of Leishmania tropica and L. major in a mixed focus of cutaneous leishmaniasis in southeast Tunisia.

    Science.gov (United States)

    Tabbabi, Ahmed; Ghrab, Jamila; Aoun, Karim; Ready, Paul Donald; Bouratbine, Aïda

    2011-08-01

    From 2009 to 2010, 3129 sandflies were caught in CDC light traps placed in various habitats in Ghomrassen, Tataouine governorate, southeast Tunisia, a mixed focus of human cutaneous leishmaniasis caused by Leishmania tropica and Leishmania major. Species diversity was quantified in anthropogenic, semi-anthropogenic and semi-natural locations. Sandflies were identified according to morphological characters and also by the comparative sequence analysis of a fragment of the mitochondrial cytochrome b gene to distinguish between two putative local vectors of L. tropica, namely Phlebotomus chabaudi and Phlebotomus riouxi. The lowest sandfly diversities were found in L. major sites, where the incriminated vector P. papatasi predominated in the burrows of the rodent reservoir hosts (Meriones) as well as inside and outside houses of human cases. In L. tropica sites, the incriminated peri-domestic vector Phlebotomus sergenti was the most abundant species inside houses, whereas P. riouxi or P. chabaudi was the dominant species in the semi-natural rocky habitats favoured by the putative rodent reservoir, Ctenodactylus gundi. All specimens of P. chabaudi identified molecularly had the diagnostic cytochrome b characters of P. riouxi, indicating either that the latter represents only a geographical variant of P. chabaudi or that these two species may sometimes hybridize. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Naturally occurring culturable aerobic gut flora of adult Phlebotomus papatasi, vector of Leishmania major in the Old World.

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    Jaba Mukhopadhyay

    Full Text Available Cutaneous leishmaniasis is a neglected, vector-borne parasitic disease and is responsible for persistent, often disfiguring lesions and other associated complications. Leishmania, causing zoonotic cutaneous leishmaniasis (ZCL in the Old World are mainly transmitted by the predominant sand fly vector, Phlebotomus papatasi. To date, there is no efficient control measure or vaccine available for this widespread insect-borne infectious disease.A survey was carried out to study the abundance of different natural gut flora in P. papatasi, with the long-term goal of generating a paratransgenic sand fly that can potentially block the development of Leishmania in the sand fly gut, thereby preventing transmission of leishmania in endemic disease foci. Sand flies, in particular, P. papatasi were captured from different habitats of various parts of the world. Gut microbes were cultured and identified using 16S ribosomal DNA analysis and a phylogenetic tree was constructed. We found variation in the species and abundance of gut flora in flies collected from different habitats. However, a few Gram-positive, nonpathogenic bacteria including Bacillus flexus and B. pumilus were common in most of the sites examined.Our results indicate that there is a wide range of variation of aerobic gut flora inhabiting sand fly guts, which possibly reflect the ecological condition of the habitat where the fly breeds. Also, some species of bacteria (B. pumilus, and B. flexus were found from most of the habitats. Important from an applied perspective of dissemination, our results support a link between oviposition induction and adult gut flora.

  4. The MyD88 protein 88 pathway is differently involved in immune responses induced by distinct substrains of Leishmania major.

    Science.gov (United States)

    Revaz-Breton, Mélanie; Ronet, Catherine; Ives, Annette; Torre, Yazmin Hauyon-La; Masina, Slavica; Tacchini-Cottier, Fabienne; Launois, Pascal

    2010-06-01

    Host resistance to Leishmania major is highly dependent on the development of a Th1 immune response. The TLR adaptator myeloid differentiation protein 88 (MyD88) has been implicated in the Th1 immune response associated with the resistant phenotype observed in C57BL/6 mice after infection with L. major. To investigate whether the MyD88 pathway is differentially used by distinct substrains of parasites, MyD88(-/-) C57BL/6 mice were infected with two substrains of L. major, namely L. major LV39 and L. major IR75. MyD88(-/-) mice were susceptible to both substrains of L. major, although with different kinetics of infection. The mechanisms involved during the immune response associated with susceptibility of MyD88(-/-) mice to L. major is however, parasite substrain-dependent. Susceptibility of MyD88(-/-) mice infected with L. major IR75 is a consequence of Th2 immune-deviation, whereas susceptibility of MyD88(-/-) mice to infection with L. major LV39 resulted from an impaired Th1 response. Depletion of regulatory T cells (Treg) partially restored IFN-gamma secretion and the Th1 immune response in MyD88(-/-) mice infected with L. major LV39, demonstrating a role of Treg activity in the development of an impaired Th1 response in these mice.

  5. The use of a water-soluble formazan complex to quantitate the cell number and mitochondrial function of Leishmania major promastigotes

    DEFF Research Database (Denmark)

    Berg, K; Zhai, L; Chen, M

    1994-01-01

    One of the methods to quantitate Leishmania major promastigotes (LmP) has been to utilize the formation of a formazan dye, which in turn is produced via conversion of an artificial substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The method has one major drawback...... as the formazan-like dye is released spontaneously into the medium, making it possible to perform several measurements on the same parasite culture without disturbing or killing the parasites. The measurements were shown to reflect the numbers of parasites as confirmed via comparative experiments using...... radioactive thymidine uptake and cell counting, respectively. The method is simple, fast, and highly reproducible and is suitable for drug screening, identification of drug-resistant isolates, and growth-kinetics studies. It is therefore contemplated that the MTS method will be a general and useful technique...

  6. The structure of tubulin-binding cofactor A from Leishmania major infers a mode of association during the early stages of microtubule assembly

    Energy Technology Data Exchange (ETDEWEB)

    Barrack, Keri L.; Fyfe, Paul K.; Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk [University of Dundee, Dow Street, Dundee DD1 5EH, Scotland (United Kingdom)

    2015-04-21

    The structure of a tubulin-binding cofactor from L. major is reported and compared with yeast, plant and human orthologues. Tubulin-binding cofactor A (TBCA) participates in microtubule formation, a key process in eukaryotic biology to create the cytoskeleton. There is little information on how TBCA might interact with β-tubulin en route to microtubule biogenesis. To address this, the protozoan Leishmania major was targeted as a model system. The crystal structure of TBCA and comparisons with three orthologous proteins are presented. The presence of conserved features infers that electrostatic interactions that are likely to involve the C-terminal tail of β-tubulin are key to association. This study provides a reagent and template to support further work in this area.

  7. The Leishmania promastigote surface antigen-2 (PSA-2) is specifically recognised by Th1 cells in humans with naturally acquired immunity to L. major

    DEFF Research Database (Denmark)

    Kemp, M; Handman, E; Kemp, K

    1998-01-01

    The promastigote surface antigen-2 (PSA-2) is a Leishmania parasite antigen, which can induce Th1-mediated protection against murine leishmaniasis when used as a vaccine. To evaluate PSA-2 as a human vaccine candidate the specific T-cell response to PSA-2 was characterised in individuals immune......-beta, and little interleukin-4, thereby showing a Th1 cytokine pattern. Parallel cultures showed clear Th1 and Th2 response patterns to purified protein derivative of tuberculin or tetanus toxoid, respectively. Flow cytometric analysis revealed that PSA-2 induced blastogenesis in the CD3 positive population...... and that these cells were the major source of interferon-gamma. The results show that Th1-like cells recognising PSA-2 are expanded during infection by L. major and that they maintain their Th1-like cytokine profile upon reactivation in vitro. Since immunity to cutaneous leishmaniasis is mediated by antigen...

  8. LIGHT Is critical for IL-12 production by dendritic cells, optimal CD4+ Th1 cell response, and resistance to Leishmania major.

    Science.gov (United States)

    Xu, Guilian; Liu, Dong; Okwor, Ifeoma; Wang, Yang; Korner, Heinrich; Kung, Sam K P; Fu, Yang-Xin; Uzonna, Jude E

    2007-11-15

    Although studies indicate LIGHT (lymphotoxin (LT)-like, exhibits inducible expression and competes with HSV glycoprotein D for herpes virus entry mediator (HVEM), a receptor expressed by T lymphocytes) enhances inflammation and T cell-mediated immunity, the mechanisms involved in this process remain obscure. In this study, we assessed the role of LIGHT in IL-12 production and development of CD4(+) Th cells type one (Th1) in vivo. Bone marrow-derived dendritic cells from LIGHT(-/-) mice were severely impaired in IL-12p40 production following IFN-gamma and LPS stimulation in vitro. Furthermore, blockade of LIGHT in vitro and in vivo with HVEM-Ig and LT beta receptor (LTbetaR)-Ig leads to impaired IL-12 production and defective polyclonal and Ag-specific IFN-gamma production in vivo. In an infection model, injection of HVEM-Ig or LTbetaR-Ig into the usually resistant C57BL/6 mice results in defective IL-12 and IFN-gamma production and severe susceptibility to Leishmania major that was reversed by rIL-12 treatment. This striking susceptibility to L. major in mice injected with HVEM-Ig or LTbetaR-Ig was also reproduced in LIGHT(-/-) --> RAG1(-/-) chimeric mice. In contrast, L. major-infected LTbeta(-/-) mice do not develop acute disease, suggesting that the effect of LTbetaR-Ig is not due to blockade of membrane LT (LTalpha1beta2) signaling. Collectively, our data show that LIGHT plays a critical role for optimal IL-12 production by DC and the development of IFN-gamma-producing CD4(+) Th1 cells and its blockade results in severe susceptibility to Leishmania major.

  9. Gluconeogenesis in Leishmania mexicana

    Science.gov (United States)

    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

  10. Comparison of Parasite Burden Using Real-Time Polymerase Chain Reaction Assay and Limiting Dilution Assay in Leishma-nia major Infected Mouse

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    Somayeh GHOTLOO

    2015-12-01

    Full Text Available Background:Limiting dilution assay is considered as the gold standard method for quantifying the number of parasites in the animal model of Leishmania infection. Nowadays, real-time PCR is being increasingly applied to quantify infectious agents. In the present study, a real-time PCR assay was developed to estimate para­site burdens in lymph nodes of Leishmania major infected BALB/C mice. Enumera­tion of parasites was also performed by limiting dilution assay and compared with the results of real-time PCR based quantification.Methods:The SYBR Green based real- time PCR assay was performed to amplify a 75 bp fragment of superoxide dismutase B1 gene in the lymph nodes of L. major infected BALB/C mice 8 weeks post infection. Mice were infected subcutaneously at the base of their tail with 2 × 105L. major promastigotes in the stationary phase of growth. To compare parasite burdens obtained by real-time PCR assay with those of limiting dilution assay, twelve 8-fold serial dilutions of the lymph node homoge­nates were prepared in the Schneider medium and incubated at 26°C.After 7 days, wells containing motile parasites were identified by direct observation under an inverted light microscope and the total number of parasites was estimated using the ELIDA software.Results:Spearman's correlation coefficient of the parasite burdens between real-time PCR and limiting dilution assay was 0.72 (Pvalue = 0.008.Conclusion:Real-time PCR assay is an appropriate replacement to existing limit­ing dilution assay in quantifying parasite burden in the experimental model of Leishma­nia infection.

  11. Crystal structure of a concentrative nucleoside transporter from Vibrio cholerae at 2.4;#8201;Å

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    Johnson, Zachary Lee; Cheong, Cheom-Gil; Lee, Seok-Yong (Duke)

    2012-07-11

    Nucleosides are required for DNA and RNA synthesis, and the nucleoside adenosine has a function in a variety of signalling processes. Transport of nucleosides across cell membranes provides the major source of nucleosides in many cell types and is also responsible for the termination of adenosine signalling. As a result of their hydrophilic nature, nucleosides require a specialized class of integral membrane proteins, known as nucleoside transporters (NTs), for specific transport across cell membranes. In addition to nucleosides, NTs are important determinants for the transport of nucleoside-derived drugs across cell membranes. A wide range of nucleoside-derived drugs, including anticancer drugs (such as Ara-C and gemcitabine) and antiviral drugs (such as zidovudine and ribavirin), have been shown to depend, at least in part, on NTs for transport across cell membranes. Concentrative nucleoside transporters, members of the solute carrier transporter superfamily SLC28, use an ion gradient in the active transport of both nucleosides and nucleoside-derived drugs against their chemical gradients. The structural basis for selective ion-coupled nucleoside transport by concentrative nucleoside transporters is unknown. Here we present the crystal structure of a concentrative nucleoside transporter from Vibrio cholerae in complex with uridine at 2.4 {angstrom}. Our functional data show that, like its human orthologues, the transporter uses a sodium-ion gradient for nucleoside transport. The structure reveals the overall architecture of this class of transporter, unravels the molecular determinants for nucleoside and sodium binding, and provides a framework for understanding the mechanism of nucleoside and nucleoside drug transport across cell membranes.

  12. Cross-protective effect of a combined L5 plus L3 Leishmania major ribosomal protein based vaccine combined with a Th1 adjuvant in murine cutaneous and visceral leishmaniasis.

    Science.gov (United States)

    Ramirez, Laura; Corvo, Laura; Duarte, Mariana C; Chávez-Fumagalli, Miguel A; Valadares, Diogo G; Santos, Diego M; de Oliveira, Camila I; Escutia, Marta R; Alonso, Carlos; Bonay, Pedro; Tavares, Carlos A P; Coelho, Eduardo A F; Soto, Manuel

    2014-01-02

    Two Leishmania major ribosomal proteins L3 (LmL3) and L5 (LmL5) have been described as protective molecules against cutaneous leishmaniasis due to infection with L. major and Leishmania braziliensis in BALB/c mice when immunized with a Th1 adjuvant (non-methylated CpG-oligodeoxynucleotides; CpG-ODN). In the present study we analyzed the cross-protective efficacy of an LmL3-LmL5-CpG ODN combined vaccine against infection with Leishmania amazonensis and Leishmania chagasi (syn. Leishmania infantum) the etiologic agents of different clinical forms of human leishmaniasis in South America. The combined vaccine was administered subcutaneously to BALB/c mice. After immunization the cellular and humoral responses elicited were analyzed. Mice were independently challenged with L. amazonensis and L. chagasi. The size of the cutaneous lesions caused by the infection with the first species, the parasite loads and the immune response in both infection models were analyzed nine weeks after challenge. Mice vaccinated with the combined vaccine showed a Th1-like response against LmL3 and LmL5. Vaccinated mice were able to delay lesion development due to L. amazonensis infection and to control parasite loads in the site of infection. A reduction of the parasite burden in the lymph nodes draining the site of infection and in the liver and spleen was observed in the vaccinated mice after a subcutaneous infection with L. chagasi. In both models of infection, protection was correlated to parasite antigen-specific production of IFN-γ and down-regulation of parasite-mediated IL-4 and IL-10 responses. The data presented here demonstrate the potential use of L. major L3 and L5 recombinant ribosomal proteins for the development of vaccines against various Leishmania species.

  13. Antileishmanial Activity of Lavandula angustifolia and Rosmarinus Officinalis Essential Oils and Nano-emulsions on Leishmania major (MRHO/IR/75/ER

    Directory of Open Access Journals (Sweden)

    Azar SHOKRI

    2017-12-01

    Full Text Available AbstractBackground: The aim of present study was to evaluate antileishmanial effects of Lavandula angustifolia (L. angustifolia and Rosmarinus officinalis (R. officinalis medicinal plants essential oils and nano-emulsions on Leishmania major (L. major. Methods: The present study was performed in Leishmaniasis Reference Lab at Mazandaran University of Medical Sciences, Iran during 2016-2017. The IC50 values were calculated in both the promastigote and amastigote stages in J774 macrophage in comparison with meglumine antimoniate (MA as positive control. In addition, cytotoxicity effects of essential oils and nano-emulsions prepared from both plants against macrophages were evaluated.Results: Both essential oil and nano-emulsion of Lavander and Rosemary showed anti-leishmania activity on promastigote with IC50=0.11 μl/mL, IC50=0.26 μl/mL, and IC50=0.08 μl/mL respectively. Moreover, during amastigote assay, Lavander and Rosemary essential oils and nano-emulsion were effective at least in concentration of 0.12 μl/mL and 0.06 µl/mL (P=0.0001 respectively, on mean infected macrophages (MIR and amastigotes in macrophages (P=0.0001. Additionally, cytotoxicity assay against macrophage revealed no toxicity on the host cells at IC50 concentrations.Conclusion: The nano-emulsions of both plants were more effective than essential oil in both MIR and amastigote. However, in comparison with MA, the Lavander essential oil is more effective in reducing MIR. Rosemary nano-emulsion reduced MIR significantly more than MA in concentration of 0.25 μl/mL (P<0.001. Further investigations are recommended to evaluate the effect of these medicinal plants in murine models.

  14. Antileishmanial Activity of Lavandula angustifolia and Rosmarinus Officinalis Essential Oils and Nano-emulsions on Leishmania major (MRHO/IR/75/ER).

    Science.gov (United States)

    Shokri, Azar; Saeedi, Majid; Fakhar, Mahdi; Morteza-Semnani, Katayoun; Keighobadi, Masoud; Hosseini Teshnizi, Saeed; Kelidari, Hamid Reza; Sadjadi, Siamak

    2017-01-01

    The aim of present study was to evaluate antileishmanial effects of Lavandula angustifolia ( L. angustifolia ) and Rosmarinus officinalis ( R. officinalis ) medicinal plants essential oils and nano-emulsions on Leishmania major (L . major). The present study was performed in Leishmaniasis Reference Lab at Mazandaran University of Medical Sciences, Iran during 2016-2017. The IC50 values were calculated in both the promastigote and amastigote stages in J774 macrophage in comparison with meglumine antimoniate (MA) as positive control. In addition, cytotoxicity effects of essential oils and nano-emulsions prepared from both plants against macrophages were evaluated. Both essential oil and nano-emulsion of Lavander and Rosemary showed anti-leishmania activity on promastigote with IC 50 =0.11 μl/mL, IC 50 =0.26 μl/mL, and IC 50 =0.08 μl/mL respectively. Moreover, during amastigote assay, Lavander and Rosemary essential oils and nano-emulsion were effective at least in concentration of 0.12 μl/mL and 0.06 μl/mL ( P =0.0001) respectively, on mean infected macrophages (MIR) and amastigotes in macrophages ( P =0.0001). Additionally, cytotoxicity assay against macrophage revealed no toxicity on the host cells at IC 50 concentrations. The nano-emulsions of both plants were more effective than essential oil in both MIR and amastigote. However, in comparison with MA, the Lavander essential oil is more effective in reducing MIR. Rosemary nano-emulsion reduced MIR significantly more than MA in concentration of 0.25 μl/mL ( P <0.001). Further investigations are recommended to evaluate the effect of these medicinal plants in murine models.

  15. Behavior of Leishmania major metacyclic promastigotes during the course of infection and immune response development in resistant versus susceptible hosts Comportamento de promastigoteas metacíclicos de Leishmania major durante o curso da infecção e da resposta imune em hospedeiros resistentes versus suscetíveis

    Directory of Open Access Journals (Sweden)

    Regina Coeli Cunha Dórea

    2003-11-01

    Full Text Available Little is known on the epitopes derived from metacyclic promastigotes of Leishmania that are important on the regulation or destruction of the parasite, as targets of immune attack in the vertebrate host. In this study we investigated an alternative method to obtain metacyclic promasigotes of Leishmania major, as evaluated by the course of infection and delayed-type hipersensitivity (DTH in resistant versus susceptible inbred mice. Non-infective (procyclic promastigotes of L. major recently transformed from tissue amastigotes were attached to a negatively charged glass-wool column, whereas metacyclic promastigotes were not bound to columns and could be easily recovered. Optimal chromatography conditions were validated through statistical analyses. Parasite average yield from glass wool columns and promastigote viability were estimated by light microscopy. Metacyclic promastigotes yielded 43.5% to 57.5%. Different patterns of cutaneous lesions were obtained in BALB/c (susceptible and C57BL/6 (resistant mice, the former with highly infective lesions induced by metacyclic promastigotes. DTH responses proved to be higher in groups of C57BL/6 mice which were infected with metacyclic promastigotes. These results indicate that the new method could be integrated with the investigation of metacyclogenesis of Leishmania in vivo.Pouco se conhece sobre os epítopos derivados de promastigotas metacíclicos de Leishmania que são importantes para a regulação ou destruição do parasita, como alvos de ação imunológica no hospedeiro vertebrado. Neste estudo, nós investigamos um método alternativo para obter promastigotas metacíclicos de Leishmania major, pela avaliação do curso da infecção e reação de hipersensibilidade do tipo retardado (HTR em hospedeiros resistentes e susceptíveis. Promastigotas não-infectantes (procíclicos de L. major, recentemente isolados de amastigotas, foram selecionados pela adesão a colunas de lã de vidro

  16. Mononuclear cells from patients recovered from cutaneous leishmaniasis respond to Leishmania major amastigote class I nuclease with a predominant Th1-like response

    Science.gov (United States)

    Farajnia, S; Mahboudi, F; Ajdari, S; Reiner, N E; Kariminia, A; Alimohammadian, M H

    2005-01-01

    The Leishmania major amastigote class I nuclease (LmaCIN) is a developmentally regulated protein that is highly expressed in the amastigote stage of L. major. This protein is homologous to the P4 nuclease of L. pifanoi, which has been shown to induce protective immune response in a murine model. To evaluate LmaCIN as a potential human vaccine candidate, cellular immune responses to recombinant LmaCIN were examined in individuals recovered from Old World cutaneous leishmaniasis. Peripheral blood mononuclear cells (PBMC) from patients recovered from L. major infection were cultured either with recombinant LmaCIN or autoclaved L. major (ALM) as control. rLmaCIN induced significant proliferation of PBMC from 90% of recovered patients. Phenotypic analysis of proliferating cells showed that CD8+ cells were the predominant cell type proliferating in response to rLmaC1N. Screening of culture supernatants for cytokines showed that rLmaCIN induced high levels of interferon (IFN)-γ (mean ± s.e.m.: 1398 ± 179 pg/ml) associated with little interleukin (IL)-10 and little or no IL-5 production. These findings show that LmaCIN is immunogenic in humans during L. major infection and that it can elicit immunological responses relevant to immunoprophylaxis of leishmaniasis. PMID:15730396

  17. In vitro activity of the essential oil of Cymbopogon citratus and its major component (citral) on Leishmania amazonensis.

    Science.gov (United States)

    Santin, Marta Regina; dos Santos, Adriana Oliveira; Nakamura, Celso Vataru; Dias Filho, Benedito Prado; Ferreira, Izabel Cristina Piloto; Ueda-Nakamura, Tânia

    2009-11-01

    Leishmaniasis causes considerable mortality throughout the world, affecting more than 12 million people. Cymbopogon citratus (DC) Stapf, Family Poaceae, is a widely used herb in tropical countries and is also known as a source of ethnomedicines. In this study, the inhibitory effect and the morphological and ultrastructural alterations on Leishmania amazonensis by the essential oil (EO) of C. citratus and its main constituent, citral, were evaluated. The results showed that the antiproliferative activity of EO on promastigotes and axenic amastigotes, and intracellular amastigote forms of L. amazonensis was significantly better than citral, and indicated a dose-dependent effect. Neither compound showed a cytotoxic effect on macrophage strain J774G8. The promastigote forms of L. amazonensis underwent remarkable morphological and ultrastructural alterations compared with untreated cultures. These alterations were visible by light, scanning, and transmission electron microscopy of promastigotes treated with EO and citral at concentrations corresponding to the IC(50) (1.7 and 8.0 microg/ml) and IC(90) (3.2 and 25 microg/ml), respectively, after 72 h of incubation. This study revealed that citral-rich essential oil from C. citratus has promising antileishmanial properties, and is a good candidate for further research to develop a new anti-protozoan drug.

  18. Experimental transmission of Leishmania infantum by two major vectors: a comparison between a viscerotropic and a dermotropic strain.

    Science.gov (United States)

    Maia, Carla; Seblova, Veronika; Sadlova, Jovana; Votypka, Jan; Volf, Petr

    2011-06-01

    We quantified Leishmania infantum parasites transmitted by natural vectors for the first time. Both L. infantum strains studied, dermotropic CUK3 and viscerotropic IMT373, developed well in Phlebotomus perniciosus and Lutzomyia longipalpis. They produced heavy late-stage infection and colonized the stomodeal valve, which is a prerequisite for successful transmission. Infected sand fly females, and especially those that transmit parasites, feed significantly longer on the host (1.5-1.8 times) than non-transmitting females. Quantitative PCR revealed that P. perniciosus harboured more CUK3 strain parasites, while in L. longipalpis the intensity of infection was higher for the IMT373 strain. However, in both sand fly species the parasite load transmitted was higher for the strain with dermal tropism (CUK3). All but one sand fly female infected by the IMT373 strain transmitted less than 600 promastigotes; in contrast, 29% of L. longipalpis and 14% of P. perniciosus infected with the CUK3 strain transmitted more than 1000 parasites. The parasite number transmitted by individual sand flies ranged from 4 up to 4.19×10(4) promastigotes; thus, the maximal natural dose found was still about 250 times lower than the experimental challenge dose used in previous studies. This finding emphasizes the importance of determining the natural infective dose for the development of an accurate experimental model useful for the evaluation of new drugs and vaccines.

  19. Methanolic Extract’s Activity of Artemisia absinthium, Vitexagnus-castus and Phytolacaamericana Against Leishmania major; in vitro and in vivo

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    Khanjani Jafroodi S.1 MSc,

    2015-06-01

    Full Text Available Aims Leishmaniasis is the most prevalent vector- borne parasitic disease in Iran. Drug treatment is the best way to treat leishmaniasis, while the common drugs are not efficient enough and inevitable side effects limit using these drugs. The aim of this study was to analyze in vitro and in vivo activity of the methanolic extract of Artemisia absinthium, Vitex agnus-castus and Phytolaca americana Against Leishmania major. Materials & Methods The methanolic extracts of Artemisia absinthium, Vitex agnuscastus and Phytolaca americana were prepared by cold percolation method. The inhibitory concentration 50 (IC50 of the plant extracts was determined against L. major promastigotes followed by efficacy evaluation of the extracts against amastigotes and in vivo assay in the BALB/c animal model. The data was analyzed with SPSS 19 software using Student’s T test and ANOVA. Findings Artemisia absinthium had the highest amount of active compounds against promastigotes of L. major (IC50=159.45 and antiprolifrative activity of Artemisia absinthium on both forms of L. major (extracellular promastigotes and intracellular amastigotes was the highest (MI=33%. Vitex agnus-castus had the least toxic effect for macrophages (8%. All extracts limited the progression of lesion size versus control group, however, only inhibitory effect of Artemisia absinthium extract was statistically significant. Conclusion Artemisia absinthium is the most effective growth inhibitor of amastigotes in animal lesions and it is safe for drug application in human and animals.

  20. The PGE2/IL-10 Axis Determines Susceptibility of B-1 Cell-Derived Phagocytes (B-1CDP to Leishmania major Infection.

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    Angélica F Arcanjo

    Full Text Available B-1 cells can be differentiated from B-2 cells because they are predominantly located in the peritoneal and pleural cavities and have distinct phenotypic patterns and activation properties. A mononuclear phagocyte derived from B-1 cells (B-1CDP has been described. As the B-1CDP cells migrate to inflammatory/infectious sites and exhibit phagocytic capacity, the microbicidal ability of these cells was investigated using the Leishmania major infection model in vitro. The data obtained in this study demonstrate that B-1CDP cells are more susceptible to infection than peritoneal macrophages, since B-1CDP cells have a higher number of intracellular amastigotes forms and consequently release a larger number of promastigotes. Exacerbated infection by L. major required lipid bodies/PGE2 and IL-10 by B-1CDP cells. Both infection and the production of IL-10 were decreased when PGE2 production was blocked by NSAIDs. The involvement of IL-10 in this mechanism was confirmed, since B-1CDP cells from IL-10 KO mice are more competent to control L. major infection than cells from wild type mice. These findings further characterize the B-1CDP cells as an important mononuclear phagocyte that plays a previously unrecognized role in host responses to L. major infection, most likely via PGE2-driven production of IL-10.

  1. Diagnostic Antigens of Leishmania.

    Science.gov (United States)

    1994-01-31

    L. major (LTM p-2), L. major (Friedlander), and Trypanosoma cruzi (MHOM/CH/00/Tulahuen C2) were used. Leishmania promastigotes and T. cruzi ...some weak hybridization was observed with L. amazonensis, but none was seen with L. braziliensis, L. guyanensis, or T cruzi . A similar, overlapping... cruzi (8) have been previously isolated by us. To address this possibility in rLt-1, a portion of the repeat was expressed separately as rLt-lr. The

  2. Structural modeling and docking studies of ribose 5-phosphate isomerase from Leishmania major and Homo sapiens: a comparative analysis for Leishmaniasis treatment.

    Science.gov (United States)

    Capriles, Priscila V S Z; Baptista, Luiz Phillippe R; Guedes, Isabella A; Guimarães, Ana Carolina R; Custódio, Fabio L; Alves-Ferreira, Marcelo; Dardenne, Laurent E

    2015-02-01

    Leishmaniases are caused by protozoa of the genus Leishmania and are considered the second-highest cause of death worldwide by parasitic infection. The drugs available for treatment in humans are becoming ineffective mainly due to parasite resistance; therefore, it is extremely important to develop a new chemotherapy against these parasites. A crucial aspect of drug design development is the identification and characterization of novel molecular targets. In this work, through an in silico comparative analysis between the genomes of Leishmania major and Homo sapiens, the enzyme ribose 5-phosphate isomerase (R5PI) was indicated as a promising molecular target. R5PI is an important enzyme that acts in the pentose phosphate pathway and catalyzes the interconversion of d-ribose-5-phosphate (R5P) and d-ribulose-5-phosphate (5RP). R5PI activity is found in two analogous groups of enzymes called RpiA (found in H. sapiens) and RpiB (found in L. major). Here, we present the first report of the three-dimensional (3D) structures and active sites of RpiB from L. major (LmRpiB) and RpiA from H. sapiens (HsRpiA). Three-dimensional models were constructed by applying a hybrid methodology that combines comparative and ab initio modeling techniques, and the active site was characterized based on docking studies of the substrates R5P (furanose and ring-opened forms) and 5RP. Our comparative analyses show that these proteins are structural analogs and that distinct residues participate in the interconversion of R5P and 5RP. We propose two distinct reaction mechanisms for the reversible isomerization of R5P to 5RP, which is catalyzed by LmRpiB and HsRpiA. We expect that the present results will be important in guiding future molecular modeling studies to develop new drugs that are specially designed to inhibit the parasitic form of the enzyme without significant effects on the human analog. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Zoonotic disease in a peripheral population: persistence and transmission of Leishmania major in a putative sink-source system in the Negev Highlands, Israel.

    Science.gov (United States)

    Berger, Ruti; Wasserberg, Gideon; Warburg, Alon; Orshan, Laor; Kotler, Burt P

    2014-08-01

    Populations at the edge of their geographic distributions are referred to as peripheral populations. Very little attention has been given to this topic in the context of persistence of infectious disease in natural populations. In this study, we examined this question using zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania major in the Negev Desert of Israel as a model system. Here, we suggest that the regional persistence of Phlebotomus papatasi populations and L. major transmission in the Sede Boqer region could be explained through processes akin to sink-source and/or mainland-island metapopulation dynamics. Given its potentially enzootically superior ecological conditions, we hypothesize that the Zin Valley ecotope constitutes the "mainland" or the "source" patch for the Sede Boqer area where L. major transmission is persistent and resistant to local extinctions (die-outs) whereas the local sand fly populations on the Zin Plateau ("island patch" or "sink patch") are more prone to local extinctions. Between 2007 and 2008, we trapped sand flies and sand rats in the two areas and compared sand fly abundance and L. major infection prevalence in both. In both 2007 and 2008, sand fly abundance was high and continuous in the Zin Wadi but low and discontinuous in the Zin Plateau. Infection prevalence of sand rats was significantly higher in the Wadi (13%) compared with the Zin Plateau (3%). Minimum infection rate in sand flies did not differ significantly between the two areas. Overall, our results are consistent with the premise that the Zin Valley population is relatively robust in terms of L. major transmission, whereas transmission is potentially more tenuous in the plateau. Understanding the biotic and abiotic processes enabling the persistence of L. major and other vector-borne diseases in peripheral disease foci is important for predicting the effect of anthropogenic land use and climate change.

  4. Crystal Structure of the Leishmania Major Phosphodiesterase LmjPDEB1 and Insight into the Design of hte Parasite-Selective Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Wang,H.; Yan, Z.; Geng, J.; Kunz, S.; Seebeck, T.; Ke, H.

    2007-01-01

    Human leishmaniasis is a major public health problem in many countries, but chemotherapy is in an unsatisfactory state. Leishmania major phosphodiesterases (LmjPDEs) have been shown to play important roles in cell proliferation and apoptosis of the parasite. Thus LmjPDE inhibitors may potentially represent a novel class of drugs for the treatment of leishmaniasis. Reported here are the kinetic characterization of the LmjPDEB1 catalytic domain and its crystal structure as a complex with 3-isobutyl-1-methylxanthine (IBMX) at 1.55 Angstroms resolution. The structure of LmjPDEB1 is similar to that of human PDEs. IBMX stacks against the conserved phenylalanine and forms a hydrogen bond with the invariant glutamine, in a pattern common to most inhibitors bound to human PDEs. However, an extensive structural comparison reveals subtle, but significant differences between the active sites of LmjPDEB1 and human PDEs. In addition, a pocket next to the inhibitor binding site is found to be unique to LmjPDEB1. This pocket is isolated by two gating residues in human PDE families, but constitutes a natural expansion of the inhibitor binding pocket in LmjPDEB1. The structure particularity might be useful for the development of parasite-selective inhibitors for the treatment of leishmaniasis.

  5. Species diversity and relative abundance of phlebotomine sand flies (Diptera: Psychodidae) on three Army installations in the southern United States and susceptibility of a domestic sand fly to infection with Old World Leishmania major.

    Science.gov (United States)

    Claborn, David M; Rowton, Edgar D; Lawyer, Phillip G; Brown, Grayson C; Keep, Lisa W

    2009-11-01

    Leishmania infections in American veterans of Iraq and Afghanistan have raised concern that veterans could serve as reservoirs of Old World parasites for domestic vector populations. A survey of sand flies on three U.S. Army facilities in the southern United States was conducted to identify potential vectors. Five species, including two new state records, are reported for Fort Hood, TX. Very few flies were detected in Fort Bragg, NC. Large numbers of a man-biting species, Lutzomyia shannoni, were trapped on Fort Campbell, KY. Weekly activity patterns for dominant species are presented. In addition, an infection experiment was conducted to determine if a domestic sand fly is susceptible to infection with Old World Leishmania major. Lu. shannoni became infected and supported Le. major up to 6 days postprandial. Metacyclogenesis and actual transmission of Le. major to an uninfected mouse did not occur because infected flies did not take subsequent blood meals.

  6. Characterization of immune response to killed leishmania major promastigotes plus BCG vaccine in Sudanese volunteers: a double-blind placebo controlled study

    International Nuclear Information System (INIS)

    Sati, Iman Nasr Eldin

    1996-12-01

    This work was examined whether intradermal immunization of healthy adult Sudanese volunteers with killed leishmania major (KLM) promastigotes plus BCG would induce antigen-specific T cell responses. Only healthy Sudanese volunteers with negative reactivity to leishmania skin test and with ≤20 mm induration of reactivity to purified protein derivative (PPD) were included in the trial. Group (A) (n=3): received a single dose (0.1ml) at a concentration of 10 mg protein of a whole cell component of KLM promastigotes/ml BCG, group (B) (n=12): received as a single dose of viable attenuated BCG alone (0.1 ml) at a concentration of 1 mg protein/ml diluent, group (C) (n=11): received the vaccine diluent only (Placebo) (o.1 ml). Study subjects were tested for their immunological and clinical responses before intervention, . Following vaccination 65% of group (A) subjects converted in their reactivity to leishmanin skin testing,non of the BCG vaccinated subjects converted in leishmanin skin test and only one subject of group (C) became leishmanin positive. Levels of Interferon-gamma (IFN-γ), interleukin-5 (IL-5) and interleukin-10 (IL-10) were measured by a double sandwich enzyme-linked immunosorbent assay (ELISA). A vaccine was considered as a positive responder in terms of cytokine production when the level of the produced cytokine was equal to the 80th percentile of the levels produced by the volunteers in the placebo group. 92% of the group vaccinated with KLM=BCG had circulating T cells. No significant of IL-5 or Il-10 was reported in any of the volunteers in the three group. Levels of anti l eishmania specific IgG were measured by ELISA in optical densities. Volunteers with mean antibody titre above the cut-off point (mean=3X standard deviation) were considered to have positive scores. Accordingly after vaccination 7.69% one volunteers in group (A) had a positive antibody response corresponding to 0% in the other two groups. No serious side effects were reported

  7. Immune modulating effects of NKT cells in a physiologically low dose Leishmania major infection model after αGalCer analog PBS57 stimulation.

    Directory of Open Access Journals (Sweden)

    Klaus G Griewank

    2014-06-01

    Full Text Available Leishmaniasis is a parasitic infection affecting ∼12 million people worldwide, mostly in developing countries. Treatment options are limited and no effective vaccines exist to date. Natural Killer T (NKT cells are a conserved innate-like lymphocyte population with immunomodulating effects in various settings. A number of reports state a role of NKT cells in different models of Leishmania infection. Here, we investigated the effect of NKT cells in a physiologically relevant, intradermal low dose infection model. After inoculation of 103 infectious-stage L. major, comparable numbers of skin-immigrating NKT cells in both susceptible BALB/c mice and resistant C57BL/6 mice were noted. Compared to their wild type counterparts, NKT cell-deficient mice on a C57BL/6 background were better able to contain infection with L. major and showed decreased IL-4 production in cytokine analysis performed 5 and 8 weeks after infection. Low doses of the NKT cell stimulating αGalCer analog PBS57 applied at the time of infection led to disease exacerbation in C57BL/6 wild-type, but not NKT-deficient mice. The effect was dependent both on the timing and amount of PBS57 administered. The effect of NKT cell stimulation by PBS57 proved to be IL-4 dependent, as it was neutralized in IL-4-deficient C57BL/6 or anti-IL-4 antibody-treated wild-type mice. In contrast to C57BL/6 mice, administration of PBS57 in susceptible BALB/c mice resulted in an improved course of disease. Our results reveal a strain- and cytokine-dependent regulatory role of NKT cells in the development of immunity to low dose L. major infections. These effects, probably masked in previous studies using higher parasite inocula, should be considered in future therapy and immunization approaches.

  8. Immune modulating effects of NKT cells in a physiologically low dose Leishmania major infection model after αGalCer analog PBS57 stimulation.

    Science.gov (United States)

    Griewank, Klaus G; Lorenz, Beate; Fischer, Michael R; Boon, Louis; Lopez Kostka, Susanna; von Stebut, Esther

    2014-06-01

    Leishmaniasis is a parasitic infection affecting ∼12 million people worldwide, mostly in developing countries. Treatment options are limited and no effective vaccines exist to date. Natural Killer T (NKT) cells are a conserved innate-like lymphocyte population with immunomodulating effects in various settings. A number of reports state a role of NKT cells in different models of Leishmania infection. Here, we investigated the effect of NKT cells in a physiologically relevant, intradermal low dose infection model. After inoculation of 103 infectious-stage L. major, comparable numbers of skin-immigrating NKT cells in both susceptible BALB/c mice and resistant C57BL/6 mice were noted. Compared to their wild type counterparts, NKT cell-deficient mice on a C57BL/6 background were better able to contain infection with L. major and showed decreased IL-4 production in cytokine analysis performed 5 and 8 weeks after infection. Low doses of the NKT cell stimulating αGalCer analog PBS57 applied at the time of infection led to disease exacerbation in C57BL/6 wild-type, but not NKT-deficient mice. The effect was dependent both on the timing and amount of PBS57 administered. The effect of NKT cell stimulation by PBS57 proved to be IL-4 dependent, as it was neutralized in IL-4-deficient C57BL/6 or anti-IL-4 antibody-treated wild-type mice. In contrast to C57BL/6 mice, administration of PBS57 in susceptible BALB/c mice resulted in an improved course of disease. Our results reveal a strain- and cytokine-dependent regulatory role of NKT cells in the development of immunity to low dose L. major infections. These effects, probably masked in previous studies using higher parasite inocula, should be considered in future therapy and immunization approaches.

  9. Nucleotides, Nucleosides, and Nucleobases

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank; Dandanell, Gert; Hove-Jensen, Bjarne

    2008-01-01

    We review literature on the metabolism of ribo- and deoxyribonucleotides, nucleosides, and nucleobases in Escherichia coli and Salmonella,including biosynthesis, degradation, interconversion, and transport. Emphasis is placed on enzymology and regulation of the pathways, at both the level of gene...

  10. Comparative assessment of a DNA and protein Leishmania donovani gamma glutamyl cysteine synthetase vaccine to cross-protect against murine cutaneous leishmaniasis caused by L. major or L. mexicana infection.

    Science.gov (United States)

    Campbell, S A; Alawa, J; Doro, B; Henriquez, F L; Roberts, C W; Nok, A; Alawa, C B I; Alsaadi, M; Mullen, A B; Carter, K C

    2012-02-08

    Leishmaniasis is a major health problem and it is estimated that 12 million people are currently infected. A vaccine which could cross-protect people against different Leishmania spp. would facilitate control of this disease as more than one species of Leishmania may be present. In this study the ability of a DNA vaccine, using the full gene sequence for L. donovani gamma glutamyl cysteine synthetase (γGCS) incorporated in the pVAX vector (pVAXγGCS), and a protein vaccine, using the corresponding recombinant L. donovani γGCS protein (LdγGCS), to protect against L. major or L. mexicana infection was evaluated. DNA vaccination gave transient protection against L. major and no protection against L. mexicana despite significantly enhancing specific antibody titres in vaccinated infected mice compared to infected controls. Vaccination with the LdγGCS protected against both species but only if the protein was incorporated into non-ionic surfactant vesicles for L. mexicana. The results of this study indicate that a L. donovani γGCS vaccine could be used to vaccinate against more than one Leishmania species but only if the recombinant protein is used. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Distribution, fauna and seasonal variation of sandflies, simultaneous detection of nuclear internal transcribed spacer ribosomal DNA gene of Leishmania major in Rhombomys opimus and Phlebotomus papatasi, in Natanz district in central part of Iran.

    Science.gov (United States)

    Parvizi, Parviz; Akhoundi, Mohammad; Mirzaei, Hanieh

    2012-01-01

    Zoonotic cutaneous leishmaniasis (ZCL) due to Leishmania major is increasing in many parts of Iran. This disease originally is a disease found in gerbils. Leishmania parasites are transmitted by sandflies that live and breed in gerbil burrows. Nested PCR amplified Leishmania ITS1-5.8S rRNA gene in both main reservoir host "Rhombomys opimus" and in the "Phlebotomus papatasi" main vector of ZCL, in Iran. Population differentiation and seasonal variation of sandflies were analyzed at a microgeographical level in order to identify any isolation by distance, habitat or seasons. Populations of sandflies were sampled from the edges of villages in Natanz, Isfahan province, Iran, using the Centers for Disease Control traps and sticky papers. Individual sandflies were identified based on external and internal morphological characters. Nested PCR protocols were used to amplify Leishmania ITS1-5.8S rRNA gene, which were shown to be species-specific via DNA sequence. A total of 4500 sandflies were collected and identified. P. papatasi, Phlebotomus sergenti and Phlebotomus jacusieli from genus Phlebotomus and Sergentomyia sintoni and Sergentomyia clydei from genus Sergentomyia were identified in this region. P. papatasi was the most abundant sandfly in the collections. Ten out of 549 female P. papatasi and four out of 19 R. opimus were found to be infected with L. major. Seasonal activity of sandflies starts in June and ends in November. Abundance of P. papatasi was in September. Finding and molecular typing of L. major in P. papatasi and R. opimus confirmed the main vector and reservoir in this region.

  12. Structure determination of glycogen synthase kinase-3 from Leishmania major and comparative inhibitor structure-activity relationships with Trypanosoma brucei GSK-3

    Energy Technology Data Exchange (ETDEWEB)

    Ojo, Kayode K; Arakaki, Tracy L; Napuli, Alberto J; Inampudi, Krishna K; Keyloun, Katelyn R; Zhang, Li; Hol, Wim G.J.; Verlind, Christophe L.M.J.; Merritt, Ethan A; Van Voorhis, Wesley C [UWASH

    2012-04-24

    Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18_V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 Å resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3β (HsGSK-3β) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found.

  13. Mechanisms of immunity to Leishmania major infection in mice: the contribution of DNA vaccines coding for two novel sets of histones (H2A-H2B or H3-H4).

    Science.gov (United States)

    Carrión, Javier

    2011-09-01

    The immune phenotype conferred by two different sets of histone genes (H2A-H2B or H3-H4) was assessed. BALB/c mice vaccinated with pcDNA3H2AH2B succumbed to progressive cutaneous leishmaniosis (CL), whereas vaccination with pcDNA3H3H4 resulted in partial resistance to Leishmania major challenge associated with the development of mixed T helper 1 (Th1)/Th2-type response and a reduction in parasite-specific Treg cells number at the site of infection. Therefore, the presence of histones H3 and H4 may be considered essential in the development of vaccine strategies against CL based on the Leishmania histones. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Naturally Occurring Culturable Aerobic Gut Flora of Adult Phlebotomus papatasi, Vector of Leishmania major in the Old World

    Science.gov (United States)

    2012-05-22

    cesspits and latrines [22]. In Egypt, breeding sites of P. papatasi have been found in a similar range of ecotopes [23]. Rabbit holes in peri- domestic ...symbionts of arthropods [50,51,52]. Here we examine the presence and distribution of different aerobic gut microbes of P. papatasi, the major vector of...the residents and/or their animals was provided before consent was obtained. Consents were listed in a written log kept by the collectors. Collected

  15. Blocking junctional adhesion molecule C enhances dendritic cell migration and boosts the immune responses against Leishmania major.

    Directory of Open Access Journals (Sweden)

    Romain Ballet

    2014-12-01

    Full Text Available The recruitment of dendritic cells to sites of infections and their migration to lymph nodes is fundamental for antigen processing and presentation to T cells. In the present study, we showed that antibody blockade of junctional adhesion molecule C (JAM-C on endothelial cells removed JAM-C away from junctions and increased vascular permeability after L. major infection. This has multiple consequences on the output of the immune response. In resistant C57BL/6 and susceptible BALB/c mice, we found higher numbers of innate immune cells migrating from blood to the site of infection. The subsequent migration of dendritic cells (DCs from the skin to the draining lymph node was also improved, thereby boosting the induction of the adaptive immune response. In C57BL/6 mice, JAM-C blockade after L. major injection led to an enhanced IFN-γ dominated T helper 1 (Th1 response with reduced skin lesions and parasite burden. Conversely, anti JAM-C treatment increased the IL-4-driven T helper 2 (Th2 response in BALB/c mice with disease exacerbation. Overall, our results show that JAM-C blockade can finely-tune the innate cell migration and accelerate the consequent immune response to L. major without changing the type of the T helper cell response.

  16. In silico analysis of six known Leishmania major antigens and in vitro evaluation of specific epitopes eliciting HLA-A2 restricted CD8 T cell response.

    Directory of Open Access Journals (Sweden)

    Negar Seyed

    2011-09-01

    Full Text Available BACKGROUND: As a potent CD8(+ T cell activator, peptide vaccine has found its way in vaccine development against intracellular infections and cancer, but not against leishmaniasis. The first step toward a peptide vaccine is epitope mapping of different proteins according to the most frequent HLA types in a population. METHODS AND FINDINGS: Six Leishmania (L. major-related candidate antigens (CPB,CPC,LmsTI-1,TSA,LeIF and LPG-3 were screened for potential CD8(+ T cell activating 9-mer epitopes presented by HLA-A*0201 (the most frequent HLA-A allele. Online software including SYFPEITHI, BIMAS, EpiJen, Rankpep, nHLApred, NetCTL and Multipred were used. Peptides were selected only if predicted by almost all programs, according to their predictive scores. Pan-A2 presentation of selected peptides was confirmed by NetMHCPan1.1. Selected peptides were pooled in four peptide groups and the immunogenicity was evaluated by in vitro stimulation and intracellular cytokine assay of PBMCs from HLA-A2(+ individuals recovered from L. major. HLA-A2(- individuals recovered from L. major and HLA-A2(+ healthy donors were included as control groups. Individual response of HLA-A2(+ recovered volunteers as percent of CD8(+/IFN-γ(+ T cells after in vitro stimulation against peptide pools II and IV was notably higher than that of HLA-A2(- recovered individuals. Based on cutoff scores calculated from the response of HLA-A2(- recovered individuals, 31.6% and 13.3% of HLA-A2(+ recovered persons responded above cutoff in pools II and IV, respectively. ELISpot and ELISA results confirmed flow cytometry analysis. The response of HLA-A2(- recovered individuals against peptide pools I and III was detected similar and even higher than HLA-A2(+ recovered individuals. CONCLUSION: Using in silico prediction we demonstrated specific response to LmsTI-1 (pool II and LPG-3- (pool IV related peptides specifically presented in HLA-A*0201 context. This is among the very few reports

  17. Separation and evaluation of natural antileishmanial potential against Leishmania major and infuntum isolated from the Tunisia strains

    Directory of Open Access Journals (Sweden)

    Salwa Bouabdallah

    2018-03-01

    Full Text Available Besides its nutritional value as a dietary supplement, Tribulus terrestris is used as a remedy for fertility disorder in Ayurveda and Chinese medicine as well as by modern herbalists. The aim of this study was to explore the biological potential (antileishmanial effect of an extract rich in saponins from Tunisian tribulus. The chloroform extract of the various parts of T. terrestris was subjected to partial purification by solvent partitioning with ethanol and n-butanol. All prepared extracts were tested for their anti-leishmanial activity. The result showed that n-butanolic extract (saponin fraction, when isolated from leaves part exhibited the best antileishmanial effect against both pathogenic parasites Leshmania L. major (GlC94 and L. infuntum (LV50 evaluated in vitro assessment through MTT assay. n-Butanolic extract had been detected, quantified and purified using the RP-HPLC finger print (Hypersil ODS coupled to UV-vis. High peak area (5116.82 at 3.03 min was detected at 205 nm.

  18. Subcutaneous immunization against Leishmania major - infection in mice: efficacy of formalin-killed promastigotes combined with adjuvants Imunização subcutânea na infecção do camundongo pela Leishmania major: eficácia dos promastigotos mortos pela formalina combinada com adjuvantes

    Directory of Open Access Journals (Sweden)

    Joshua M. Mutiso

    2010-04-01

    Full Text Available Formalin-killed promastigotes (FKP of Leishmania major, in combination with Montanide ISA 720 (MISA, BCG or alum were used in vaccination of an inbred murine model against cutaneous leishmaniasis (CL. Significant and specific increases in anti-FKP IgG responses were detected for both alum-FKP and BCG-FKP compared to MISA-FKP (p Promastigotos mortos pela formalina (FKP de Leishmania major combinados com Montanide ISA 720 (MISA, BCG ou alumen foram usados na vacinação de modelo murino cutâneo de leishmaniose (CL. Aumento significante e específico de resposta IgG anti FKP foram detectados tanto no FKP com alumen como naquele com BCG comparados ao MISA-FKP (p < 0,001. Aumento significante da proliferação esplênica de linfócitos de memória foi obtida nos camundongos vacinados com MISA-FKP quando comparados aos grupos vacinados com alumen-FKP ou BCG-FKP (p < 0,01. As maiores respostas por interferon-gama foram observadas no grupo BCG-FKP seguido pelo MISA-FKP enquanto que o alumen-FKP deu a menor resposta. No grupo MISA-FKP foram obtidas reduções significantes do tamanho das lesões quando comparado aos grupos vacinados com BCG/adjuvante de alumen-FKP. Embora o grupo BCG-FKP tenha mostrado a maior resposta por interferon-gama, não houve controle das lesões cutâneas. Redução significante no número de parasitas foi observada tanto no grupo vacinado com MISA-FKP como no BCG-FKP (p < 0,001. Houve boa correlação entre a carga parasitária e o nível de interferon-gama indicando que a resposta do interferon-gama é parâmetro sensível do estado imunológico. Em conclusão, MISA-FKP é a forma mais eficaz de vacina contra a leishmaniose cutânea murina.

  19. Spatial and simultaneous seroepidemiology of anti-Leishmania spp. antibodies in dog owners and their dogs from randomly selected households in a major city of southern Brazil.

    Science.gov (United States)

    Benitez, Aline do Nascimento; Martins, Felippe Danyel Cardoso; Mareze, Marcelle; Nino, Beatriz de Souza Lima; Caldart, Eloiza Teles; Ferreira, Fernanda Pinto; Mitsuka-Breganó, Regina; Freire, Roberta Lemos; Galhardo, Juliana Arena; Martins, Camila Marinelli; Biondo, Alexander Welker; Navarro, Italmar Teodorico

    2018-06-01

    Although leishmaniasis has been described as a classic example of a zoonosis requiring a comprehensive approach for control, to date, no study has been conducted on the spatial distribution of simultaneous Leishmania spp. seroprevalence in dog owners and dogs from randomly selected households in urban settings. Accordingly, the present study aimed to simultaneously identify the seroprevalence, spatial distribution and associated factors of infection with Leishmania spp. in dog owners and their dogs in the city of Londrina, a county seat in southern Brazil with a population of half a million people and ranked 18th in population and 145th in the human development index (HDI) out of 5570 Brazilian cities. Overall, 564 households were surveyed and included 597 homeowners and their 729 dogs. Anti-Leishmania spp. antibodies were detected by ELISA in 9/597 (1.50%) dog owners and in 32/729 (4.38%) dogs, with significantly higher prevalence (p = 0.0042) in dogs. Spatial analysis revealed associations between seropositive dogs and households located up to 500 m from the local railway. No clusters were found for either owner or dog case distributions. In summary, the seroepidemiological and spatial results collectively show a lack of association of the factors for infection, and the results demonstrated higher exposure for dogs than their owners. However, railway areas may provide favorable conditions for the maintenance of infected phlebotomines, thereby causing infection in nearby domiciled dogs. In such an urban scenario, local sanitary barriers should be focused on the terrestrial routes of people and surrounding areas, particularly railways, via continuous vector surveillance and identification of phlebotomines infected by Leishmania spp. Copyright © 2018. Published by Elsevier B.V.

  20. In vitro cytocidal effects of the essential oil from Croton cajucara (red sacaca) and its major constituent 7- hydroxycalamenene against Leishmania chagasi

    OpenAIRE

    Rodrigues, Igor A; Azevedo, Mariana M B; Chaves, Francisco C M; Bizzo, Humberto R; Corte-Real, Suzana; Alviano, Daniela S; Alviano, Celuta S; Rosa, Maria S S; Vermelho, Alane B

    2013-01-01

    Background Visceral leishmaniasis is the most serious form of leishmaniasis and can be lethal if left untreated. Currently available treatments for these parasitic diseases are frequently associated to severe side effects. The leaves of Croton cajucara are used as an infusion in popular medicine to combat several diseases. Previous studies have demonstrated that the linalool-rich essential oil from C. cajucara (white sacaca) is extremely efficient against the tegumentary specie Leishmania ama...

  1. Evaluation of the polymerase chain reaction in the diagnosis of cutaneous leishmaniasis due to Leishmania major: a comparison with direct microscopy of smears and sections from lesions

    DEFF Research Database (Denmark)

    Andresen, K; Gaafar, A; El-Hassan, A M

    1996-01-01

    We have compared the sensitivity of the polymerase chain reaction (PCR) as a diagnostic tool against conventional microscopical diagnostic techniques in patients with cutaneous leishmaniasis from the Sudan. Twenty-eight patients were diagnosed according to clinical criteria followed......%, respectively. The PCR should be considered as a valuable and sensitive diagnostic tool in the diagnosis of cutaneous leishmaniasis; it has the added advantage of identification of the species of Leishmania causing the lesion....

  2. Leishmania metacaspase: an arginine-specific peptidase.

    Science.gov (United States)

    Martin, Ricardo; Gonzalez, Iveth; Fasel, Nicolas

    2014-01-01

    The purpose of this chapter is to give insights into metacaspase of Leishmania protozoan parasites as arginine-specific cysteine peptidase. The physiological role of metacaspase in Leishmania is still a matter of debate, whereas its peptidase enzymatic activity has been well characterized. Among the different possible expression systems, metacaspase-deficient yeast cells (Δyca1) have been instrumental in studying the activity of Leishmania major metacaspase (LmjMCA). Here, we describe techniques for purification of LmjMCA and its activity measurement, providing a platform for further identification of LmjMCA substrates.

  3. Evaluation of immune responses and analysis of the effect of vaccination of the Leishmania major recombinant ribosomal proteins L3 or L5 in two different murine models of cutaneous leishmaniasis.

    Science.gov (United States)

    Ramírez, Laura; Santos, Diego M; Souza, Ana P; Coelho, Eduardo A F; Barral, Aldina; Alonso, Carlos; Escutia, Marta R; Bonay, Pedro; de Oliveira, Camila I; Soto, Manuel

    2013-02-18

    Four new antigenic proteins located in Leishmania ribosomes have been characterized: S4, S6, L3 and L5. Recombinant versions of the four ribosomal proteins from Leishmania major were recognized by sera from human and canine patients suffering different clinical forms of leishmaniasis. The prophylactic properties of these proteins were first studied in the experimental model of cutaneous leishmaniasis caused by L. major inoculation into BALB/c mice. The administration of two of them, LmL3 or LmL5 combined with CpG-oligodeoxynucleotides (CpG-ODN) was able to protect BALB/c mice against L. major infection. Vaccinated mice showed smaller lesions and parasite burden compared to mice inoculated with vaccine diluent or vaccine adjuvant. Protection was correlated with an antigen-specific increased production of IFN-γ paralleled by a decrease of the antigen-specific IL-10 mediated response in protected mice relative to non-protected controls. Further, it was demonstrated that BALB/c mice vaccinated with recombinant LmL3 or LmL5 plus CpG-ODN were also protected against the development of cutaneous lesions following inoculation of L. braziliensis. Together, data presented here indicate that LmL3 or LmL5 ribosomal proteins combined with Th1 inducing adjuvants, may be relevant components of a vaccine against cutaneous leishmaniasis caused by distinct species. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Microbial transformation of nucleosides

    Science.gov (United States)

    Lamba, S. S.

    1979-01-01

    A study involving the use of coulter counter in studying the effects of neomycin on E. coli, S. aureus and A. aerogenes was completed. The purpose of this was to establish proper technique for enumeration of cells per ml. It was found that inhibitory effects on growth of E. coli and A. aerogenes, both gram negative organisms, were directly related to the concentration of neomycin used. However, in case S. aureus, a gram positive organism, a decreased inhibition was noted at higher concentrations. A paper entitled, Use of Coulter Counter in Studying Effect of Drugs on Cells in Culture 1 - Effects of Neomycin on E. coli, S. aureus and A. aerogenes, is attached in the appendix. Laboratory procedures were also established to study the effects of nucleoside antibiotic cordycepin on He La cell grown in suspension cultures.

  5. A comparative study of the effects of venoms from five rear-fanged snake species on the growth of Leishmania major: identification of a protein with inhibitory activity against the parasite.

    Science.gov (United States)

    Peichoto, María E; Tavares, Flávio L; Dekrey, Gregory; Mackessy, Stephen P

    2011-07-01

    Leishmania parasites of several species cause cutaneous and visceral disease to millions of people worldwide, and treatment for this vector-borne protozoan parasite typically involves administration of highly toxic antimonial drugs. Snake venoms are one of the most concentrated enzyme sources in nature, displaying a broad range of biological effects, and several drugs now used in humans were derived from venoms. In this study, we compared the effects of the venoms of the South American rear-fanged snakes Philodryas baroni (PbV), Philodryas olfersii olfersii (PooV) and Philodryas patagoniensis (PpV), and the North American rear-fanged snakes Hypsiglena torquata texana (HttV) and Trimorphodon biscutatus lambda (TblV), on the growth of Leishmania major, a causative agent of cutaneous leishmaniasis. Different concentrations of each venom were incubated with the log-phase promastigote stage of L. major. TblV showed significant anti-leishmanial activity (IC₅₀ of 108.6 μg/mL) at its highest concentrations; however, it induced parasite proliferation at intermediate concentrations. PpV was not very active in decreasing the parasitic growth, and a high final concentration (1.7 mg/mL) was necessary to inhibit proliferation by only 51.5% ± 3.6%. PbV, PooV and HttV, at final concentrations of 562, 524 and 438 μg/mL respectively, had no significant effect on L. major growth. The phospholipase A₂ of TblV (trimorphin) was isolated and assayed as for crude venom, and it also exhibited dose-dependent biphasic effects on the parasite culture, with potent cytotoxicity at higher concentrations (IC₅₀ of 0.25 μM; 3.6 μg/mL) and stimulation of proliferation at very low concentrations. Anti-leishmanial activity of TblV appears to be solely due to the action of trimorphin. This is the first report of anti-leishmanial activity of rear-fanged snake venoms, and these results suggest novel possibilities for discovering new protein-based drugs that might be used as possible agents

  6. Adenylosuccinato lyase (ADSL) de leishmania major friedlin: sua relevância na via de recuperação de purino-nucleotídeos

    OpenAIRE

    Monique Mantovani

    2006-01-01

    Muitas espécies de Leishmania são responsáveis por sérias doenças cutâneas e viscerais, que exibem alta incidência em regiões tropicais e subtropicais. Os medicamentos disponíveis são potencialmente carcinogênicos, requerem múltiplas administrações e a hospitalização do paciente. Um programa efetivo de desenvolvimento de novos fármacos requer a validação genética e bioquímica dos alvos. Neste contexto, uma das diferenças metabólicas mais marcantes entre os protozoários parasitas e o hospedeir...

  7. Evaluation of the polymerase chain reaction in the diagnosis of cutaneous leishmaniasis due to Leishmania major: a comparison with direct microscopy of smears and sections from lesions

    DEFF Research Database (Denmark)

    Andresen, K; Gaafar, A; El-Hassan, A M

    1996-01-01

    We have compared the sensitivity of the polymerase chain reaction (PCR) as a diagnostic tool against conventional microscopical diagnostic techniques in patients with cutaneous leishmaniasis from the Sudan. Twenty-eight patients were diagnosed according to clinical criteria followed by microscopi......We have compared the sensitivity of the polymerase chain reaction (PCR) as a diagnostic tool against conventional microscopical diagnostic techniques in patients with cutaneous leishmaniasis from the Sudan. Twenty-eight patients were diagnosed according to clinical criteria followed......%, respectively. The PCR should be considered as a valuable and sensitive diagnostic tool in the diagnosis of cutaneous leishmaniasis; it has the added advantage of identification of the species of Leishmania causing the lesion....

  8. In vitro cytocidal effects of the essential oil from Croton cajucara (red sacaca) and its major constituent 7- hydroxycalamenene against Leishmania chagasi.

    Science.gov (United States)

    Rodrigues, Igor A; Azevedo, Mariana M B; Chaves, Francisco C M; Bizzo, Humberto R; Corte-Real, Suzana; Alviano, Daniela S; Alviano, Celuta S; Rosa, Maria S S; Vermelho, Alane B

    2013-10-02

    Visceral leishmaniasis is the most serious form of leishmaniasis and can be lethal if left untreated. Currently available treatments for these parasitic diseases are frequently associated to severe side effects. The leaves of Croton cajucara are used as an infusion in popular medicine to combat several diseases. Previous studies have demonstrated that the linalool-rich essential oil from C. cajucara (white sacaca) is extremely efficient against the tegumentary specie Leishmania amazonensis. In this study, we investigated the effects of the 7-hydroxycalamenene-rich essential oil from the leaves of C. cajucara (red sacaca) against Leishmania chagasi, as well as on the interaction of these parasites with host cells. Promastigotes were treated with different concentrations of the essential oil for determination of its minimum inhibitory concentration (MIC). In addition, the effects of the essential oil on parasite ultrastructure were analyzed by transmission electron microscopy. To evaluate its efficacy against infected cells, mouse peritoneal macrophages infected with L. chagasi promastigotes were treated with the inhibitory and sub-inhibitory concentrations of the essential oil. The minimum inhibitory concentrations of the essential oil and its purified component 7-hydroxycalamenene against L. chagasi were 250 and 15.6 μg/mL, respectively. Transmission electron microscopy analysis revealed important nuclear and kinetoplastic alterations in L. chagasi promastigotes. Pre-treatment of macrophages and parasites with the essential oil reduced parasite/macrophage interaction by 52.8%, while it increased the production of nitric oxide by L. chagasi-infected macrophages by 80%. These results indicate that the 7-hydroxycalamenene-rich essential oil from C. cajucara is a promising source of leishmanicidal compounds.

  9. In vitro cytocidal effects of the essential oil from Croton cajucara (red sacaca) and its major constituent 7- hydroxycalamenene against Leishmania chagasi

    Science.gov (United States)

    2013-01-01

    Background Visceral leishmaniasis is the most serious form of leishmaniasis and can be lethal if left untreated. Currently available treatments for these parasitic diseases are frequently associated to severe side effects. The leaves of Croton cajucara are used as an infusion in popular medicine to combat several diseases. Previous studies have demonstrated that the linalool-rich essential oil from C. cajucara (white sacaca) is extremely efficient against the tegumentary specie Leishmania amazonensis. In this study, we investigated the effects of the 7-hydroxycalamenene-rich essential oil from the leaves of C. cajucara (red sacaca) against Leishmania chagasi, as well as on the interaction of these parasites with host cells. Methods Promastigotes were treated with different concentrations of the essential oil for determination of its minimum inhibitory concentration (MIC). In addition, the effects of the essential oil on parasite ultrastructure were analyzed by transmission electron microscopy. To evaluate its efficacy against infected cells, mouse peritoneal macrophages infected with L. chagasi promastigotes were treated with the inhibitory and sub-inhibitory concentrations of the essential oil. Results The minimum inhibitory concentrations of the essential oil and its purified component 7-hydroxycalamenene against L. chagasi were 250 and 15.6 μg/mL, respectively. Transmission electron microscopy analysis revealed important nuclear and kinetoplastic alterations in L. chagasi promastigotes. Pre-treatment of macrophages and parasites with the essential oil reduced parasite/macrophage interaction by 52.8%, while it increased the production of nitric oxide by L. chagasi-infected macrophages by 80%. Conclusion These results indicate that the 7-hydroxycalamenene-rich essential oil from C. cajucara is a promising source of leishmanicidal compounds. PMID:24088644

  10. 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

  11. 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.

  12. Nucleoside composition of Heloderma venoms.

    Science.gov (United States)

    Aird, Steven D

    2008-06-01

    Venoms of Heloderma horridum and Heloderma suspectum were analyzed for the possible presence of purine and pyrimidine nucleosides. Adenosine, cytidine, guanosine, hypoxanthine, inosine, and uridine were found in mug quantities. These amounts are much smaller than those seen in many elapid or viperine venoms, but greater and more varied than those found in crotaline venoms. While their contribution to the hypotension induced by Heloderma venoms may be minor, venom nucleosides nonetheless act in concert with kallikreins/hemorrhagins, alkaline phosphomonoesterase, 5'-nucleotidase, helodermin, helospectins, helothermine, and serotonin. The use of nucleosides as toxins is therefore a generalized squamate strategy, rather than the exclusive province of snakes. Both Heloderma venoms were found to be devoid of NADase and phosphodiesterase activities. Enzymes to release endogenous purines in the prey, are not significant components of Heloderma venoms.

  13. Identification and characterization of new Leishmania promastigote surface antigens, LaPSA-38S and LiPSA-50S, as major immunodominant excreted/secreted components of L. amazonensis and L. infantum.

    Science.gov (United States)

    Bras-Gonçalves, Rachel; Petitdidier, Elodie; Pagniez, Julie; Veyrier, Renaud; Cibrelus, Prisca; Cavaleyra, Mireille; Maquaire, Sarah; Moreaux, Jérôme; Lemesre, Jean-Loup

    2014-06-01

    We have previously demonstrated that sera from dogs vaccinated with excreted/secreted antigens (ESA) of Leishmania infantum promastigotes (LiESAp) mainly recognized an immunodominant antigen of 54 kDa. An anti-LiESAp-specific IgG2 humoral response was observed and associated to Th1-type response in vaccinated dogs. This response was highly correlated with a long-lasting and strong LiESAp-vaccine protection toward L. infantum experimental infection. In addition, it was also shown that dogs from the vaccinated group developed a selective IgG2 response against an immunodominant antigen of 45 kDa of Leishmania amazonensis ESA promastigotes (LaESAp). In order to identify and characterize these immunodominant antigens, a mouse monoclonal antibody (mAb F5) was produced by immunization against LaESAp. It was found to recognize the major antigenic targets of both LaESAp and LiESAp. Analysis with mAb F5 of L. amazonensis amastigote and promastigote cDNA expression libraries enabled the identification of clones encoding proteins with significant structural homology to the promastigote surface antigens named PSA-2/gp-46. Among them, one clone presented a full-length cDNA and encoded a novel L. amazonensis protein of 38.6 kDa calculated molecular mass (LaPSA-38S) sharing an amino acid sequence consistent with that of the PSA polymorphic family and a N-terminal signal peptide, characteristic of a secreted protein. We then screened a L. infantum promastigote DNA cosmid library using a cDNA probe derived from the LaPSA-38S gene and identified a full-length clone of a novel excreted/secreted protein of L. infantum with a calculated molecular mass of 49.2 kDa and named LiPSA-50S. The fact that a significant immunological reactivity was observed against PSA, suggests that these newly identified proteins could have an important immunoregulatory influence on the immune response. This hypothesis is supported by the fact that (i) these proteins were naturally excreted/secreted by viable

  14. Deoxyribonucleoside kinases activate nucleoside antibiotics in severely pathogenic bacteria

    DEFF Research Database (Denmark)

    Sandrini, Michael; Shannon, O.; Clausen, A.R.

    2007-01-01

    Common bacterial pathogens are becoming progressively more resistant to traditional antibiotics, representing a major public-health crisis. Therefore, there is a need for a variety of antibiotics with alternative modes of action. In our study, several nucleoside analogs were tested against...

  15. A pilot study to control Lutzomyia umbratilis (Diptera: Psychodidae, the major vector of Leishmania brazileiensis guyanensis, in a peri-urban rainforest of Manaus, Amazonas State, Brazil

    Directory of Open Access Journals (Sweden)

    P. D. Ready

    1985-03-01

    Full Text Available In the second half of 1980, 112 (or ca. 16% of the inhabitants of the new settlement of São José, city of Manaus, contracted cutaneous leishmaniasis whilst clearing their properties of terra firme rainforest. With the aid of SUCAM, the authors carried out a pilot study to investigate the feasibility of reducing populations of Lutzomyia umbratilis, the local silvatic vector of Leishmania braziliensis guyanensis, by spraying insecticide on its favoured diurnal resting sites, the bases of the larger forest trees. Most manvector contact is at these resting sites and, therefore, it was encouraging to record a marked reduction of the tree-base populations of L. umbratilis for 21 days following just one application of D.D.T. emulsion in an area 200m square. Most of the treated trunks were not occupied by L. umbratilis for at least eleven months. Suggestions for extending the pilot study are made, and the need for collaboration with a clinical team is emphasized. Leishmania b. guyanensis is the aetiological agent of [quot ]pain bois[quot ], which is hyperendemic from French Guiana to central Amazônia. In the absence of proven vaccines or methods of vector control, some simple methods for limiting transmission of Le. b. guyanensis to man are listed.No segundo semestre de 1980, 112 (ou aproximadamente 16% dos habitantes do recém estabelecido bairro de São José, cidade de Manaus, contraíram leishmaniose, durante o desmatamento que realizavam em seus lotes localizados na floresta tipo "terra firme". Com a ajuda da SUCAM, os AA. realizaram um estudo piloto para investigar a viabilidade de redução das populações de Lutzomyia umbratilis, o vetor silvático de Leishmania braziliensis guyanensis, borrifando inseticidas na base das árvores grandes da floresta que são os lugares preferidos de repouso diurno destes insetos. A maioria do contato homem-vetor ocorre nestes lugares de repouso diurno (o vetor sendo mais ativo à noite nas copas das

  16. Innate Immunity to Leishmania Infection: Within Phagocytes

    Directory of Open Access Journals (Sweden)

    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.

  17. Double-Blind Randomized Efficacy Field Trial of Alum Precipi¬tated Autoclaved Leishmania major (Alum-ALM Vaccine Mixed With BCG Plus Imiquimod Vs. Placebo Control Group

    Directory of Open Access Journals (Sweden)

    Mohammad BARATI

    2015-10-01

    Full Text Available Background: Canine visceral leishmaniasis (CVL is not only an emerging veterinary concern but also a public health threat in endemic areas. The aim of this study was to assess the efficacy, immunogenicity and safety of two doses of aluminum hydroxide (alum precipitated Leishmania major (Alum-ALM mixed with BCG plus imiquimod against CVL.Methods: A total of 560 ownership dogs were serologically tested and 234 healthy dogs with no clinical signs of CVL, no anti-Leishmania antibodies and negative leish­manin skin test were selected and double-blind randomly injected intradermally either with 0.1 ml Alum-ALM (200µg protein mixed with BCG (2 × 106 CFUs plus imiquimod (121 dogs or with 0.1 ml of normal saline (113 dogs.Results: The follow-up examinations showed that there was no side effect associated with the vaccination except one case. Strong skin test conversion were seen in vac­cinated group (30.3% compared to the control group (6.6% at 22-24 weeks after the booster injection (p<0.001. The seroconversion was 16.3% (18/110 in vaccinated group and 26.4% (28/106 in control group after two transmission cycles but the differ­ence was not significant (P=0.095. The efficacy rate based on seroconversion was 40.4 %.Conclusion: Two injections of Alum-ALM mixed with BCG and imiquimod is safe, although decreases the seroconversion rate of CVL, but the overall efficacy was low.

  18. Nucleoside Inhibitors of Zika Virus

    Czech Academy of Sciences Publication Activity Database

    Eyer, L.; Nencka, Radim; Huvarová, I.; Palus, Martin; Alves, M. J.; Gould, E. A.; De Clercq, E.; Růžek, Daniel

    2016-01-01

    Roč. 214, č. 5 (2016), s. 707-711 ISSN 0022-1899 Institutional support: RVO:61388963 ; RVO:60077344 Keywords : Zika virus * flavivirus * nucleoside analogue * antiviral * therapy Subject RIV: CC - Organic Chemistry; EE - Microbiology, Virology (BC-A) Impact factor: 6.273, year: 2016

  19. Validation of Recombinant Salivary Protein PpSP32 as a Suitable Marker of Human Exposure to Phlebotomus papatasi, the Vector of Leishmania major in Tunisia

    Science.gov (United States)

    Bettaieb, Jihene; Abdeladhim, Maha; Hadj Kacem, Saoussen; Abdelkader, Rania; Gritli, Sami; Chemkhi, Jomaa; Aslan, Hamide; Kamhawi, Shaden; Ben Salah, Afif; Louzir, Hechmi; Valenzuela, Jesus G.; Ben Ahmed, Melika

    2015-01-01

    Background During a blood meal, female sand flies, vectors of Leishmania parasites, inject saliva into the host skin. Sand fly saliva is composed of a large variety of components that exert different pharmacological activities facilitating the acquisition of blood by the insect. Importantly, proteins present in saliva are able to elicit the production of specific anti-saliva antibodies, which can be used as markers for exposure to vector bites. Serological tests using total sand fly salivary gland extracts are challenging due to the difficulty of obtaining reproducible salivary gland preparations. Previously, we demonstrated that PpSP32 is the immunodominant salivary antigen in humans exposed to Phlebotomus papatasi bites and established that humans exposed to P. perniciosus bites do not recognize it. Methodology/Principal Findings Herein, we have validated, in a large cohort of 522 individuals, the use of the Phlebotomus papatasi recombinant salivary protein PpSP32 (rPpSP32) as an alternative method for testing exposure to the bite of this sand fly. We also demonstrated that screening for total anti-rPpSP32 IgG antibodies is sufficient, being comparable in efficacy to the screening for IgG2, IgG4 and IgE antibodies against rPpSP32. Additionally, sera obtained from dogs immunized with saliva of P. perniciosus, a sympatric and widely distributed sand fly in Tunisia, did not recognize rPpSP32 demonstrating its suitability as a marker of exposure to P. papatasi saliva. Conclusions/Significance Our data indicate that rPpSP32 constitutes a useful epidemiological tool to monitor the spatial distribution of P. papatasi in a particular region, to direct control measures against zoonotic cutaneous leishmaniasis, to assess the efficiency of vector control interventions and perhaps to assess the risk of contracting the disease. PMID:26368935

  20. Searching for New Chemotherapies for Tropical Diseases: Ruthenium-Clotrimazole Complexes Display High in vitro Activity Against Leishmania major and Trypanosoma cruzi and Low Toxicity Toward Normal Mammalian Cells

    Science.gov (United States)

    Martínez, Alberto; Carreon, Teresia; Iniguez, Eva; Anzellotti, Atilio; Sánchez, Antonio; Tyan, Marina; Sattler, Aaron; Herrera, Linda; Maldonado, Rosa A.; Sánchez-Delgado, Roberto A.

    2012-01-01

    Eight new ruthenium complexes of clotrimazole (CTZ) with high antiparasitic activity have been synthesized, cis,fac-[RuIICl2(DMSO)3(CTZ)] (1), cis,cis,trans-[RuIICl2(DMSO)2(CTZ)2] (2), Na[RuIIICl4(DMSO)(CTZ)] (3) and Na[trans-RuIIICl4(CTZ)2] (4), [RuII(η6-p-cymene)Cl2(CTZ)] (5), [RuII(η6-p-cymene)(bipy)(CTZ)][BF4]2 (6), [RuII(η6-p-cymene)(en)(CTZ)][BF4]2 (7) and [RuII(η6-p-cymene)(acac)(CTZ)][BF4] (8) (bipy = bipyridine; en = ethlylenediamine; acac = acetylacetonate). The crystal structures of compounds 4-8 are described. Complexes 1-8 are active against promastigotes of Leishmania major and epimastigotes of Trypanosoma cruzi. Most notably complex 5 increases the activity of CTZ by factors of 110 and 58 against L. major and T. cruzi, with no appreciable toxicity to human osteoblasts, resulting in nanomolar and low micromolar lethal doses and therapeutic indexes of 500 and 75, respectively. In a high-content imaging assay on L. major infected intraperitoneal mice macrophages, complex 5 showed significant inhibition on the proliferation of intracellular amastigotes (IC70 = 29 nM), while complex 8 displayed some effect at a higher concentration (IC40 = 1 μM). PMID:22448965

  1. An outbreak of Leishmania major from an endemic to a non-endemic region posed a public health threat in Iraq from 2014-2017: Epidemiological, molecular and phylogenetic studies.

    Science.gov (United States)

    Al-Bajalan, Mariwan M M; Al-Jaf, Sirwan M A; Niranji, Sherko S; Abdulkareem, Dler R; Al-Kayali, Khudhair K; Kato, Hirotomo

    2018-03-01

    Cutaneous leishmaniasis (CL) is a neglected worldwide, zoonotic, vector-borne, tropical disease that is a threat to public health. This threat may spread from endemic to non-endemic areas. Current research has exploited epidemiological, molecular and phylogenetical studies to determine the danger of an outbreak of CL in the borderline area between northern and central Iraq from 2014-2017. For the first time, using sequence analysis of the cytochrome b gene, the occurrence of CL in the borderline area between northern and central Iraq was confirmed to be due to Leishmania major. The phylogenetic analysis indicated that it was closely related to the L. major MRHO/IR/75/ER strain in Iran. In conclusion, the genotype confirmation of the L. major strain will improve our understanding of the epidemiology of the disease. This is important for facilitating control programs to prevent the further spread of CL. Furthermore, this area could be considered as a model for further research on the risk of global CL epidemics in other non-endemic countries where both reservoir hosts and sandfly vectors are present.

  2. Human cellular and humoral immune responses to Phlebotomus papatasi salivary gland antigens in endemic areas differing in prevalence of Leishmania major infection.

    Directory of Open Access Journals (Sweden)

    Wafa Kammoun-Rebai

    2017-10-01

    Full Text Available Sand fly saliva compounds are able to elicit specific immune responses that have a significant role in Leishmania parasite establishment and disease outcome. Characterizing anti-saliva immune responses in individuals living in well defined leishmaniasis endemic areas would provide valuable insights regarding their effect on parasite transmission and establishment in humans.We explored the cellular and humoral immune responses to Phlebotomus (P. papatasi salivary gland extracts (SGE in individuals living in cutaneous leishmaniasis (CL old or emerging foci (OF, EF. OF was characterized by a higher infection prevalence as assessed by higher proportions of leishmanin skin test (LST positive individuals compared to EF. Subjects were further subdivided into healed, asymptomatic or naïve groups. We showed anti-SGE proliferation in less than 30% of the individuals, regardless of the immune status, in both foci. IFN-γ production was higher in OF and only observed in immune individuals from OF and naïve subjects from EF. Although IL-10 was not detected, addition of anti-human IL-10 antibodies revealed an increase in proliferation and IFN-γ production only in individuals from OF. The percentage of seropositive individuals was similar in immune and naïves groups but was significantly higher in OF. No correlation was observed between anti-saliva immune responses and LST response. High anti-SGE-IgG responses were associated with an increased risk of developing ZCL. No differences were observed for anti-SGE humoral or cellular responses among naïve individuals who converted or not their LST response or developed or not ZCL after the transmission season.These data suggest that individuals living in an old focus characterized by a frequent exposure to sand fly bites and a high prevalence of infection, develop higher anti-saliva IgG responses and IFN-γ levels and a skew towards a Th2-type cellular response, probably in favor of parasite establishment

  3. 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+...

  4. Th1 Cell Development Induced by Cysteine Proteinases A and B in Localized Cutaneous Leishmaniasis Due to Leishmania guyanensis

    Science.gov (United States)

    Pascalis, Hervé; Lavergne, Anne; Bourreau, Eliane; Prévot-Linguet, Ghislaine; Kariminia, Amina; Pradinaud, Roger; Rafati, Sima; Launois, Pascal

    2003-01-01

    The cysteine proteinases CPA and CPB from Leishmania major induced Th1 responses in patients with leishmaniasis due to Leishmania guyanensis. Furthermore, cysteine proteinases induced neither interleukin 4 (IL-4) nor IL-13 and low levels of IL-10 in controls and patients. The results suggest that CPs would be quite good candidates for a vaccine against different Leishmania species. PMID:12704171

  5. Investigation of Antileishmanial Effect of Alcoholic Extract and Essential Oil of Medicinal Plant Leaf Black Alfalfa (Medicago Lupulina, on The Number of Clinical Isolates of Leishmania Major Promastigotes in Vitro

    Directory of Open Access Journals (Sweden)

    E Gharirvand Eskandari

    2016-05-01

    Full Text Available Introduction: Leishmaniasis has created enormous global health problems. Side effects, drug resistance and the lack of effective vaccines had led to the new effective compounds effective of plants. The aim of this study was to introduce a traditional medicinal plant called Black alfalfa (Medicago Lupulina that can be used as a valuable resource against cutaneous leishmaniasis. Methods: In this experimental study, alcoholic extract was prepared by maceration and essential oil by distillation water method. Leishmania major promastigotes were cultured at 25 ± 2° C in N.N.N culturemedium, then in Schneider and next were cultured in RPMI- 1640. afterward, using MTT (Methyl Thiazole Tetrazolium, the IC50 (Inhibitory Concentrations 50% for extracts, essence and Glucantime were determined. The results using Tukey and t-test were analyzed and were presented by software SPSS16. MTT assay were repeated. 3 times for each sample. Results: IC50 for alfalfa leaf extract and essential oil of black alfalfa and Glucantime against L. major promastigotes was determined after 24, 48 and 72 hours, 240, 130 and 69 micrograms per ml, and 801, 340 and 190 micrograms per ml, also 26, 19 and 11 micrograms per ml , respectively. There was a significant differences between the IC50 plant extract and essential oil and Glucantime after 24, 48 and 72 hours. Conclusion: Alcoholic extracts and essential oil the plant had significant anti leishmaniasis effects in vitro. In this way, it can be considered as an anti-leishmaniasis among the herbs.

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

    OpenAIRE

    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...

  7. Diversity among Leishmania isolates from the Sudan: isoenzyme homogeneity of L. donovani versus heterogeneity of L. major

    DEFF Research Database (Denmark)

    Ibrahim, M E; Evans, D A; Theander, T G

    1995-01-01

    Leishmania isolates from patients in the Sudan suffering from either visceral or cutaneous leishmaniasis were characterized using a battery of 12 enzymes. Aspartate aminotransferase separated the L. donovani isolates into 2 distinct zymodemes, but the overall results showed no significant...

  8. Natural infection of bats with Leishmania in Ethiopia.

    Science.gov (United States)

    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.

  9. Molecular identification of Leishmania species in Taybad district, Iran

    Directory of Open Access Journals (Sweden)

    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.

  10. Marine Nucleosides: Structure, Bioactivity, Synthesis and Biosynthesis

    Directory of Open Access Journals (Sweden)

    Ri-Ming Huang

    2014-12-01

    Full Text Available Nucleosides are glycosylamines that structurally form part of nucleotide molecules, the building block of DNA and RNA. Both nucleosides and nucleotides are vital components of all living cells and involved in several key biological processes. Some of these nucleosides have been obtained from a variety of marine resources. Because of the biological importance of these compounds, this review covers 68 marine originated nucleosides and their synthetic analogs published up to June 2014. The review will focus on the structures, bioactivities, synthesis and biosynthetic processes of these compounds.

  11. High quality long-term CD4+ and CD8+ effector memory populations stimulated by DNA-LACK/MVA-LACK regimen in Leishmania major BALB/c model of infection.

    Science.gov (United States)

    Sánchez-Sampedro, Lucas; Gómez, Carmen Elena; Mejías-Pérez, Ernesto; Sorzano, Carlos Oscar S; Esteban, Mariano

    2012-01-01

    Heterologous vaccination based on priming with a plasmid DNA vector and boosting with an attenuated vaccinia virus MVA recombinant, with both vectors expressing the Leishmania infantum LACK antigen (DNA-LACK and MVA-LACK), has shown efficacy conferring protection in murine and canine models against cutaneus and visceral leishmaniasis, but the immune parameters of protection remain ill defined. Here we performed by flow cytometry an in depth analysis of the T cell populations induced in BALB/c mice during the vaccination protocol DNA-LACK/MVA-LACK, as well as after challenge with L. major parasites. In the adaptive response, there is a polyfunctional CD4(+) and CD8(+) T cell activation against LACK antigen. At the memory phase the heterologous vaccination induces high quality LACK-specific long-term CD4(+) and CD8(+) effector memory cells. After parasite challenge, there is a moderate boosting of LACK-specific CD4(+) and CD8(+) T cells. Anti-vector responses were largely CD8(+)-mediated. The immune parameters induced against LACK and triggered by the combined vaccination DNA/MVA protocol, like polyfunctionality of CD4(+) and CD8(+) T cells with an effector phenotype, could be relevant in protection against leishmaniasis.

  12. Locked and unlocked nucleosides in functional nucleic acids

    DEFF Research Database (Denmark)

    Doessing, Holger; Vester, Birte

    2011-01-01

    Nucleic acids are able to adopt a plethora of structures, many of which are of interest in therapeutics, bio- or nanotechnology. However, structural and biochemical stability is a major concern which has been addressed by incorporating a range of modifications and nucleoside derivatives. This rev......Nucleic acids are able to adopt a plethora of structures, many of which are of interest in therapeutics, bio- or nanotechnology. However, structural and biochemical stability is a major concern which has been addressed by incorporating a range of modifications and nucleoside derivatives....... This review summarizes the use of locked nucleic acid (LNA) and un-locked nucleic acid (UNA) monomers in functional nucleic acids such as aptamers, ribozymes, and DNAzymes....

  13. Interferon-¿ production by human T cells and natural killer cells in vitro in response to antigens from the two intracellular pathogens Mycobacterium tuberculosis and Leishmania major

    DEFF Research Database (Denmark)

    Kemp, K; Hviid, L; Kharazmi, A

    1997-01-01

    Acquired resistance to both mycobacteria and Leishmania is primarily mediated by interferon-gamma (IFN-gamma), which triggers mechanisms leading to the death of the microorganism in macrophages. In this study, cell activation and IFN-gamma production was investigated in human peripheral blood...... mononuclear cells (PBMC) from individuals previously sensitized to tuberculin and without known exposure to Leishmania parasites. Immune staining for intracellular IFN-gamma and surface markers allowed flow cytometric identification of the cellular sources of IFN-gamma in cell cultures incubated with purified...

  14. [Determination of Nucleosides and HPLC Fingerprints of Cordyceps].

    Science.gov (United States)

    Wang, Bing; Li, Ning; Dong, Ting-xia; Zhan, Hua-qiang

    2015-05-01

    To establish the HPLC fingerprints method of Cordyceps and to determine the contents of uridine, inosine, guanosine and adenosine. The HPLC separation was performed on a Grace Prevail C18 column( 150 mm x 4.6 mm, 5 μm) in a gradient elution mode with a mixture consisting of water and methanol at a flow rate of 1.0 mL/min, the detection wavelength was set at 260 nm, the column temperature was 25 degrees C. The contents of four nucleosides were determined in Cordyceps from different habitats, and the HPLC fingerprint of Cordyceps was set up with 13 common peaks. Among of them, uridine, inosine, guanosine and adenosine were identified. The similarities of ten fingerprints were greater than 0.95 with good separation of each chromatographic peak, and met the requirement of the fingerprints. There were similar results in cluster analysis and principal component analysis of the major nucleosides and the fingerprints of 10 batches of Cordyceps. The results of sample classification in principal component analysis showed a good similarity with cluster analysis. This method showed the information of chemical composition in Cordyceps, with good repeatability and similarity between samples, indicating that the stable chemical distribution and proportion of the major nucleosides in the medical materials. Fingerprints, principal component analysis and cluster analysis, which are applied to identify the different sources of Cordyceps, provide an experimental basis for establishing the characteristics evaluation methodology of medicinal materials.

  15. Regulator and effector functions of T-cell subsets in human Leishmania infections

    DEFF Research Database (Denmark)

    Kemp, M

    1997-01-01

    the cytokine production by CD4+ T cells has been identified as a major factor in determining the outcome of the infection. In these models Th1 cells producing IFN-gamma provide protection against the infection whereas Th2 cells producing IL-4 and IL-10 aggravate the disease. The fatal outcome of Leishmania...... reactions to Leishmania parasites in humans can be associated with both protection and pathogenesis. Many individuals without previous exposure to Leishmania parasites have T cells which can respond to Leishmania antigens. These cells have the potential to generate either Th1 or Th2 like responses. During...... infection with Leishmania parasites humans develop specific T-cell recognition of well-characterized parasite antigens. T cells producing disease-exacerbating factors such as IL-4 in response to Leishmania antigen stimulation have been identified in humans as well as in mice. Both Th1 like and Th2 like...

  16. 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.

  17. Hledání genů kontrolujících odpověď myši na parazitického prvoka Leishmania major

    Czech Academy of Sciences Publication Activity Database

    Schlegel, David; Havelková, Helena; Demant, P.; Lipoldová, Marie

    X, č. 24 (2002), s. 1210-6267 [Genetická konference - Perspektivy genetiky - genomy a genová exprese. 05.02.2002-06.02.2002, Brno] R&D Projects: GA ČR GA310/00/0760 Institutional research plan: CEZ:AV0Z5052915 Keywords : chromosome * Leishmania * mouse Subject RIV: EB - Genetics ; Molecular Biology

  18. Nucleoside antibiotics: biosynthesis, regulation, and biotechnology.

    Science.gov (United States)

    Niu, Guoqing; Tan, Huarong

    2015-02-01

    The alarming rise in antibiotic-resistant pathogens has coincided with a decline in the supply of new antibiotics. It is therefore of great importance to find and create new antibiotics. Nucleoside antibiotics are a large family of natural products with diverse biological functions. Their biosynthesis is a complex process through multistep enzymatic reactions and is subject to hierarchical regulation. Genetic and biochemical studies of the biosynthetic machinery have provided the basis for pathway engineering and combinatorial biosynthesis to create new or hybrid nucleoside antibiotics. Dissection of regulatory mechanisms is leading to strategies to increase the titer of bioactive nucleoside antibiotics. Copyright © 2014. Published by Elsevier Ltd.

  19. 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 ...

  20. Involvement of Leishmania donovani major surface glycoprotein ...

    Indian Academy of Sciences (India)

    Unknown

    plasmid pGEX-gp63 or control vector was introduced into Escherichia coli ... (B) Map of pGP63-2′; coding region inverted. (C) Map of pRH2; ... Control hybridizations showed that β-tubulin and other unrelated. mRNAs are expressed normally in the vector controls and the antisense transfectants (figure 2B). Western blot ...

  1. SOME RECENT FINDINGS IN THE BIOTECHNOLOGY OF BIOLOGICALLY IMPORTANT NUCLEOSIDES

    Directory of Open Access Journals (Sweden)

    A. Mikhailopulo

    2013-08-01

    Full Text Available Some recent findings in the biotechnology of biologically important nucleosides will be discussed, viz., (i a new strategy of the cascade one-pot transformation of D-pentoses into nucleosides based on the extension and deepening of the knowledge of the mechanism of functioning of the ribokinase, phosphopentomutase, and uridine, thymidine and purine nucleoside (PNP phosphorylases, and the role of different factors (structural, electronic, stereochemical in the glycoside bond formation, (ii the modern chemistries of the chemo-enzymatic syntheses of nucleosides, (iii the transglycosylation reaction using natural and sugar modified nucleosides as donors of carbohydrate residues and heterocyclic bases as acceptors catalyzed by nucleoside phosphorylases (NP.

  2. Isolation of Leishmania tropica from an Ethiopian cutaneous leishmaniasis patient

    NARCIS (Netherlands)

    Hailu, Asrat; Di Muccio, Trentina; Abebe, Tamrat; Hunegnaw, Mesfin; Kager, Piet A.; Gramiccia, Marina

    2006-01-01

    Cutaneous leishmaniasis (CL) in the Old World is caused mainly by three species of Leishmania: L. major, L. tropica and L. aethiopica, and sporadically by L. infantum and L. donovani. In Ethiopia, zoonotic cutaneous leishmaniasis, caused by L. aethiopica, is a major public health problem affecting

  3. Genomic Organization of Leishmania Species

    Directory of Open Access Journals (Sweden)

    B Kazemi

    2011-09-01

    Full Text Available Leishmania is a protozoan parasite belonging to the family Trypanosomatidae, which is found among 88 different countries. The parasite lives as an amastigote in vertebrate macro­phages and as a promastigote in the digestive tract of sand fly. It can be cultured in the laboratory us­ing appropriate culture media. Although the sexual cycle of Leishmania has not been observed during the promastigote and amastigote stages, it has been reported by some researchers. Leishma­nia has eukaryotic cell organization. Cell culture is convenient and cost effective, and because posttranslational modifications are common processes in the cultured cells, the cells are used as hosts for preparing eukaryotic recombinant proteins for research. Several transcripts of rDNA in the Leishmania genome are suitable regions for conducting gene transfer. Old World Leishmania spp. has 36 chromosomes, while New World Leishmania spp. has 34 or 35 chromo­somes. The genomic organization and parasitic characteristics have been investigated. Leishmania spp. has a unique genomic organization among eukaryotes; the genes do not have introns, and the chromosomes are smaller with larger numbers of genes confined to a smaller space within the nucleus. Leishmania spp. genes are organized on one or both DNA strands and are transcribed as polycistronic (prokaryotic-like transcripts from undefined promoters. Regulation of gene expres­sion in the members of Trypanosomatidae differs from that in other eukaryotes. The trans-splic­ing phenomenon is a necessary step for mRNA processing in lower eukaryotes and is observed in Leishmania spp. Another particular feature of RNA editing in Leishmania spp. is that mitochon­drial genes encoding respiratory enzymes are edited and transcribed. This review will discuss the chromosomal and mitochondrial (kinetoplast genomes of Leishmania spp. as well as the phenome­non of RNA editing in the kinetoplast genome.

  4. Curcumin overcomes the inhibitory effect of nitric oxide on Leishmania.

    Science.gov (United States)

    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.

  5. 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.

  6. 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

    2016-01-01

    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

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  11. Identification and characterization of new Leishmania promastigote surface antigens, LaPSA-38S and LiPSA-50S, as major immunodominant excreted/secreted components of L. amazonensis and L. infantum

    OpenAIRE

    Bras Goncalves, Rachel; Petitdidier, Elodie; Pagniez, Julie; Veyrier, Renaud; Cibrelus, P.; Cavaleyra, Mireille; Maquaire, S.; Moreaux, J.; Lemesre, Jean-Loup

    2014-01-01

    We have previously demonstrated that sera from dogs vaccinated with excreted/secreted antigens (ESA) of Leishmania infantum promastigotes (LiESAp) mainly recognized an immunodominant antigen of 54 kDa. An anti-LiESAp-specific IgG2 humoral response was observed and associated to Th1-type response in vaccinated dogs. This response was highly correlated with a long-lasting and strong LiES-Ap-vaccine protection toward L. infantum experimental infection. In addition, it was also shown that dogs fr...

  12. 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.

  13. Cross-protective efficacy from a immunogen firstly identified in Leishmania infantum against tegumentary leishmaniasis.

    Science.gov (United States)

    Martins, V T; Lage, D P; Duarte, M C; Costa, L E; Chávez-Fumagalli, M A; Roatt, B M; Menezes-Souza, D; Tavares, C A P; Coelho, E A F

    2016-02-01

    Experimental vaccine candidates have been evaluated to prevent leishmaniasis, but no commercial vaccine has been proved to be effective against more than one parasite species. LiHyT is a Leishmania-specific protein that was firstly identified as protective against Leishmania infantum. In this study, LiHyT was evaluated as a vaccine to against two Leishmania species causing tegumentary leishmaniasis (TL): Leishmania major and Leishmania braziliensis. BALB/c mice were immunized with rLiHyT plus saponin and lately challenged with promastigotes of the two parasite species. The immune response generated was evaluated before and 10 weeks after infection, as well as the parasite burden at this time after infection. The vaccination induced a Th1 response, which was characterized by the production of IFN-γ, IL-12 and GM-CSF, as well as by high levels of IgG2a antibodies, after in vitro stimulation using both the protein and parasite extracts. After challenge, vaccinated mice showed significant reductions in their infected footpads, as well as in the parasite burden in the tissue and organs evaluated, when compared to the control groups. The anti-Leishmania Th1 response was maintained after infection, being the IFN-γ production based mainly on CD4(+) T cells. We described one conserved Leishmania-specific protein that could compose a pan-Leishmania vaccine. © 2016 John Wiley & Sons Ltd.

  14. Pyrrolidine analogues of nucleosides and nucleotides

    Czech Academy of Sciences Publication Activity Database

    Rejman, Dominik; Pohl, Radek; Kovačková, Soňa; Kočalka, Petr; Švenková, Alžběta; Šanderová, Hana; Krásný, Libor; Rosenberg, Ivan

    -, č. 52 (2008), s. 577-578 ISSN 0261-3166. [Joint Symposium of the International Roundtable on Nucleosides, Nucleotides and Nucleic Acids /18./ and the International Symposium on Nucleic Acid Chemistry /35./. Kyoto, 08.09.2008-12.09.2008] R&D Projects: GA MŠk(CZ) LC06077; GA MŠk 2B06065; GA MZd NR9138 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50200510; CEZ:AV0Z50520514 Keywords : pyrrolidine * nucleoside * nucleotide Subject RIV: CC - Organic Chemistry

  15. Synthesis and turnover of purine nucleoside phosphorylase in human lymphocytes

    International Nuclear Information System (INIS)

    Snyder, F.F.; Kwan, E.; Mably, E.R.; Neote, K.

    1986-01-01

    The authors study the synthesis of purine nucleoside phosphorylase during phytohumagglutinin induced T cell transformation and have examined the turnover of purine nucleoside phosphorylase in thte human lymphoblast WI-L2. A polyclonal antibody to purine nucleoside phosphorylase in the synthetic and turnover studies is used. Fluorography of purine nucleoside phosphorylase immunoprecipitates from WI-L2 cells are shown. Lymphoblast proteins were labelled during 16hour cultrue with 4,5-tritium-leucine. Samples were 20 ug of total protein, immunoprecipitate of 200 ug protein with control serum and purine nucleoside phosphorylase antiserum

  16. Sand fly evolution and its relationship to Leishmania transmission

    Directory of Open Access Journals (Sweden)

    PD Ready

    2000-08-01

    Full Text Available The evolutionary relationships of sand flies and Leishmania are discussed in this report, which draws distinctions between co-association, co-evolution and co-speciation (or co-cladogenesis. Examples focus on Phlebotomus vectors of Le. infantum and Le. major in the Mediterranean subregion.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Vector transmission of leishmania abrogates vaccine-induced protective immunity.

    Directory of Open Access Journals (Sweden)

    Nathan C Peters

    2009-06-01

    Full Text Available Numerous experimental vaccines have been developed to protect against the cutaneous and visceral forms of leishmaniasis caused by infection with the obligate intracellular protozoan Leishmania, but a human vaccine still does not exist. Remarkably, the efficacy of anti-Leishmania vaccines has never been fully evaluated under experimental conditions following natural vector transmission by infected sand fly bite. The only immunization strategy known to protect humans against natural exposure is "leishmanization," in which viable L. major parasites are intentionally inoculated into a selected site in the skin. We employed mice with healed L. major infections to mimic leishmanization, and found tissue-seeking, cytokine-producing CD4+ T cells specific for Leishmania at the site of challenge by infected sand fly bite within 24 hours, and these mice were highly resistant to sand fly transmitted infection. In contrast, mice vaccinated with a killed vaccine comprised of autoclaved L. major antigen (ALM+CpG oligodeoxynucleotides that protected against needle inoculation of parasites, showed delayed expression of protective immunity and failed to protect against infected sand fly challenge. Two-photon intra-vital microscopy and flow cytometric analysis revealed that sand fly, but not needle challenge, resulted in the maintenance of a localized neutrophilic response at the inoculation site, and removal of neutrophils following vector transmission led to increased parasite-specific immune responses and promoted the efficacy of the killed vaccine. These observations identify the critical immunological factors influencing vaccine efficacy following natural transmission of Leishmania.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Mucosal Leishmaniasis caused by Leishmania (Viannia braziliensis and Leishmania (Viannia guyanensis in the Brazilian Amazon.

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    Jorge Augusto de Oliveira Guerra

    Full Text Available BACKGROUND: Leishmania (Viannia braziliensis is a parasite recognized as the most important etiologic agent of mucosal leishmaniasis (ML in the New World. In Amazonia, seven different species of Leishmania, etiologic agents of human Cutaneous Leishmaniasis, have been described. Isolated cases of ML have been described for several different species of Leishmania: L. (V. panamensis, L. (V. guyanensis and L. (L. amazonensis. METHODOLOGY: Leishmania species were characterized by polymerase chain reaction (PCR of tissues taken from mucosal biopsies of Amazonian patients who were diagnosed with ML and treated at the Tropical Medicine Foundation of Amazonas (FMTAM in Manaus, Amazonas state, Brazil. Samples were obtained retrospectively from the pathology laboratory and prospectively from patients attending the aforementioned tertiary care unit. RESULTS: This study reports 46 cases of ML along with their geographical origin, 30 cases caused by L. (V. braziliensis and 16 cases by L. (V. guyanensis. This is the first record of ML cases in 16 different municipalities in the state of Amazonas and of simultaneous detection of both species in 4 municipalities of this state. It is also the first record of ML caused by L. (V. guyanensis in the states of Pará, Acre, and Rondônia and cases of ML caused by L. (V. braziliensis in the state of Rondônia. CONCLUSIONS/SIGNIFICANCE: L. (V. braziliensis is the predominant species that causes ML in the Amazon region. However, contrary to previous studies, L. (V. guyanensis is also a significant causative agent of ML within the region. The clinical and epidemiological expression of ML in the Manaus region is similar to the rest of the country, although the majority of ML cases are found south of the Amazon River.

  1. Tetrofuranose nucleoside phosphonic acids: Synthesis and properties

    Czech Academy of Sciences Publication Activity Database

    Poláková, Ivana; Buděšínský, Miloš; Točík, Zdeněk; Rosenberg, Ivan

    2011-01-01

    Roč. 76, č. 5 (2011), s. 503-536 ISSN 0010-0765 R&D Projects: GA AV ČR KAN200520801; GA ČR GA203/09/0820; GA MŠk(CZ) LC06061; GA MŠk(CZ) LC06077 Institutional research plan: CEZ:AV0Z40550506 Keywords : tetrofuranosyl phosphonate * nucleotide analogues * phosphonomethoxy nucleosides * sugar hydroxyphosphonates Subject RIV: CC - Organic Chemistry Impact factor: 1.283, year: 2011

  2. Synthesis and Biological Activity of Reversed Pyrimidine Nucleosides

    Directory of Open Access Journals (Sweden)

    Nataša Župančić

    2015-03-01

    Full Text Available An efficient approach to reversed nucleosides which enables their synthesis in gram quantities is described. N-1′-Pyrimidine reversed nucleosides were prepared by treating of the sodium salt of pyrimidine bases with protected 5-tosyl ribose. Additionally, N-1′,N-3′-disubstituted reversed nucleosides were isolated in the condensation reactions with the 5-halogen pyrimidines. Using the Sonogashira coupling of 5′-iodouracil reversed nucleoside with ethynyltrimethyl silane gave 5′-ethynyl derivative which was further transformed into 5′-acetyl reversed nucleoside. Biological activity of deprotected reversed nucleosides was validated on the panel of six human carcinoma cell lines (HeLa, MIAPaCa2, Hep2, NCI-H358, CaCo-2, and HT-29. 5′-Iodouracil derivative displayed moderate growth inhibition activity against human colon carcinoma (CaCo-2 cells.

  3. Evidence for Rosettes as an Unrecognized Stage in the Life Cycle of Leishmania Parasites

    OpenAIRE

    Iovannisci, David M.; Plested, C. Paul; Moe, Gregory R.

    2010-01-01

    Leishmania parasites, which afflict 12 million people in 88 countries, exist as promastigotes transmitted by insect vectors and as amastigotes residing in mammalian macrophages. Promastigote cells arranged in rosettes have also been described but universally disregarded as a distinct stage in the life cycle. We present evidence that only rosettes of Leishmania major promastigotes express cell surface polysialic acid (PSA) and PSA containing de-N-acetyl neuraminic acid (NeuPSA). Expression of ...

  4. Nucleoside transport in primary cultured rabbit tracheal epithelial cells.

    Science.gov (United States)

    Mathias, Neil R; Wu, Sharon K; Kim, Kwang-Jin; Lee, Vincent H L

    2005-01-01

    The present study aimed at elucidating the mechanisms of nucleoside transport in primary cultured rabbit tracheal epithelial cells (RTEC) grown on a permeable filter support. Uptake of (3)H-uridine, the model nucleoside substrate, from the apical fluid of primary cultured RTEC was examined with respect to its dependence on Na(+), substrate concentration, temperature and its sensitivity to inhibitors, other nucleosides and antiviral nucleoside analogs. Apical (3)H-uridine uptake in primary cultured RTEC was strongly dependent on an inward Na(+) gradient and temperature. Ten micromolar nitro-benzyl-mercapto-purine-ribose (NBMPR) (an inhibitor of es-type nucleoside transport in the nanomolar range) did not further inhibit this process. (3)H-uridine uptake from apical fluid was inhibited by basolateral ouabain (10 microM) and apical phloridzin (100 microM), indicating that uptake may involve a secondary active transport process. Uridine uptake was saturable with a K(m) of 3.4 +/- 1.8 microM and the V(max) of 24.3 +/- 5.2 pmoles/mg protein/30 s. Inhibition studies indicated that nucleoside analogs that have a substitution on the nucleobase competed with uridine uptake from apical fluid, but those with modifications on the ribose sugar including acyclic analogs were ineffective. The pattern of inhibition of apical (3)H-uridine, (3)H-inosine and (3)H-thymidine uptake into RTEC cells by physiological nucleosides was consistent with multiple systems: A pyrimidine-selective transport system (CNT1); a broad nucleoside substrate transport system that excludes inosine (CNT4) and an equilibrative NBMPR-insensitive nucleoside transport system (ei type). These results indicate that the presence of apically located nucleoside transporters in the epithelial cells lining the upper respiratory tract can lead to a high accumulation of nucleosides in the trachea. At least one Na(+)-dependent, secondary, active transport process may mediate the apical absorption of nucleosides or

  5. Practical Considerations For Developing Nucleoside Reverse Transcriptase Inhibitors

    OpenAIRE

    Hurwitz, Selwyn J.; Schinazi, Raymond F.

    2012-01-01

    Nucleoside reverse transcriptase inhibitors (NRTI) remain a cornerstone of current antiretroviral regimens in combinations usually with a non-nucleoside reverse transcriptase inhibitor (NNRTI), a protease inhibitor (PI), or an integrase inhibitor (INI). The antiretroviral efficacy and relative safety of current NRTI results from a tight and relatively specific binding of their phosphorylated nucleoside triphosphates (NRTI-TP) with the HIV-1 reverse transcriptase which is ess...

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  8. Synthesis and Antiviral Activity of Novel 4'-Branched Carbocyclic C-Nucleoside

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Ying Lan; Hong, Joon Hee [Chosun University, Gwangju (Korea, Republic of)

    2005-09-15

    C-Nucleosides have received considerable attention due not only to the chemical stability but also to the interesting biological activities of naturally occurring compounds such as showdomycin, formycins, oxazinomycin, etc. Also, several biologically active synthetic C-nucleosides such as pseudoisocytidine, thiazofurin, and 9-deazaadenosine have been reported. Carbocyclic nucleosides are another class of metabolically stable nucleosides in which a methylene group replaces the oxygen in the furan ring of the natural nucleosides. Another interesting feature of carbocyclic nucleosides is that a number of carbocyclic adenosine analogues are assumed to exert their antiviral action via the inhibition of S-adenosyl-homocysteine hydrolase. Moreover, this mechanism might be exploited in a combination therapy in association with the nucleosides with a different mechanism of action. On the basis of these interesting chemical and biological properties of C-nucleosides and carbocyclic nucleosides, it was of interest to synthesize hybrid nucleosides, carbocyclic C-nucleosides. Although some carbocyclic and C-nucleosides are naturally occurring, so far no natural carbocyclic C-nucleosides have been reported. The history of synthesis of carbocyclic C-nucleosides dates back to the 1960s. Despite the long history of carbocyclic C-nucleosides, only a few carbocyclic C-nucleosides have been synthesized, probably due to the synthetic difficulties of these nucleosides. Herein, we would like to report the synthesis procedure of a novel 4'-branched carbocyclic C-nucleoside.

  9. Structure of grouper iridovirus purine nucleoside phosphorylase

    International Nuclear Information System (INIS)

    Kang, You-Na; Zhang, Yang; Allan, Paula W.; Parker, William B.; Ting, Jing-Wen; Chang, Chi-Yao; Ealick, Steven E.

    2010-01-01

    The crystal structure of purine nucleoside phosphorylase from grouper iridovirus was solved at 2.38 Å resolution. Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides to the corresponding free bases and ribose 1-phosphate. The crystal structure of grouper iridovirus PNP (givPNP), corresponding to the first PNP gene to be found in a virus, was determined at 2.4 Å resolution. The crystals belonged to space group R3, with unit-cell parameters a = 193.0, c = 105.6 Å, and contained four protomers per asymmetric unit. The overall structure of givPNP shows high similarity to mammalian PNPs, having an α/β structure with a nine-stranded mixed β-barrel flanked by a total of nine α-helices. The predicted phosphate-binding and ribose-binding sites are occupied by a phosphate ion and a Tris molecule, respectively. The geometrical arrangement and hydrogen-bonding patterns of the phosphate-binding site are similar to those found in the human and bovine PNP structures. The enzymatic activity assay of givPNP on various substrates revealed that givPNP can only accept 6-oxopurine nucleosides as substrates, which is also suggested by its amino-acid composition and active-site architecture. All these results suggest that givPNP is a homologue of mammalian PNPs in terms of amino-acid sequence, molecular mass, substrate specificity and overall structure, as well as in the composition of the active site

  10. Two nucleoside transporters in Lactococcus lactis with different substrate specificities

    DEFF Research Database (Denmark)

    Martinussen, Jan; Sørensen, Claus; Jendresen, Christian Bille

    2010-01-01

    , and the utilization of nucleotides is dependent on exogenous phosphatases. The composition of transporters with specificity for purine and pyrimidine nucleosides and nucleobases is subject to variation. The ability of Lactococcus lactis to transport different nucleosides across the cell membrane was characterized...

  11. 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

  12. Detection of Leishmania RNA virus in Leishmania parasites.

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Dichotomy of the T cell response to Leishmania antigens in patients suffering from cutaneous leishmaniasis; absence or scarcity of Th1 activity is associated with severe infections

    DEFF Research Database (Denmark)

    Gaafar, A; Kharazmi, A; Ismail, A

    1995-01-01

    The T cell response was studied in 25 patients suffering from cutaneous leishmaniasis caused by Leishmania major with severe (n = 10) and mild (n = 15) disease manifestations. Peripheral blood mononuclear cells (PBMC) from the patients were activated by sonicates of Leishmania promastigotes (LMP...

  16. 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.

  17. An Efficient and Facile Methodology for Bromination of Pyrimidine and Purine Nucleosides with Sodium Monobromoisocyanurate (SMBI

    Directory of Open Access Journals (Sweden)

    Roger Stromberg

    2013-10-01

    Full Text Available An efficient and facile strategy has been developed for bromination of nucleosides using sodium monobromoisocyanurate (SMBI. Our methodology demonstrates bromination at the C-5 position of pyrimidine nucleosides and the C-8 position of purine nucleosides. Unprotected and also several protected nucleosides were brominated in moderate to high yields following this procedure.

  18. Vitamin E Phosphate Nucleoside Prodrugs: A Platform for Intracellular Delivery of Monophosphorylated Nucleosides

    Directory of Open Access Journals (Sweden)

    Richard Daifuku

    2018-02-01

    Full Text Available Vitamin E phosphate (VEP nucleoside prodrugs are designed to bypass two mechanisms of tumor resistance to therapeutic nucleosides: nucleoside transport and kinase downregulation. Certain isoforms of vitamin E (VE have shown activity against solid and hematologic tumors and result in chemosensitization. Because gemcitabine is one of the most common chemotherapeutics for the treatment of cancer, it was used to demonstrate the constructs utility. Four different VE isoforms were conjugated with gemcitabine at the 5′ position. Two of these were δ-tocopherol-monophosphate (MP gemcitabine (NUC050 and δ-tocotrienol-MP gemcitabine (NUC052. NUC050 was shown to be able to deliver gemcitabine-MP intracellularly by a nucleoside transport independent mechanism. Its half-life administered IV in mice was 3.9 h. In a mouse xenograft model of non-small cell lung cancer (NSCLC NCI-H460, NUC050 at a dose of 40 mg/kg IV qwk × 4 resulted in significant inhibition to tumor growth on days 11–31 (p < 0.05 compared to saline control (SC. Median survival was 33 days (NUC050 vs. 25.5 days (SC ((hazard ratio HR = 0.24, p = 0.017. Further, NUC050 significantly inhibited tumor growth compared to historic data with gemcitabine at 135 mg/kg IV q5d × 3 on days 14–41 (p < 0.05. NUC052 was administered at a dose of 40 mg/kg IV qwk × 2 followed by 50 mg/kg qwk × 2. NUC052 resulted in inhibition to tumor growth on days 14–27 (p < 0.05 and median survival was 34 days (HR = 0.27, p = 0.033. NUC050 and NUC052 have been shown to be safe and effective in a mouse xenograft of NSCLC.

  19. Serological reactivity of different antigenic preparations of Leishmania (Leishmania amazonensis and the Leishmania braziliensis complex Reatividade sorológica frente a diferentes preparações antigênicas de Leishmania (Leishmania amazonensis e do complexo Leishmania braziliensis

    Directory of Open Access Journals (Sweden)

    Adriano Gomes-Silva

    2008-04-01

    Full Text Available Total antigen from Leishmania (Leishmania amazonensis and isolates from the Leishmania braziliensis complex, along with their respective antigenic fractions obtained by affinity chromatography on concanavalin-A-Sepharose and jacalin-agarose columns evaluated using immunoenzymatic ELISA assay. For this, serum samples from 229 patients were used, grouped as American tegmental leishmaniasis (nº=58, visceral leishmaniasis (nº=28, Chagas disease (nº=49, malaria (nº=32, tuberculosis (nº=13 and healthy volunteers (nº=49. Samples from American tegmentary leishmaniasis showed higher reactivity with antigens isolated from the Leishmania braziliensis complex than with antigens from Leishmania amazonensis (pAntígeno total de Leishmania (Leishmania amazonensis e isolado do complexo Leishmania brazilienis, assim como suas respectivas frações antigênicas obtidas por cromatografia de afinidade em coluna de concanavalina-A ligada a sepharose e Jacalina ligada a agarose foram avaliadas por ensaio imunoenzimático ELISA. Para tanto, foram utilizadas amostras de soros de 229 pacientes agrupadas em leishmaniose tegumentar americana (nº=58, leishmaniose visceral (nº=28, doença de Chagas (nº=49, malaria (nº=32, tuberculose (nº=13 e voluntários saudáveis (nº=49. Houve maior reatividade das amostras de leishmaniose tegumentar americana com a utilização dos antígenos obtidos do isolado do complexo Leishmania braziliensis quando comparado com antígenos de Leishmania amazonensis (p<0,001. Observou-se ainda que a sensibilidade do teste ELISA variou de 60 a 95% entre os antígenos obtidos do isolado do complexo Leishmania braziliensis. Houve acentuada reatividade inespecífica das amostras de soros com a utilização das frações antigênicas ligantes de Concanavalina-A e Jacalina de ambos os complexos Leishmania em comparação aos demais antígenos (p<0,001. Os resultados apresentados no presente trabalho sugerem que a utilização de antígenos hom

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

    Science.gov (United States)

    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.

  1. Natural and engineered biosynthesis of nucleoside antibiotics in Actinomycetes.

    Science.gov (United States)

    Chen, Wenqing; Qi, Jianzhao; Wu, Pan; Wan, Dan; Liu, Jin; Feng, Xuan; Deng, Zixin

    2016-03-01

    Nucleoside antibiotics constitute an important family of microbial natural products bearing diverse bioactivities and unusual structural features. Their biosynthetic logics are unique with involvement of complex multi-enzymatic reactions leading to the intricate molecules from simple building blocks. Understanding how nature builds this family of antibiotics in post-genomic era sets the stage for rational enhancement of their production, and also paves the way for targeted persuasion of the cell factories to make artificial designer nucleoside drugs and leads via synthetic biology approaches. In this review, we discuss the recent progress and perspectives on the natural and engineered biosynthesis of nucleoside antibiotics.

  2. In Silico Investigation of Flavonoids as Potential Trypanosomal Nucleoside Hydrolase Inhibitors

    Directory of Open Access Journals (Sweden)

    Christina Hung Hung Ha

    2015-01-01

    Full Text Available Human African Trypanosomiasis is endemic to 37 countries of sub-Saharan Africa. It is caused by two related species of Trypanosoma brucei. Current therapies suffer from resistance and public accessibility of expensive medicines. Finding safer and effective therapies of natural origin is being extensively explored worldwide. Pentamidine is the only available therapy for inhibiting the P2 adenosine transporter involved in the purine salvage pathway of the trypanosomatids. The objective of the present study is to use computational studies for the investigation of the probable trypanocidal mechanism of flavonoids. Docking experiments were carried out on eight flavonoids of varying level of hydroxylation, namely, flavone, 5-hydroxyflavone, 7-hydroxyflavone, chrysin, apigenin, kaempferol, fisetin, and quercetin. Using AutoDock 4.2, these compounds were tested for their affinity towards inosine-adenosine-guanosine nucleoside hydrolase and the inosine-guanosine nucleoside hydrolase, the major enzymes of the purine salvage pathway. Our results showed that all of the eight tested flavonoids showed high affinities for both hydrolases (lowest free binding energy ranging from −10.23 to −7.14 kcal/mol. These compounds, especially the hydroxylated derivatives, could be further studied as potential inhibitors of the nucleoside hydrolases.

  3. 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...... that licochalcone A in concentrations that are nontoxic to host cells exhibits a strong antileishmanial activity and that appropriate substituted chalcones might be a new class of antileishmanial drugs....

  4. Characterization of Leishmania (Leishmania) amazonensis promastigotes resistant to pentamidine.

    Science.gov (United States)

    Coelho, Adriano C; Gentil, Luciana G; da Silveira, José Franco; Cotrim, Paulo C

    2008-09-01

    Pentamidine is a second-line agent used in the treatment of leishmaniasis and its mode of action and mechanism of resistance is not well understood. It was previously demonstrated that transfection of promastigotes and amastigotes with the ABC transporter PRP1 gene confers resistance to pentamidine. To further clarify this point, we generated Leishmania amazonensis mutants resistant to pentamidine. Our results indicated that this ABC transporter is not associated with pentamidine resistance in lines generated by drug pressure through amplification or overexpression mechanisms of PRP1 gene.

  5. Medicinal Chemistry of Fluorinated Cyclic and Acyclic Nucleoside Phosphonates

    Czech Academy of Sciences Publication Activity Database

    Baszczyňski, Ondřej; Janeba, Zlatko

    2013-01-01

    Roč. 33, č. 6 (2013), s. 1304-1344 ISSN 0198-6325 Institutional support: RVO:61388963 Keywords : nucleoside phosphosphonates * fluorine * antiviral * anticancer * antiparasitic Subject RIV: CC - Organic Chemistry Impact factor: 8.131, year: 2013

  6. Impact of unnatural nucleosides on the control of microbial growth.

    Science.gov (United States)

    Hatano, Akihiko; Nishimura, Makoto; Souta, Ikuo

    2009-06-01

    This research investigated the antimicrobial activities of unnatural nucleosides. We tested the MIC and MBC of 17 synthetic nucleoside analogues against 10 microbial strains. These nucleoside analogues were classified into four groups according to their structural characteristics. Inhibition was observed with compounds 1-1, 3-1, and 4-3. In particular, 5'-deoxythymidine (3-1) was most effective at 50 micro g/mL against Bacillus subtilis and Staphylococcus aureus. This analogue has had the hydroxyl group at the 5' position replaced with a hydrogen atom. All compounds had weak effects against various species of mold. The MBC of 5'-deoxythymidine was 50 g/mL in 0.5 h against S. aureus. These results showed that 5'-deoxythymidine had the most effective antimicrobial activity of the 17 different unnatural nucleosides. The inhibitory effect of 3-1 suggests that it may be useful as an antibacterial agent in medical situations.

  7. Current prodrug strategies for improving oral absorption of nucleoside analogues

    Directory of Open Access Journals (Sweden)

    Youxi Zhang

    2014-04-01

    Full Text Available Nucleoside analogues are first line chemotherapy in various severe diseases: AIDS (acquired immunodeficiency disease syndrome, cytomegalovirus infections, cancer, etc. However, many nucleoside analogues exhibit poor oral bioavailability because of their high polarity and low intestinal permeability. In order to get around this drawback, prodrugs have been utilized to improve lipophilicity by chemical modification of the parent drug. Alternatively, prodrugs targeting transporters present in the intestine have been applied to promote the transport of the nucleoside analogues. Valacyclovir and valganciclovir are two classic valine ester prodrugs transported by oligopeptide transporter 1. The ideal prodrug achieves delivery of a parent drug by attaching a non-toxic moiety that is stable during transport, but is readily degraded to the parent drug once at the target. This article presents advances of prodrug approaches for enhancing oral absorption of nucleoside analogues.

  8. Meteorite-catalyzed synthesis of nucleosides and other prebiotic compounds

    Czech Academy of Sciences Publication Activity Database

    Ferus, Martin; Knížek, Antonín; Civiš, Svatopluk

    2015-01-01

    Roč. 112, č. 23 (2015), s. 7109-7110 ISSN 0027-8424 Institutional support: RVO:61388955 Keywords : meteorite-catalzzed synthesis * nucleosides * prebiotic compounds Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 9.423, year: 2015

  9. Thermus thermophilus Strains Active in Purine Nucleoside Synthesis

    Directory of Open Access Journals (Sweden)

    Marcos Almendros

    2009-03-01

    Full Text Available Several strains of Thermus thermophilus were tested in order to detect purine nucleoside synthase activity using pyrimidine nucleosides as the sugar-donor and adenine or hypoxanthine as bases. High productivity values (t =1 hr were obtained while completely avoiding adenosine-deaminase degradation of the products. N-2-deoxy-ribosyltransferase activity is described for the first time in hyperthermophilic bacteria.

  10. [Cutaneous leishmaniasis due to Leishmania infantum associated with HIV].

    Science.gov (United States)

    Diatta, B-A; Diallo, M; Diadie, S; Faye, B; Ndiaye, M; Hakim, H; Diallo, S; Seck, B; Niang, S-O; Kane, A; Dieng, M-T

    2016-10-01

    In Senegal, reported cases of cutaneous leishmaniasis are often due to Leishmania major. Immunosuppression related to HIV infection contributes to the emergence of leishmaniasis in humans and to cutaneous localization of viscerotropic species. We report the first observed case in Senegal of opportunistic cutaneous leishmaniasis due to Leishmania infantum associated with HIV. A 5-year-old boy presented crusted ulcerative lesions of the scalp and left forearm, together with axillary and cervical lymphadenopathy present for two months. Direct parasitological examination of the scalp and arm lesions, coupled with liquid aspiration of lymph nodes and bone marrow, enabled identification of amastigote forms of Leishmania. Polymerase chain reaction performed on skin, lymph node and bone marrow biopsy samples allowed identification of L. infantum. The child was positive for HIV1. Treatment of HIV infection and leishmaniasis resulted in clinical improvement. Co-infection with cutaneous leishmaniasis due to L. infantum and HIV is a complex combination in terms of the related therapeutic issues. The clinical and laboratory outcomes depend on restoration of immunity and on the efficacy, safety and availability of anti-leishmaniasis drugs. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  11. Synthesis of coumarin or ferrocene labeled nucleosides via Staudinger ligation

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    Kois Pavol

    2006-11-01

    Full Text Available Abstract Background Reaction of azides with triaryl phosphines under mild conditions gives iminophosphoranes which can react with almost any kind of electrophilic reagent, e.g. aldehydes/ketones to form imines or esters to form amides. This so-called Staudinger ligation has been employed in a wide range of applications as a general tool for bioconjugation including specific labeling of nucleic acids. Results A new approach for the preparation of labeled nucleosides via intermolecular Staudinger ligation is described. Reaction of azidonucleosides with triphenylphosphine lead to iminophosphorane intermediates, which react subsequently with derivatives of coumarin or ferrocene to form coumarin or ferrocene labeled nucleosides. Fluorescent properties of coumarin labeled nucleosides are determined. Conclusion New coumarin and ferrocene labeled nucleosides were prepared via intermolecular Staudinger ligation. This reaction joins the fluorescent coumarin and biospecific nucleoside to the new molecule with promising fluorescent and electrochemical properties. The isolated yields of products depend on the structure of azidonucleoside and carboxylic acids. A detailed study of the kinetics of the Staudinger ligation with nucleoside substrates is in progress.

  12. Cathepsin B-like and cell death in the unicellular human pathogen Leishmania.

    Science.gov (United States)

    El-Fadili, A K; Zangger, H; Desponds, C; Gonzalez, I J; Zalila, H; Schaff, C; Ives, A; Masina, S; Mottram, J C; Fasel, N

    2010-09-02

    In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H(2)O(2)), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes.

  13. Biodistribution of meglumine antimoniate in healthy and Leishmania (Leishmania infantum chagasi-infected BALB/c mice

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    Samanta Etel Treiger Borborema

    2013-08-01

    Full Text Available Pentavalent antimonials such as meglumine antimoniate (MA are the primary treatments for leishmaniasis, a complex disease caused by protozoan parasites of the genus Leishmania . Despite over 70 years of clinical use, their mechanisms of action, toxicity and pharmacokinetics have not been fully elucidated. Radiotracer studies performed on animals have the potential to play a major role in pharmaceutical development. The aims of this study were to prepare an antimony radiotracer by neutron irradiation of MA and to determine the biodistribution of MA in healthy and Leishmania (Leishmania infantum chagasi-infected mice. MA (Glucantime(r was neutron irradiated inside the IEA-R1 nuclear reactor, producing two radioisotopes, 122Sb and 124Sb, with high radionuclidic purity and good specific activity. This irradiated compound presented anti-leishmanial activity similar to that of non-irradiated MA in both in vitro and in vivo evaluations. In the biodistribution studies, healthy mice showed higher uptake of antimony in the liver than infected mice and elimination occurred primarily through biliary excretion, with a small proportion of the drug excreted by the kidneys. The serum kinetic curve was bi-exponential, with two compartments: the central compartment and another compartment associated with drug excretion. Radiotracers, which can be easily produced by neutron irradiation, were demonstrated to be an interesting tool for answering several questions regarding antimonial pharmacokinetics and chemotherapy.

  14. Radioimmunoassays for the modified nucleosides N[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine and 2-methylthioadenosine.

    Science.gov (United States)

    Vold, B S

    1979-01-01

    Radioimmunoassays were established for the modified nucleosides N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine, t6A, and 2-methylthioadenosine, ms2A. The assays depended on the production of antisera specific for t6A and ms2A that have not been previously reported. The nitrocellulose membrane filtration and saturated ammonium sulfate RIA techniques were compared for efficiency. Various radioactive antigens were employed to establish which type of antigen would give the best binding. The tritium post-labeling procedure of Randerath and Randerath was used to obtain labeled nucleosides of high enough specific activity to be useful for RIAs when the labeled nucleoside was not available commerically. The specificity of the antibodies toward nucleosides and purified tRNAs is reported. Although the titer of the t6A antiserum was low, the specificity was very sharp. An interesting finding was that threonine, a major structural component of the side-chain modification of t6A, was completely infective as an inhibitor. PMID:493139

  15. In-silico Leishmania Target Selectivity of Antiparasitic Terpenoids

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    Ifedayo Victor Ogungbe

    2013-07-01

    Full Text Available Neglected Tropical Diseases (NTDs, like leishmaniasis, are major causes of mortality in resource-limited countries. The mortality associated with these diseases is largely due to fragile healthcare systems, lack of access to medicines, and resistance by the parasites to the few available drugs. Many antiparasitic plant-derived isoprenoids have been reported, and many of them have good in vitro activity against various forms of Leishmania spp. In this work, potential Leishmania biochemical targets of antiparasitic isoprenoids were studied in silico. Antiparasitic monoterpenoids selectively docked to L. infantum nicotinamidase, L. major uridine diphosphate-glucose pyrophosphorylase and methionyl t-RNA synthetase. The two protein targets selectively targeted by germacranolide sesquiterpenoids were L. major methionyl t-RNA synthetase and dihydroorotate dehydrogenase. Diterpenoids generally favored docking to L. mexicana glycerol-3-phosphate dehydrogenase. Limonoids also showed some selectivity for L. mexicana glycerol-3-phosphate dehydrogenase and L. major dihydroorotate dehydrogenase while withanolides docked more selectively with L. major uridine diphosphate-glucose pyrophosphorylase. The selectivity of the different classes of antiparasitic compounds for the protein targets considered in this work can be explored in fragment- and/or structure-based drug design towards the development of leads for new antileishmanial drugs.

  16. 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.

  17. Intranasal immunization with LACK-DNA promotes protective immunity in hamsters challenged with Leishmania chagasi.

    Science.gov (United States)

    DE Oliveira Gomes, Daniel Claudio; DA Silva Costa Souza, Beatriz Lilian; DE Matos Guedes, Herbert Leonel; Lopes, Ulisses Gazos; Rossi-Bergmann, Bartira

    2011-12-01

    LACK (Leishmania analogue of the receptor kinase C) is a conserved protein in protozoans of the genus Leishmania which is associated with the immunopathogenesis and susceptibility of BALB/c mice to L. major infection. Previously, we demonstrated that intranasal immunization with a plasmid carrying the LACK gene of Leishmania infantum (LACK-DNA) promotes protective immunity in BALB/c mice against Leishmania amazonensis and Leishmania chagasi. In the present study, we investigated the protective immunity achieved in hamsters intranasally vaccinated with 2 doses of LACK-DNA (30 μg). Compared with controls (PBS and pCI-neo plasmid), animals vaccinated with LACK-DNA showed significant reduction in parasite loads in the spleen and liver, increased lymphoproliferative response and increased nitric oxide (NO) production by parasite antigen-stimulated splenocytes. Furthermore, hamsters vaccinated with LACK-DNA presented high IgG and IgG2a serum levels when compared to control animals. Our results showed that intranasal vaccination with LACK-DNA promotes protective immune responses in hamsters and demonstrated the broad spectrum of intranasal LACK-DNA efficacy in different host species, confirming previous results in murine cutaneous and visceral leishmaniasis.

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

    Science.gov (United States)

    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.

  19. Evidence for rosettes as an unrecognized stage in the life cycle of Leishmania parasites.

    Science.gov (United States)

    Iovannisci, David M; Plested, C Paul; Moe, Gregory R

    2010-01-01

    Leishmania parasites, which afflict 12 million people in 88 countries, exist as promastigotes transmitted by insect vectors and as amastigotes residing in mammalian macrophages. Promastigote cells arranged in rosettes have also been described but universally disregarded as a distinct stage in the life cycle. We present evidence that only rosettes of Leishmania major promastigotes express cell surface poly-alpha2,8 N-acetyl neuraminic acid (PSA) and PSA containing de-N-acetyl neuraminic acid (NeuPSA). Expression of rosette-specific PSA antigens was mosaic, with individual promastigotes expressing PSA, NeuPSA or both. A 50 kDa protein was detected by Western blot analysis of a detergent-insoluble cell fraction with both PSA and NeuPSA-reactive antibodies. Frequencies of rosette formation as well as cell surface PSA/NeuPSA expression were temperature dependent. Rosettes also engaged in an unusual swarming behavior, congregating into extended clusters. Distinct structures resembling cellular fusion bodies were formed in and released from rosettes. The results indicate that rosettes are an unrecognized stage in the life cycle of Leishmania. We hypothesize that rosettes initiate mating in Leishmania during which PSA/NeuPSA expression plays an important role. Recognizing rosettes as a distinct form of the Leishmania life cycle opens new possibilities for treatment or prevention of disease and, possibly, in vitro genetic recombination without passage of cells through insect vectors.

  20. Serological evidence of Leishmania donovani infection in apparently healthy dogs using direct agglutination test (DAT) and rk39 dipstick tests in Kafta Humera, north-west Ethiopia

    NARCIS (Netherlands)

    Kalayou, S.; Tadelle, H.; Bsrat, A.; Abebe, N.; Haileselassie, M.; Schallig, H. D. F. H.

    2011-01-01

    Leishmania (Kinetoplastida: Trypanosomatidae) are protozoan parasites of significant medical and veterinary importance. Over the last decade, visceral leishmaniasis (VL) has emerged as a major opportunistic infection associated with HIV/AIDS in North Western Ethiopia. This paper reports on

  1. Leishmania carbon metabolism in the macrophage phagolysosome- feast or famine? [version 1; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    Malcolm J. McConville

    2015-10-01

    Full Text Available 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.

  2. Activity of Brazilian and Bulgarian propolis against different species of Leishmania

    Directory of Open Access Journals (Sweden)

    Gérzia Maria de Carvalho Machado

    2007-02-01

    Full Text Available Extracts of propolis samples collected in Brazil and Bulgaria were assayed against four Leishmania species - Leishmania amazonensis, L. braziliensis, L. chagasi from the New World, and L. major from the Old World - associated to different clinical forms of leishmaniasis. The composition of the extracts has been previously characterized by high temperature high resolution gas chromatography coupled to mass spectrometry. Considering the chemical differences among the extracts and the behavior of the parasites, it was observed significant differences in the leishmanicidal activities with IC50/1 day values in the range of 2.8 to 229.3 µg/ml . An overall analysis showed that for all the species evaluated, Bulgarian extracts were more active than the ethanol Brazilian extract. As the assayed propolis extracts have their chemical composition determined it merits further investigation the effect of individual components or their combinations on each Leishmania species.

  3. [Identification of Leishmania species in patients and phlebotomines in transmission areas in a region of Peru].

    Science.gov (United States)

    Córdova, Ofelia; Vargas, Franklin; Hashiguchi, Yoshihisa; Kato, Hirotomo; Gómez, Eduardo

    2011-01-01

    To identify the species of Leishmania present in the skin lesions of patients and Lutzomyias living in endemic areas of La Libertad, Peru. Molecular methods based on PCR and RFLP were used, which allowed to have efficient data with small amounts of samples (small specimens), due to their high sensitivity and ease of application in the field work. The results of PCR of clinical samples of patients and insect vectors showed the presence of Leishmania (V.) peruviana as a major causative agent of andean leishmaniasis transmitted by Lutzomyia peruensis. The presence of Leishmania (V.) guyanensis in Lutzomyia ayacuchensis, was found as well. The presence of L. (V.) peruviana and L. (V.) guyanensis in the Andean areas under study was found. These findings remark the need of a wider research about the geographical distribution of L. (V.) guyanensis and clinical features related to the infection in endemic areas of cutaneous leishmaniasis.

  4. Leishmania infantum Parasites Subvert the Host Inflammatory Response through the Adenosine A2A Receptor to Promote the Establishment of Infection

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    Mikhael H. F. Lima

    2017-07-01

    Full Text Available Adenosine is an endogenously released purine nucleoside that signals through four widely expressed G protein-coupled receptors: A1, A2A, A2B, and A3. Of these, A2AR is recognized as mediating major adenosine anti-inflammatory activity. During cutaneous leishmaniasis, adenosine induces immunosuppression, which promotes the establishment of infection. Herein, we demonstrated that A2AR signaling is exploited by Leishmania infantum parasites, the etiologic agent that causes Visceral Leishmaniasis, to successfully colonize the vertebrate host. A2AR gene-deleted mice exhibited a well-developed cellular reaction with a strong Th1 immune response in the parasitized organs. An intense infiltration of activated neutrophils into the disease-target organs was observed in A2AR−/− mice. These cells were characterized by high expression of CXCR2 and CD69 on their cell surfaces and increased cxcl1 expression. Interestingly, this phenotype was mediated by IFN-γ on the basis that a neutralizing antibody specific to this cytokine prevented neutrophilic influx into parasitized organs. In evaluating the immunosuppressive effects, we identified a decreased number of CD4+ FOXP3+ T cells and reduced il10 expression in A2AR−/− infected mice. During ex vivo cell culture, A2AR−/− splenocytes produced smaller amounts of IL-10. In conclusion, we demonstrated that the A2AR signaling pathway is detrimental to development of Th1-type adaptive immunity and that this pathway could be associated with the regulatory process. In particular, it promotes parasite surveillance.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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

  7. 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.

  8. Synthesis and Antiviral Activity of 3-Aminoindole Nucleosides of 2-Acetamido-2-deoxy-D-glucose

    Energy Technology Data Exchange (ETDEWEB)

    Abdelrahman, Adel A. H.; Elessawy, Farag A.; Barakat, Yousif A. [Menoufia Univ., Shebin El-Koam (Egypt); Ellatif, Mona M. Abd [The British Univ. in Egypt, Cairo (Egypt)

    2012-10-15

    A new method for the construction of 3-aminoindole nucleosides of 2-acetamido-2-deoxy-D-glucose based is presented. Nitration and acetylation of the indole nucleosides by acetic anhydride-nitric acid mixture followed by reduction using silver catalyst (SNSM) impregnated on silica gel, afforded the corresponding amino indole nucleosides. The nucleosides were tested for antiviral activity against hepatitis B virus (HBV) to show different degrees of antiviral activities or inhibitory actions.

  9. Advanced Prodrug Strategies in Nucleoside and Non-Nucleoside Antiviral Agents: A Review of the Recent Five Years

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    Hanadi Sinokrot

    2017-10-01

    Full Text Available Background: Poor pharmacokinetic profiles and resistance are the main two drawbacks from which currently used antiviral agents suffer, thus make them excellent targets for research, especially in the presence of viral pandemics such as HIV and hepatitis C. Methods: The strategies employed in the studies covered in this review were sorted by the type of drug synthesized into ester prodrugs, targeted delivery prodrugs, macromolecular prodrugs, other nucleoside conjugates, and non-nucleoside drugs. Results: Utilizing the ester prodrug approach a novel isopropyl ester prodrug was found to be potent HIV integrase inhibitor. Further, employing the targeted delivery prodrug zanamivir and valine ester prodrug was made and shown a sole delivery of zanamivir. Additionally, VivaGel, a dendrimer macromolecular prodrug, was found to be very efficient and is now undergoing clinical trials. Conclusions: Of all the strategies employed (ester, targeted delivery, macromolecular, protides and nucleoside analogues, and non-nucleoside analogues prodrugs, the most promising are nucleoside analogues and macromolecular prodrugs. The macromolecular prodrug VivaGel works by two mechanisms: envelope mediated and receptor mediated disruption. Nucleotide analogues have witnessed productive era in the recent past few years. The era of non-interferon based treatment of hepatitis (through direct inhibitors of NS5A has dawned.

  10. Synthesis of O-Amino Sugars and Nucleosides.

    Science.gov (United States)

    Chen, Na; Xie, Juan

    2018-03-12

    Nucleic acids and carbohydrates are essential biomolecules involved in numerous biological and pathological processes. Development of multifunctional building blocks based on nucleosides and sugars is in high demand for the generation of novel oligonucleotide mimics and glycoconjugates for biomedical applications. Recently, aminooxyl-functionalized compounds have attracted increasing research interest because of their easy derivatization through oxime ligation or N -oxyamide formation reactions. Various biological applications have been reported for O -amino carbohydrate- and nucleoside-derived compounds. Here, we report our efforts in the design and synthesis of glyco-, glycosyl, nucleoside- and nucleo-aminooxy acid derivatives from readily available sugars and amino acids, and their use for the generation of N -oxyamide-linked oligosaccharides, glycopeptides, glycolipids, oligonucleosides and nucleopeptides as novel glycoconjugates or oligonucleotide mimics. Delicate and key points in the synthesis will be emphasized.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Leishmania in phlebotomid sandflies. VII. On the taxonomic status of Leishmania peruviana, causative agent of Peruvian 'uta', as indicated by its development in the sandfly, Lutzomyia longipalpis.

    Science.gov (United States)

    Lainson, R; Ready, P D; Shaw, J J

    1979-12-31

    The name Leishmania peruviana was given by Velez (1913) to the parasite responsible for a form of cutaneous leishmaniasis known as 'uta'; this disease occurs in the Peruvian Andes. Clinical similarities between uta and 'oriental sore', which is caused by Leishmania tropica of the Eastern Hemisphere, have, however, led to the suggestion that uta is simply due to L. tropica, which was introduced into Latin America by African slaves or European immigrants. Leishmania species are divisible into three distinct sections, according to their pattern of development in their natural (phlebotomine) vectors. One of these sections, the PERIPYLARIA, contains the subspecies of Leishmania braziliensis, and is characterized by parasites that undergo a phase of development attached to the wall of the hindgut (pylorus and ileum), in addition to multiplication in the midgut and subsequent invasion of the foregut. Such development is unknown in any other group of leishmaniae, including those groups of the section SUPRAPYLARIA, which includes parasites of the L. tropica complex. Three isolates of L. peruviana were studied in laboratory-bred sandflies, Lutzomyia longipalpis (Lutz & Neiva), and all showed consistent and prolific development of rounded or stumpy flagellates attached to the wall of the hindgut and, in some instances, growth of free, elongate promastigotes throughout the midgut. Development of both L. tropica and L. major, in the same insect, was restricted to massive development of free flagellates in the midgut, up to the cardial valve. From the behaviour of L. peruviana in the sandfly, its slow growth in hamster skin and the small size of its amastigotes, it is concluded that this parasite is (a) distinctly different from both L. tropica and L. major, and (b) closely related to subspecies of L. braziliensis within the section PERIPYLARIA. On this evidence it is also concluded that L. peruviana is indigenous to the American continent. The specific name is best retained for

  13. A novel A2 allele found in Leishmania (Leishmania infantum chagasi Novo alelo do gene A2 descrito em Leishmania (Leishmania infantum chagasi

    Directory of Open Access Journals (Sweden)

    Trícia Maria Ferreira de Sousa Oliveira

    2011-03-01

    Full Text Available Visceral leishmaniasis (VL is a widely spread zoonotic disease. In Brazil the disease is caused by Leishmania (Leishmania infantum chagasi. Peridomestic sandflies acquire the etiological agent by feeding on blood of infected reservoir animals, such as dogs or wildlife. The disease is endemic in Brazil and epidemic foci have been reported in densely populated cities all over the country. Many clinical features of Leishmania infection are related to the host-parasite relationship, and many candidate virulence factors in parasites that cause VL have been studied such as A2 genes. The A2 gene was first isolated in 1994 and then in 2005 three new alleles were described in Leishmania (Leishmania infantum. In the present study we amplified by polymerase chain reaction (PCR and sequenced the A2 gene from the genome of a clonal population of L. (L. infantum chagasi VL parasites. The L. (L. infantum chagasi A2 gene was amplified, cloned, and sequenced in. The amplified fragment showed approximately 90% similarity with another A2 allele amplified in Leishmania (Leishmania donovani and in L.(L. infantum described in literature. However, nucleotide translation shows differences in protein amino acid sequence, which may be essential to determine the variability of A2 genes in the species of the L. (L. donovani complex and represents an additional tool to help understanding the role this gene family may have in establishing virulence and immunity in visceral leishmaniasis. This knowledge is important for the development of more accurate diagnostic tests and effective tools for disease control.A leishmaniose visceral (LV é uma zoonose amplamente disseminada, causada no Brasil pela Leishmania (Leishmania infantum chagasi. Flebotomíneos vetores adquirem o agente etiológico, alimentando-se do sangue de animais contaminados, como cachorros ou animais selvagens. A doença é endêmica no Brasil, e focos de epidemia são relatados em cidades densamente povoadas

  14. Synthesis of some novel hydrazono acyclic nucleoside analogues

    Directory of Open Access Journals (Sweden)

    Mohammad N. Soltani Rad

    2010-05-01

    Full Text Available The syntheses of novel hydrazono acyclic nucleosides similar to miconazole scaffolds are described. In this series of acyclic nucleosides, pyrimidine as well as purine and other azole derivatives replaced the imidazole function in miconazole and the ether group was replaced with a hydrazone moiety using phenylhydrazine. To interpret the dominant formation of (E-hydrazone derivatives rather than (Z-isomers, PM3 semiempirical quantum mechanic calculations were carried out which indicated that the (E-isomers had the lower heats of formation.

  15. New prodrugs based on phospholipid-nucleoside conjugates

    Energy Technology Data Exchange (ETDEWEB)

    MacCoss, M.

    1982-02-03

    A method is described for the preparation of defined, isomerically pure phospholipid-nucleoside conjugates as a prodrug in which the drug (araC) is attached to the phospholipid by a monophosphate linkage. Key intermediates in the process involve selective blocking and deblocking of the nucleoside derivative. These particular monophosphate-linked derivatives represent a new class of prodrug, which are useful by themselves or in combination with diphosphate linked derivatives. Several new compositions involving diphosphate linked derivatives are described in which the products are isomerically pure and having defined fatty acid chain lengths.

  16. Prolinol-based nucleoside phosphonic acids: Synthesis and properties

    Czech Academy of Sciences Publication Activity Database

    Vaněk, Václav; Buděšínský, Miloš; Liboska, Radek; Hurychová, Vladimíra; Rosenberg, Ivan

    -, č. 52 (2008), s. 537-538 ISSN 0261-3166. [Joint Symposium of the International Roundtable on Nucleosides, Nucleotides and Nucleic Acids /18./ and the International Symposium on Nucleic Acid Chemistry /35./. Kyoto, 08.09.2008-12.09.2008] R&D Projects: GA MŠk(CZ) LC06061; GA MŠk(CZ) LC06077; GA MŠk LC512 Institutional research plan: CEZ:AV0Z40550506 Keywords : oligonucleotide phosphonate * nucleoside analogue * pyrrolidine * prolinol Subject RIV: CC - Organic Chemistry

  17. Leishmania

    Indian Academy of Sciences (India)

    NII

    ... adverse side effect like cardiac failure & acute pancreatitis. Amphotericin B: Costly also very toxic causes cardiotoxicity and nephrotoxicity, require hospitalization. Miltefosine: Originally anticancer drug used in VL, toxic and very costly. No vaccine and appropriate diagnostic procedure are available against leishmaniasis.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Two nucleoside uptake systems in Lactococcus lactis: Competition between purine nucleosides and cytidine allows for modulation of intracellular nucleotide pools

    DEFF Research Database (Denmark)

    Martinussen, Jan; Wadskov-Hansen, Steen Lyders Lerche; Hammer, Karin

    2003-01-01

    and with an H3BO3-LiOH buffer for separation in the second dimension. We report here the sizes of the ribo- and deoxyribonucleotide pools in laboratory strain MG1363 during growth in a defined medium. We found that purine and pyrimidine-requiring strains may be used to establish physiological conditions...... in batch fermentations with altered nucleotide pools and growth rates by addition of nucleosides in different combinations. Addition of cytidine together with inosine to a purine-requiring strain leads to a reduction in the internal purine nucleotide pools and a decreased growth rate. This effect...... was found between uridine and either cytidine or inosine. These findings suggest that there are two different high-affinity nucleoside transporters, one system responsible for uridine uptake and another system responsible for the uptake of all purine nucleosides and cytidine....

  20. Leishmania species and zymodemes isolated from endemic areas of cutaneous leishmaniasis in Jordan

    Science.gov (United States)

    Nimri, Laila; Soubani, Radwan; Gramiccia, Marina

    2002-01-01

    Background Cutaneous leishmaniasis (CL) is endemic in the Middle Eastern countries. New cases are emerging in areas previously free of the disease. In Jordan, the diagnosis of cases during the 1960s and 1970s was mainly reported in military hospitals in Amman. Endemicity of the disease was ascertained after reporting a total of 524 cases during 1973–1978. Results Leishmania major and Leishmania tropica were isolated from seventy-six autochthonous and imported cases of CL, during eight-year period. The highest infection rates recorded were in the central part of Jordan (60.5%), in males (72.4%) and in the age group 21–30 years (30.5%). Lesions were on the exposed sites of the body, mainly on the face (40%). Both Leishmania spp. were isolated from all parts of the country, although L. major was the predominant species (75% of cases) in all areas except in the north part of Jordan. Isoenzyme characterization of the isolates identified four previously undescribed zymodemes (Z). Four Leishmania major zymodemes were found, one of which was a new zymodeme (ZMON-103 variant in GLUD220); L. major ZMON-103 was the most common zymodeme. Four Leishmania tropica zymodemes were identified, of which three were previously unreported. Of these, ZMON-54 var PGD96–97 was isolated from autochthonous cases, whereas ZMON-59 var MDH100 and ZMON-75 var FH110 were obtained from both autochthonous and imported cases, or from an imported CL case, respectively. Conclusion The findings of this study indicate the emergence of the CL disease in new areas. New foci are reported, where the sporadic nature of the cases indicates recent spread of the disease to these areas and the urge for the implementation of control measures. PMID:12473179

  1. Leishmania major-phlebotomus duboscqi interactions: inhibition of ...

    African Journals Online (AJOL)

    Methods: Laboratory reared sand flies were allowed to feed beneath a blood filled membrane feeder containing 1 x106 amastigotes in 20µl mixed with 0.5 ml of defibrinated rabbit blood with a 1:100 dilution of anti-LPG MABS. Control blood contained a similar number of amastigotes but no MABS. At least five female ...

  2. Leishmania aethiopica field isolates bearing an endosymbiontic dsRNA virus induce pro-inflammatory cytokine response.

    Directory of Open Access Journals (Sweden)

    Haroun Zangger

    2014-04-01

    Full Text Available Infection with Leishmania parasites causes mainly cutaneous lesions at the site of the sand fly bite. Inflammatory metastatic forms have been reported with Leishmania species such as L. braziliensis, guyanensis and aethiopica. Little is known about the factors underlying such exacerbated clinical presentations. Leishmania RNA virus (LRV is mainly found within South American Leishmania braziliensis and guyanensis. In a mouse model of L. guyanensis infection, its presence is responsible for an hyper-inflammatory response driven by the recognition of the viral dsRNA genome by the host Toll-like Receptor 3 leading to an exacerbation of the disease. In one instance, LRV was reported outside of South America, namely in the L. major ASKH strain from Turkmenistan, suggesting that LRV appeared before the divergence of Leishmania subgenera. LRV presence inside Leishmania parasites could be one of the factors implicated in disease severity, providing rationale for LRV screening in L. aethiopica.A new LRV member was identified in four L. aethiopica strains (LRV-Lae. Three LRV-Lae genomes were sequenced and compared to L. guyanensis LRV1 and L. major LRV2. LRV-Lae more closely resembled LRV2. Despite their similar genomic organization, a notable difference was observed in the region where the capsid protein and viral polymerase open reading frames overlap, with a unique -1 situation in LRV-Lae. In vitro infection of murine macrophages showed that LRV-Lae induced a TLR3-dependent inflammatory response as previously observed for LRV1.In this study, we report the presence of an immunogenic dsRNA virus in L. aethiopica human isolates. This is the first observation of LRV in Africa, and together with the unique description of LRV2 in Turkmenistan, it confirmed that LRV was present before the divergence of the L. (Leishmania and (Viannia subgenera. The potential implication of LRV-Lae on disease severity due to L. aethiopica infections is discussed.

  3. Aryl nucleoside H-phosphonates. Part 15: Synthesis, properties and, anti-HIV activity of aryl nucleoside 5'-alpha-hydroxyphosphonates.

    Science.gov (United States)

    Szymańska, Agnieszka; Szymczak, Marzena; Boryski, Jerzy; Stawiński, Jacek; Kraszewski, Adam; Collu, Gabriella; Sanna, Giseppina; Giliberti, Gabriele; Loddo, Roberta; La Colla, Paolo

    2006-03-15

    Aryl nucleoside 5'-H-phosphonates 4 bearing AZT or 2',3'-dideoxyuridine moieties were subjected to reaction with various aromatic aldehydes to produce nucleoside 5'-alpha-hydroxyphosphonate derivatives 2 as potential anti-HIV agents. Stability of the title compounds in cell culture media was investigated and three distinct decomposition pathways were identified. The anti-HIV activity of hydroxyphosphonates 2 correlates well with the type and extent of their chemical or enzymatic degradation in culture medium (RPMI 1640 containing 10% FBS), suggesting that aryl nucleoside 5'-hydroxyphosphonates 2 act as depot forms of the parent antiviral nucleosides.

  4. Amino Acid Ester Prodrugs of Nucleoside and Nucleotide Antivirals

    Czech Academy of Sciences Publication Activity Database

    Krečmerová, Marcela

    2017-01-01

    Roč. 17, č. 10 (2017), s. 818-833 ISSN 1389-5575 Grant - others:AV ČR(CZ) M200551201 Institutional support: RVO:61388963 Keywords : acyclic nucleoside analogues * antiherpetics * antiretrovirals * cidofovir * peptidomimetics * prodrugs Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 2.661, year: 2016

  5. Antiviral acyclic nucleoside phosphonates: New structures and prodrugs

    Czech Academy of Sciences Publication Activity Database

    Krečmerová, Marcela; Tichý, Tomáš; Pomeisl, Karel; Andrei, G.; Balzarini, J.; Snoeck, R.

    2016-01-01

    Roč. 1, č. 2 (2016), s. 37 [PharmaMed-2016. International Conference on Medicinal and Pharmaceutical Chemistry . 05.12.2016-07.12.2016, Dubai] R&D Projects: GA ČR(CZ) GA14-00522S Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * prodrugs * antivirals * 5-azacytosine Subject RIV: CC - Organic Chemistry

  6. New clinical perspectives on non-nucleoside reverse transcriptase ...

    African Journals Online (AJOL)

    New clinical perspectives on non-nucleoside reverse transcriptase inhibitors. S Miller. Abstract. No Abstract. Full Text: EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT · AJOL African Journals Online. HOW TO USE AJOL... for Researchers · for Librarians · for Authors ...

  7. Two convergent approaches toward novel carbocyclic C-nucleosides

    Czech Academy of Sciences Publication Activity Database

    Nencka, Radim; Šála, Michal; Dejmek, Milan; Dračínský, Martin; Holý, Antonín; Hřebabecký, Hubert

    -, č. 23 (2010), s. 4119-4130 ISSN 0039-7881 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : carbocyclic C-nucleosides * convergent approach * uracil * pyrimidines Subject RIV: CC - Organic Chemistry Impact factor: 2.260, year: 2010

  8. Synthesis of (Purin-6-yl)methylphosphonate Bases and Nucleosides

    Czech Academy of Sciences Publication Activity Database

    Hasník, Zbyněk; Pohl, Radek; Hocek, Michal

    2010-01-01

    Roč. 51, č. 18 (2010), s. 2464-2466 ISSN 0040-4039 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : purines * nucleosides * phosphonates * cross-coupling Subject RIV: CC - Organic Chemistry Impact factor: 2.618, year: 2010

  9. new clinical perspectives on non- nucleoside reverse transcriptase ...

    African Journals Online (AJOL)

    NUCLEOSIDE REVERSE TRANSCRIPTASE. INHIBITORS. TilE SOUTIIERN AFRICAN JOU NAL OF IIIV MEDICINE metabolised in the liver via the P450 cytochrome system, a pathway shared with many other compounds. This creates a risk for drug/drug interactions and the potential to modify the efficacy and/or toxicity of ...

  10. Reactivity Studies of 2,6-Ditriazolylpurine Nucleosides with Nucleophiles

    OpenAIRE

    Kovaļovs, A; Novosjolova, I; Bizdēna, Ē; Turks, M

    2012-01-01

    Reaction of 2,6-ditriazolylpurine nucleosides with nucleophiles is mild and efficient route to C6 derivatization of purine base. To explore scope and limitations of the method, we studied reactivity of various N- and S-nucleophiles as well as kinetics for selected reactions.

  11. Synthesis, bioanalysis and pharmacology of nucleoside and nucleotide analogs

    NARCIS (Netherlands)

    Jansen, R.S.

    2009-01-01

    Nucleoside analogs are an important class of drugs in anticancer and antiviral therapy. The compounds are, however, only active after intracellular conversion to their mono-, di- and triphosphate nucleotide form. In this thesis the development of sensitive liquid chromatography coupled to tandem

  12. Norbornane as the novel pseudoglycone moiety in nucleosides

    Czech Academy of Sciences Publication Activity Database

    Šála, Michal; Hřebabecký, Hubert; Dračínský, Martin; Masojídková, Milena; De Palma, A.; Neyts, J.; Holý, Antonín

    2009-01-01

    Roč. 65, č. 45 (2009), s. 9291-9299 ISSN 0040-4020 R&D Projects: GA MŠk 1M0508; GA AV ČR 1QS400550501 Institutional research plan: CEZ:AV0Z40550506 Keywords : carbocyclic nucleosides * norbornane * coxsackie virus Subject RIV: CC - Organic Chemistry Impact factor: 3.219, year: 2009

  13. Activity of Thymus capitellatus volatile extract, 1,8-cineole and borneol against Leishmania species.

    Science.gov (United States)

    Machado, M; Dinis, A M; Santos-Rosa, M; Alves, V; Salgueiro, L; Cavaleiro, C; Sousa, M C

    2014-02-24

    In the search for new leishmanicidal agents, Thymus capitellatus Hoffmanns. & Link (family Lamiaceae) volatile extract and its major compounds, 1,8-cineole and borneol, were tested against Leishmania infantum, Leishmania tropica and Leishmania major. Plant volatile extract (essential oil) was analysed by GC and GC-MS and the activity of essential oil on Leishmania promastigotes viability was assessed using tetrazolium-dye colorimetric method (MTT). The MTT test was also used to assess the cytotoxicity of essential oil on macrophages and bovine aortic endothelial cells. Effects on parasites were also analyzed by flow cytometry in order to assess mitochondrial transmembrane electrochemical gradient (JC-1), analyze phosphatidylserine externalization (annexin V-FITC, propidium iodide) and evaluate cell cycle (DNase-free, RNase, PI). Morphological and ultrastructural studies were performed by light, scanning and transmission electron microscopy. T. capitellatus volatile extract exhibited anti-parasite activity on Leishmania species, with IC50 values ranging from 35 to 62 μg/ml. However, major compounds 1,8-cineole and borneol did not showed biological activity suggesting that these monoterpenes are not responsible for the antileishmanial activity of T. capitellatus essential oil. Appearance of aberrant-shaped cells, mitochondrial swelling and autophagosomal structures were some of the ultrastructural alterations exhibited among treated promastigote cells. T. capitellatus promoted leishmanicidal effect by triggering a programmed cell death as evidenced by externalization of phosphatidylserine, loss of mitochondrial membrane potential, and cell-cycle arrest at the G(0)/G(1) phase. The volatile extract did not induced cytotoxic effects on mammalian cells. Taken together, these results suggest that T. capitellatus may represent a valuable source for therapeutic control of leishmaniasis in humans and animals. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Molecular detection and identification of Leishmania infection in naturally infected sand flies in a focus of cutaneous leishmaniasis in northern Morocco

    OpenAIRE

    Es-Sette, Nargys; Ajaoud, Malika; Laamrani-Idrissi, Abderrahman; Mellouki, Fouad; Lemrani, Meryem

    2014-01-01

    Background Cutaneous leishmaniasis is an infectious disease caused by various species of the flagellate protozoan Leishmania. During the past 20 years, cutaneous leishmaniasis has emerged as a major public health threat in Morocco. The main objective of this study was to study the occurrence of Leishmania infection in vectors and to identify sand fly blood meal sources in an endemic locality of cutaneous leishmaniasis within Sefrou province, where the vectors of leishmaniasis were still unkno...

  15. Genetics of host response to Leishmania tropica in mice - different control of skin pathology, chemokine reaction, and iInvasion into spleen and liver

    Czech Academy of Sciences Publication Activity Database

    Kobets, Tetyana; Havelková, Helena; Grekov, Igor; Volkova, Valeriya; Vojtíšková, Jarmila; Slapničková, Martina; Kurey, Irina; Sohrabi, Yahya; Svobodová, M.; Demant, P.; Lipoldová, Marie

    2012-01-01

    Roč. 6, č. 6 (2012), e1667 ISSN 1935-2735 R&D Projects: GA ČR GA310/08/1697; GA ČR GD310/08/H077; GA MŠk(CZ) LC06009 Institutional support: RVO:68378050 Keywords : leishmaniasis * Leishmania tropica * Leishmania major Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.569, year: 2012

  16. Identification of Leishmania Species Causing Cutaneous Leishmaniasis Using Random Amplified Polymorphic DNA (RAPD-PCR in Kharve, Iran

    Directory of Open Access Journals (Sweden)

    Fatemeh Saadabadi

    2013-05-01

    Full Text Available Background: Leishmaniasis, especially cutaneous leishmaniasis, is considered an important health problem in many parts of Iran including Kharve, Khorasan Razavi province. Cutaneous leishmaniasis is caused by various species of Leishmania, each having a different secondary host. Thus, identifying the parasites’ specie is of paramount importance for containment strategy planning. The morphological differentiation of Leishmania species is not possible, rendering the molecular methods as the sole means to this purpose. Therefore, to identify the causative agent of cutaneous leishmaniasis in Kharve, Random Amplified Polymorphic DNA-PCR (RAPD-PCR was used. Methods: The disease was first confirmed by direct smears. Samples were gathered from 22 patients with established cutaneous leishmaniasis. The samples were immediately cultured in NNN medium, followed by sub-culture in RPMI-1640. Afterwards, DNA was extracted and amplified using RAPD-PCR. Electrophoresis patterns from each isolate were compared with reference strains of Leishmania major (L. major and Leishmania tropica (L. tropica. Results: The results of this study indicated that the parasite causing cutaneous leishmaniasis in Kharve is L. tropica. Conclusion: It seems that L. tropica is the only causative agent of cutaneous leishmaniasis in Kharve, and RAPD-PCR is a suitable tool for Leishmania characterization in epidemiological studies.

  17. Molecular characterization of leishmania infection from naturally infected sand flies caught in a focus of cutaneous leishmaniasis (eastern iran.

    Directory of Open Access Journals (Sweden)

    Mohammad Akhoundi

    2013-12-01

    Full Text Available Cutaneous leishmaniasis due to Leishmania major is a serious and increasing problem affecting many rural areas of 17 out of 31 provinces in Iran. Little is known about sand fly fauna and leishmaniases in Eastern Iran and no study has been carried out in Sarbisheh County. The aim of this study was to determine sand flies composition and probable Leishmania infection to find the probable vectors of leishmaniasis in Sarbisheh district.Sand flies were caught using both sticky papers and CDC light traps in August 2010. They were identified morphologically and analyzed for Leishmania infection by amplification of ITS-rDNA.Totally, 842 specimens were caught and 8 species recorded. They belonged to the genera Phlebotomus and Sergentomyia: P. (Phlebotomus papatasi, P. (Paraphlebotomus sergenti, P. (Pa. caucasicus, P. (Pa. mongolensis, P. (Pa. jacusieli, S. (Sergentomyia dentata, S. (Se. sintoni and S. (Sintonius clydei. All collected females were processed for Leishmania DNA detection by PCR amplifying of Internal Transcribed Spacer1 (partial sequence, 5.8S (complete sequence and ITS2 (partial sequence fragments. Thirteen females were positive for Leishmania DNA. The sequencing of the 430 bp amplicons indicated that 9 P. papatasi and 3 females belonging to the Caucasicus group carried L. major DNA whereas one P. sergenti carried L. tropica DNA.Phlebotomus papatasi and P. sergenti are, like in several places, the probable vectors of cutaneous leishmaniases in this emerging or unknown focus of cutaneous leishmaniases.

  18. Different susceptibilities of Leishmania spp. promastigotes to the Annona muricata acetogenins annonacinone and corossolone, and the Platymiscium floribundum coumarin scoparone.

    Science.gov (United States)

    Vila-Nova, Nadja Soares; de Morais, Selene Maia; Falcão, Maria José Cajazeiras; Alcantara, Terezinha Thaize Negreiros; Ferreira, Pablito Augusto Travassos; Cavalcanti, Eveline Solon Barreira; Vieira, Icaro Gusmão Pinto; Campello, Cláudio Cabral; Wilson, Mary

    2013-03-01

    Leishmaniasis is a zoonotic disease that can manifest itself in visceral and cutaneous form. The aim of this study was to search for new leishmanicidal compounds. Preliminarily, Artemia salina assay was applied to compounds from two plants found in Northeastern Brazil, Platymiscium floribundum and Annona muricata. Then these compounds were tested against three Leishmania species (Leishmania donovani, Leishmania mexicana and Leishmania major). A screening assay using luciferase-expressing promastigote form were used to measure the viability of promastigote One coumarin, scoparone, isolated from P. floribundum and two acetogenins, annonacinone and corossolone isolated from A. muricata showed leishmanicidal activity in all species tested. Nevertheless, Leishmania species indicated different susceptibilities in relation to the tested compounds: L. mexicana was more sensitive to scoparone followed by L. major and L. donovani. The three species presented similar inhibition to corossolone and annonacinone. Acetogenin annonacinone (EC(50)=6.72-8.00 μg/mL) indicated high leishmanicidal activity; corossolone (EC(50)=16.14-18.73 μg/mL) and scoparone (EC(50)=9.11-27.51 μg/mL) moderate activity. A. saline larvae were less sensitive to the coumarin scoparone and acetogenin corossolone was the most toxic. In conclusion, the leishmanicidal activity demonstrated by the coumarin and acetogenins indicate these compounds for further studies aiming the development of new leishmanicidal agents. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Efficient synthesis and biological properties of the 2‘-trifluoromethyl analogues of acyclic nucleosides and acyclic nucleoside phosphonates

    Czech Academy of Sciences Publication Activity Database

    Jansa, Petr; Kolman, Viktor; Kostinová, Alexandra; Dračínský, Martin; Mertlíková-Kaiserová, Helena; Janeba, Zlatko

    2011-01-01

    Roč. 76, č. 10 (2011), s. 1187-1198 ISSN 0010-0765 R&D Projects: GA MV VG20102015046 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleosides * nucleotides * phosphorus * fluorine * biological activity * antibiotics Subject RIV: CC - Organic Chemistry Impact factor: 1.283, year: 2011

  20. Susceptibility of spiny rats (Proechimys semispinosus to Leishmania (Viannia panamensis and Leishmania (Leishmania chagasi

    Directory of Open Access Journals (Sweden)

    BL Travi

    2002-09-01

    Full Text Available The role of Proechimys semispinosus as reservoir of Leishmania (Viannia panamensis on the Colombian Pacific coast was experimentally evaluated. The susceptibility to L. chagasi also was assessed to determine the utility of this rodent as a model for studying reservoir characteristics in the laboratory. Wild-caught animals were screened for natural trypanosomatid infections, and negative individuals were inoculated intradermally (ID in the snout or feet with 10(7 promastigotes of L. panamensis. L. chagasi was inoculated intracardially (10(7 promastigotes or ID in the ear (10(8 promastigotes. PCR-hybridization showed that 15% of 33 spiny rats were naturally infected with L. Viannia sp. Animals experimentally infected with L. panamensis developed non-ulcerated lesions that disappeared by the 7th week post-infection (p.i. and became more resistant upon reinfection. Infectivity to sand flies was low (1/20-1/48 infected/fed flies and transient, and both culture and PCR-hybridization showed that L. panamensis was cleared by the 13th week p.i. Animals inoculated with L. chagasi became subclinically infected and were non-infective to sand flies. Transient infectivity to vectors of spiny rats infected with L. panamensis, combined with population characteristics, e.g., abundance, exploitation of degraded habitats and high reproductive rates, could make them epidemiologically suitable reservoirs.

  1. Interaction of Leishmania PTS2 receptor peroxin 7 with the glycosomal protein import machinery.

    Science.gov (United States)

    Pilar, Ana Victoria C; Madrid, Kleber P; Jardim, Armando

    2008-03-01

    Leishmania proteins containing a peroxisomal targeting signal sequence 2 (PTS2) are selectively trafficked to the glycosome by associating with the peroxin 7 receptor protein (PEX7). The L. major PEX7 (LmPEX7) encodes a approximately 41 kDa protein that exhibits limited sequence identity with PEX7 homologues from other eukaryotic organisms. Functional characterization of recombinant and native LmPEX7 revealed that this receptor bound the PTS2 protein fructose-1,6-bisphosphate aldolase. Moreover, LmPEX7 also formed a tight association with the Leishmania PEX5, the cytosolic PTS1 receptor, and PEX14, a glycosomal peripheral membrane protein required for protein import into the glycosome. Mapping studies revealed that the Leishmania PEX7 binds to a domain on LdPEX5 encompassing residues 111-148 and to a site on LdPEX14 spanning residues 120-148. Finally, subcellular localization studies revealed that Leishmania PEX7 has a dual distribution within the cytosolic compartment and glycosomal lumen.

  2. Design and Validation of Real-Time PCR: Quantitative Diagnosis of Common Leishmania Species in Iran.

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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

  4. Exposure to Leishmania braziliensis triggers neutrophil activation and apoptosis.

    Directory of Open Access Journals (Sweden)

    Sarah A C Falcão

    2015-03-01

    Full Text Available BACKGROUND: Neutrophils are the first line of defense against invading pathogens and are rapidly recruited to the sites of Leishmania inoculation. During Leishmania braziliensis infection, depletion of inflammatory cells significantly increases the parasite load whereas co-inoculation of neutrophils plus L. braziliensis had an opposite effect. Moreover, the co-culture of infected macrophages and neutrophils also induced parasite killing leading us to ask how neutrophils alone respond to an L. braziliensis exposure. Herein we focused on understanding the interaction between neutrophils and L. braziliensis, exploring cell activation and apoptotic fate. METHODS AND FINDINGS: Inoculation of serum-opsonized L. braziliensis promastigotes in mice induced neutrophil accumulation in vivo, peaking at 24 h. In vitro, exposure of thyoglycollate-elicited inflammatory or bone marrow neutrophils to L. braziliensis modulated the expression of surface molecules such as CD18 and CD62L, and induced the oxidative burst. Using mCherry-expressing L. braziliensis, we determined that such effects were mainly observed in infected and not in bystander cells. Neutrophil activation following contact with L. braziliensis was also confirmed by the release of TNF-α and neutrophil elastase. Lastly, neutrophils infected with L. braziliensis but not with L. major displayed markers of early apoptosis. CONCLUSIONS: We show that L. braziliensis induces neutrophil recruitment in vivo and that neutrophils exposed to the parasite in vitro respond through activation and release of inflammatory mediators. This outcome may impact on parasite elimination, particularly at the early stages of infection.

  5. [Status of the taxonomy of Leishmania from the Mediterranean basin].

    Science.gov (United States)

    Gradoni, L; Gramiccia, M; Pozio, E

    1984-12-01

    Isoenzyme analysis, carried out on some hundreds of Leishmania isolates from the Mediterranean basin by specialized identification Centres, has provided new data on the taxonomy of this parasite in the area. Three complexes of zymodemes have been identified: L. infantum s.l., L. tropica s.l. and L. major s.l. L. infantum is the agent of human and canine visceral leishmaniasis and it is distributed throughout the Mediterranean area. Furthermore, this parasite is the only responsible for human cutaneous leishmaniasis in Central-Western Europe, including Italy. The distribution of L. tropica, agent of urban, or anthroponotic, cutaneous leishmaniasis, appears to be very limited, being restricted to Middle East, Tunisia and Greece. On the other hand, L. major, which causes rural, or zoonotic, cutaneous leishmaniasis, is widespread throughout North Africa and Middle East. In some countries, such as Tunisia and Israel, leishmaniases are caused by all the three complexes.

  6. The diverse and dynamic nature of Leishmania parasitophorous vacuoles studied by multidimensional imaging.

    Directory of Open Access Journals (Sweden)

    Fernando Real

    Full Text Available An important area in the cell biology of intracellular parasitism is the customization of parasitophorous vacuoles (PVs by prokaryotic or eukaryotic intracellular microorganisms. We were curious to compare PV biogenesis in primary mouse bone marrow-derived macrophages exposed to carefully prepared amastigotes of either Leishmania major or L. amazonensis. While tight-fitting PVs are housing one or two L. major amastigotes, giant PVs are housing many L. amazonensis amastigotes. In this study, using multidimensional imaging of live cells, we compare and characterize the PV biogenesis/remodeling of macrophages i hosting amastigotes of either L. major or L. amazonensis and ii loaded with Lysotracker, a lysosomotropic fluorescent probe. Three dynamic features of Leishmania amastigote-hosting PVs are documented: they range from i entry of Lysotracker transients within tight-fitting, fission-prone L. major amastigote-housing PVs; ii the decrease in the number of macrophage acidic vesicles during the L. major PV fission or L. amazonensis PV enlargement; to iii the L. amazonensis PV remodeling after homotypic fusion. The high content information of multidimensional images allowed the updating of our understanding of the Leishmania species-specific differences in PV biogenesis/remodeling and could be useful for the study of other intracellular microorganisms.

  7. The First Synthesis of Dually Modified Southern-Mimicking Nucleoside: 4'-Methyl Branched and Bicyclo [3.1.0] Hexane Locked Nucleoside

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kwan Woo; Hong, Joon Hee [Chosun University, Gwangju (Korea, Republic of)

    2004-05-15

    This paper reports the stereoselective synthesis of a novel nucleoside, 4'-methyl branched and bicyclo [3.1.0] hexane locked-nucleoside 12, using a sequential [3,3]-sigmatropic rearrangement/carbene cycloaddition reaction/Curtius reaction strategy with a high stereoselectivity.

  8. 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

  9. Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania amazonensis promastigotes

    Directory of Open Access Journals (Sweden)

    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.

  10. High Resolution Melting Analysis Targeting hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species.

    Directory of Open Access Journals (Sweden)

    Ricardo Andrade Zampieri

    2016-02-01

    Full Text Available Protozoan parasites of the genus Leishmania cause a large spectrum of clinical manifestations known as Leishmaniases. These diseases are increasingly important public health problems in many countries both within and outside endemic regions. Thus, an accurate differential diagnosis is extremely relevant for understanding epidemiological profiles and for the administration of the best therapeutic protocol.Exploring the High Resolution Melting (HRM dissociation profiles of two amplicons using real time polymerase chain reaction (real-time PCR targeting heat-shock protein 70 coding gene (hsp70 revealed differences that allowed the discrimination of genomic DNA samples of eight Leishmania species found in the Americas, including Leishmania (Leishmania infantum chagasi, L. (L. amazonensis, L. (L. mexicana, L. (Viannia lainsoni, L. (V. braziliensis, L. (V. guyanensis, L. (V. naiffi and L. (V. shawi, and three species found in Eurasia and Africa, including L. (L. tropica, L. (L. donovani and L. (L. major. In addition, we tested DNA samples obtained from standard promastigote culture, naturally infected phlebotomines, experimentally infected mice and clinical human samples to validate the proposed protocol.HRM analysis of hsp70 amplicons is a fast and robust strategy that allowed for the detection and discrimination of all Leishmania species responsible for the Leishmaniases in Brazil and Eurasia/Africa with high sensitivity and accuracy. This method could detect less than one parasite per reaction, even in the presence of host DNA.

  11. Leishmania Uses Mincle to Target an Inhibitory ITAM Signaling Pathway in Dendritic Cells that Dampens Adaptive Immunity to Infection.

    Science.gov (United States)

    Iborra, Salvador; Martínez-López, María; Cueto, Francisco J; Conde-Garrosa, Ruth; Del Fresno, Carlos; Izquierdo, Helena M; Abram, Clare L; Mori, Daiki; Campos-Martín, Yolanda; Reguera, Rosa María; Kemp, Benjamin; Yamasaki, Sho; Robinson, Matthew J; Soto, Manuel; Lowell, Clifford A; Sancho, David

    2016-10-18

    C-type lectin receptors sense a diversity of endogenous and exogenous ligands that may trigger differential responses. Here, we have found that human and mouse Mincle bind to a ligand released by Leishmania, a eukaryote parasite that evades an effective immune response. Mincle-deficient mice had milder dermal pathology and a tenth of the parasite burden compared to wild-type mice after Leishmania major intradermal ear infection. Mincle deficiency enhanced adaptive immunity against the parasite, correlating with increased activation, migration, and priming by Mincle-deficient dendritic cells (DCs). Leishmania triggered a Mincle-dependent inhibitory axis characterized by SHP1 coupling to the FcRγ chain. Selective loss of SHP1 in CD11c + cells phenocopies enhanced adaptive immunity to Leishmania. In conclusion, Leishmania shifts Mincle to an inhibitory ITAM (ITAMi) configuration that impairs DC activation. Thus, ITAMi can be exploited for immune evasion by a pathogen and may represent a paradigm for ITAM-coupled receptors sensing self and non-self. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Plants used in the treatment of leishmanial ulcers due to Leishmania (Viannia) braziliensis in an endemic area of Bahia, Brazil.

    Science.gov (United States)

    França, F; Lago, E L; Marsden, P D

    1996-01-01

    This paper records the plants used in the treatment of cutaneous leishmaniasis due to Leishmania (Viannia) braziliensis (L(V)b) among the rural population of a cocoa-producing coastal area of Bahia state, Brazil. An enquiry conducted among a hundred patients identified 49 plants species used to treat skin ulceration caused by this Leishmania species. The principal plants used are caju-branco (Anacardium occidentale, Anacardiaceae), used by 65% of the population, folha-fogo (Clidemia hirta,Melastomataceae) 39%, alfavaca-grossa (Plectranthus amboinicus, Lamiaceae) 33%, mastruz (Chenopodium ambrosioides, Chenopodiaceae) 31%, erva-de-santa-maria (Solanum americanum, Solanaceae) (25%) and transagem (Plantago major, Plantaginaceae) 2%.

  13. Plants used in the treatment of leishmanial ulcers due to Leishmania (Viannia braziliensis in an endemic area of Bahia, Brazil

    Directory of Open Access Journals (Sweden)

    Flávio França

    1996-06-01

    Full Text Available This paper records the plants used in the treatment of cutaneous leishmaniasis due to Leishmania (Viannia braziliensis (L(Vb among the rural population of a cocoa- producing coastal area of Bahia state, Brazil. An enquiry conducted among a hundred patients identified 49 plant species used to treat skin ulceration caused by this Leishmania species. The principal plants used are caju-branco (Anacardium occidentale - Anacardiaceae, used by 65% of the population, folha-fogo (Clidemia hirta - Melastomataceae 39%, alfavaca-grossa (Plectranthus amboinicus - Lamiaceae 33%, mastruz (Chenopodium ambrosioides - Chenopodiaceae 31%, erva-de-santa-maria (Solatium americanum - Solanaceae (25% and transagem (Plantago major - Plantaginaceae. 2%.

  14. Identification of species of leishmania isolated from patients with cutaneous leishmaniasis in Kermanshah; using RAPD-PCR technique

    Directory of Open Access Journals (Sweden)

    Yazdan Hamzavi

    2010-09-01

    Full Text Available Annually many numbers of pationts with Cutaneous Leishmaniasis (CL have been reported in Kermanshah province- IRAN. The study aimed to identify species of Leishmania isolated from patients with CT in Kermanshah. Seven isolates of Leishmania obtained from patients with CL, without any travelling to other provinces, were cultured in NNN medium. After mass production of leptomonads in RPMI 1640 medium DNA was purified and the species were diagnosed using RAPD-PCR technique. The study of electrophoretic fingerprints of the product of RAPD-PCR in seven isolates showed that Leshmania major was the causative agent of CL patients in Kermanshah province. More studies in this field recommended.

  15. Evaluation of Anti-HIV-1 Mutagenic Nucleoside Analogues*

    Science.gov (United States)

    Vivet-Boudou, Valérie; Isel, Catherine; El Safadi, Yazan; Smyth, Redmond P.; Laumond, Géraldine; Moog, Christiane; Paillart, Jean-Christophe; Marquet, Roland

    2015-01-01

    Because of their high mutation rates, RNA viruses and retroviruses replicate close to the threshold of viability. Their existence as quasi-species has pioneered the concept of “lethal mutagenesis” that prompted us to synthesize pyrimidine nucleoside analogues with antiviral activity in cell culture consistent with an accumulation of deleterious mutations in the HIV-1 genome. However, testing all potentially mutagenic compounds in cell-based assays is tedious and costly. Here, we describe two simple in vitro biophysical/biochemical assays that allow prediction of the mutagenic potential of deoxyribonucleoside analogues. The first assay compares the thermal stabilities of matched and mismatched base pairs in DNA duplexes containing or not the nucleoside analogues as follows. A promising candidate should display a small destabilization of the matched base pair compared with the natural nucleoside and the smallest gap possible between the stabilities of the matched and mismatched base pairs. From this assay, we predicted that two of our compounds, 5-hydroxymethyl-2′-deoxyuridine and 5-hydroxymethyl-2′-deoxycytidine, should be mutagenic. The second in vitro reverse transcription assay assesses DNA synthesis opposite nucleoside analogues inserted into a template strand and subsequent extension of the newly synthesized base pairs. Once again, only 5-hydroxymethyl-2′-deoxyuridine and 5-hydroxymethyl-2′-deoxycytidine are predicted to be efficient mutagens. The predictive potential of our fast and easy first line screens was confirmed by detailed analysis of the mutation spectrum induced by the compounds in cell culture because only compounds 5-hydroxymethyl-2′-deoxyuridine and 5-hydroxymethyl-2′-deoxycytidine were found to increase the mutation frequency by 3.1- and 3.4-fold, respectively. PMID:25398876

  16. Evaluation of anti-HIV-1 mutagenic nucleoside analogues.

    Science.gov (United States)

    Vivet-Boudou, Valérie; Isel, Catherine; El Safadi, Yazan; Smyth, Redmond P; Laumond, Géraldine; Moog, Christiane; Paillart, Jean-Christophe; Marquet, Roland

    2015-01-02

    Because of their high mutation rates, RNA viruses and retroviruses replicate close to the threshold of viability. Their existence as quasi-species has pioneered the concept of "lethal mutagenesis" that prompted us to synthesize pyrimidine nucleoside analogues with antiviral activity in cell culture consistent with an accumulation of deleterious mutations in the HIV-1 genome. However, testing all potentially mutagenic compounds in cell-based assays is tedious and costly. Here, we describe two simple in vitro biophysical/biochemical assays that allow prediction of the mutagenic potential of deoxyribonucleoside analogues. The first assay compares the thermal stabilities of matched and mismatched base pairs in DNA duplexes containing or not the nucleoside analogues as follows. A promising candidate should display a small destabilization of the matched base pair compared with the natural nucleoside and the smallest gap possible between the stabilities of the matched and mismatched base pairs. From this assay, we predicted that two of our compounds, 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine, should be mutagenic. The second in vitro reverse transcription assay assesses DNA synthesis opposite nucleoside analogues inserted into a template strand and subsequent extension of the newly synthesized base pairs. Once again, only 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine are predicted to be efficient mutagens. The predictive potential of our fast and easy first line screens was confirmed by detailed analysis of the mutation spectrum induced by the compounds in cell culture because only compounds 5-hydroxymethyl-2'-deoxyuridine and 5-hydroxymethyl-2'-deoxycytidine were found to increase the mutation frequency by 3.1- and 3.4-fold, respectively. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. C-Nucleosides: Synthetic Strategies and Biological Applications

    Czech Academy of Sciences Publication Activity Database

    Štambaský, J.; Hocek, Michal; Kočovský, P.

    2009-01-01

    Roč. 109, č. 12 (2009), s. 6729-6764 ISSN 0009-2665 R&D Projects: GA MŠk LC512; GA AV ČR IAA400550902 Grant - others:NIH(US) 1R03TW007372-01 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleosides * nucleobases * biological activity * extension of genetic alphabet Subject RIV: CC - Organic Chemistry Impact factor: 35.957, year: 2009

  18. Antiviral acyclic nucleoside phosphonates: A novel group of immunomodulatroy agents

    Czech Academy of Sciences Publication Activity Database

    Zídek, Zdeněk; Franková, Daniela; Holý, A.

    2002-01-01

    Roč. 22, č. 1 (2002), s. 166-167 ISSN 1079-9907. [Cytokines and Interferons 2002. Turin - Italy, 06.10.2002-10.10.2002] R&D Projects: GA ČR GA305/00/0048 Institutional research plan: CEZ:AV0Z5008914 Keywords : nucleoside phosphonates Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 1.885, year: 2002

  19. Comparative Analysis of the Omics Technologies Used to Study Antimonial, Amphotericin B, and Pentamidine Resistance in Leishmania

    Directory of Open Access Journals (Sweden)

    Gagandeep Kaur

    2014-01-01

    Full Text Available Leishmaniasis is a serious threat in developing countries due to its endemic nature and debilitating symptoms. Extensive research and investigations have been carried out to learn about the mechanism of drug resistance in Leishmania but results obtained in the laboratory are not in agreement with those obtained from the field. Also the lack of knowledge about the mode of action for a number of drugs makes the study of drug resistance more complex. A major concern in recent times has been regarding the role of parasitic virulence in drug resistance for Leishmania. Researchers have employed various techniques to unravel the facts about resistance and virulence in Leishmania. With advent of advanced and more specific means of detection, further hints about probable mechanisms of conferring resistance are expected. This review aims to provide a consolidated picture along with a comparative account of the work done so far to study the mechanism of antimony, amphotericin B, and pentamidine resistance using various techniques.

  20. UHPLC-TQ-MS Coupled with Multivariate Statistical Analysis to Characterize Nucleosides, Nucleobases and Amino Acids in Angelicae Sinensis Radix Obtained by Different Drying Methods.

    Science.gov (United States)

    Zhu, Shaoqing; Guo, Sheng; Duan, Jin-Ao; Qian, Dawei; Yan, Hui; Sha, Xiuxiu; Zhu, Zhenhua

    2017-06-01

    To explore the nutrients in roots of Angelica sinensis (Angelicae Sinensis Radix, ASR), a medicinal and edible plant, and evaluate its nutritional value, a rapid and reliable UHPLC-TQ-MS method was established and used to determine the potential nutritional compounds, including nucleosides, nucleobases and amino acids, in 50 batches of ASR samples obtained using two drying methods. The results showed that ASR is a healthy food rich in nucleosides, nucleobases and amino acids, especially arginine. The total average content of nucleosides and nucleobases in all ASR samples was 3.94 mg/g, while that of amino acids reached as high as 61.79 mg/g. Principle component analysis showed that chemical profile differences exist between the two groups of ASR samples prepared using different drying methods, and the contents of nutritional compounds in samples dried with the tempering-intermittent drying processing method (TIDM) were generally higher than those dried using the traditional solar processing method. The above results suggest that ASR should be considered an ideal healthy food and TIDM could be a suitable drying method for ASR when taking nucleosides, nucleobases and amino acids as the major consideration for their known human health benefits.

  1. Variants of a Leishmania surface antigen derived from a multigenic family.

    Science.gov (United States)

    Murray, P J; Spithill, T W

    1991-12-25

    The promastigote surface antigen-2 (PSA-2) complex comprises a group of immunogenic surface antigens linked to the surface of the Leishmania major promastigote with glycosylphosphatidylinositol anchors. The L. major genome contains at least 14 PSA-2 genes on a 950-kilobase chromosome and comprising approximately 20% of the length of this chromosome. The sequence of three independent, but incomplete, PSA-2 cDNAs and one genomic fragment encoding a complete PSA-2 coding sequence were compared. PSA-2 genes encode polypeptides exhibiting 22-25aa tandem repeat elements, threonine-rich segments which vary between genes, a conserved COOH-terminal cysteine-rich region, and a conserved GPI anchor signal sequence. PSA-2 genes appear to be transcribed in a complex manner with multiple RNAs. The complex genomic organization of PSA-2 genes is present in other members of the genus suggesting that PSA-2 function is important for the biology of Leishmania.

  2. Nucleoside-Lipid-Based Nanocarriers for Sorafenib Delivery

    Science.gov (United States)

    Benizri, Sebastien; Ferey, Ludivine; Alies, Bruno; Mebarek, Naila; Vacher, Gaelle; Appavoo, Ananda; Staedel, Cathy; Gaudin, Karen; Barthélémy, Philippe

    2018-01-01

    Although the application of sorafenib, a small inhibitor of tyrosine protein kinases, to cancer treatments remains a worldwide option in chemotherapy, novel strategies are needed to address the low water solubility (use of nanocarriers is currently investigated in order to overcome these drawbacks. In this contribution, we report a new type of sorafenib-based nanoparticles stabilized by hybrid nucleoside-lipids. The solid lipid nanoparticles (SLNs) showed negative or positive zeta potential values depending on the nucleoside-lipid charge. Transmission electron microscopy of sorafenib-loaded SLNs revealed parallelepiped nanoparticles of about 200 nm. Biological studies achieved on four different cell lines, including liver and breast cancers, revealed enhanced anticancer activities of Sorafenib-based SLNs compared to the free drug. Importantly, contrast phase microscopy images recorded after incubation of cancer cells in the presence of SLNs at high concentration in sorafenib (> 80 μM) revealed a total cancer cell death in all cases. These results highlight the potential of nucleoside-lipid-based SLNs as drug delivery systems.

  3. Synthesis of pyrimidine containing nucleoside β-(R/S)-hydroxyphosphonate analogues

    OpenAIRE

    Meurillon, Maïa; Chaloin, Laurent; Peyrottes, Suzanne; Périgaud, Christian

    2011-01-01

    A concise route to nucleoside β-hydroxyphosphonate analogues is described. The use of a nucleoside β-ketophosphonate as the key intermediate allowed both the (R) and (S) isomers of β-hydroxyphosphonate analogues in the pyrimidine series to be accessed. Such derivatives may be considered as stable mimics of 5′-monophosphate nucleosides and, therefore, could be the starting point for the development of potential therapeutic agents.

  4. Synthesis and antiproliferative evaluation of novel azido nucleosides and their phosphoramidate derivatives

    Czech Academy of Sciences Publication Activity Database

    Xavier, N.M.; Goncalves-Pereira, R.; Jorda, Radek; Řezníčková, Eva; Kryštof, Vladimír; Oliveira, M.C.

    2017-01-01

    Roč. 89, č. 9 (2017), s. 1267-1281 ISSN 0033-4545 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : anticancer activity * azido nucleosides * bioactive molecules * ics-28 * N-glycosylation * nucleoside phosphoramidates * nucleoside/nucleotide analogs * Staudinger-phosphite reaction Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Organic chemistry Impact factor: 2.626, year: 2016

  5. The protective effect against Leishmania infection conferred by sand fly bites is limited to short-term exposure

    Czech Academy of Sciences Publication Activity Database

    Rohoušová, I.; Hostomská, J.; Vlková, M.; Kobets, Tetyana; Lipoldová, Marie; Volf, P.

    2011-01-01

    Roč. 41, č. 5 (2011), s. 481-485 ISSN 0020-7519 R&D Projects: GA MŠk(CZ) LC06009 Institutional research plan: CEZ:AV0Z50520514 Keywords : Phlebotomus duboscqi * Leishmania major * anti -saliva IgG Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.393, year: 2011

  6. The protective effect against Leishmania infection conferred by sand fly bites is limited to short-term exposure

    Czech Academy of Sciences Publication Activity Database

    Rohoušová, I.; Hostomská, J.; Vlková, M.; Kobets, Tetyana; Lipoldová, Marie; Volf, P.

    2011-01-01

    Roč. 41, č. 5 (2011), s. 481-485 ISSN 0020-7519 R&D Projects: GA MŠk(CZ) LC06009 Institutional research plan: CEZ:AV0Z50520514 Keywords : Phlebotomus duboscqi * Leishmania major * anti-saliva IgG Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.393, year: 2011

  7. Vaccination with a plasmid DNA cocktail encoding the nucleosomal histones of Leishmania confers protection against murine cutaneous leishmaniosis.

    Science.gov (United States)

    Iborra, Salvador; Soto, Manuel; Carrión, Javier; Alonso, Carlos; Requena, Jose M

    2004-09-28

    Leishmania histones are relevant immunogens for the host immune system during both Leishmania infection and disease. In the present paper we have evaluated the prophylactic value of the four Leishmania infantum histones forming the nucleosomal core in the murine model of cutaneous leishmaniasis. In a first stage, the immune response elicited by the intramuscular injection of a mixture of four plasmid DNAs, encoding the L. infantum histones H2A, H2B, H3 and H4, was determined in BALB/c mice. It was found that the immunized animals developed a specific Th1 immune response, which was associated with an antigen-specific production of interferon (IFN-gamma) and a limited humoral response against histones (dominated by antibodies of the IgG2a isotype). According to the pure Th1-type immune response elicited by the DNA vaccination with Leishmania histones, vaccinated mice showed a solid immunity that efficiently controlled the Leishmania major infection. The protection in mice vaccinated with histone-DNAs was associated with a low humoral response against leishmanial antigens, an enhanced IFN-gamma production and little, if any, IL-4 production. The relative contribution of both CD8(+) and CD4(+) T cells to the IFN-gamma production, and the IL-12 dependence were also evaluated. All these data indicated that DNA vaccination with Leishmania histones genes results in a specific Th1-like response during L. major infection, and that both CD4(+) and CD8(+) T cells contribute to the resistance of vaccinated mice to cutaneous leishmaniasis.

  8. Leishmania genome analysis and high-throughput immunological screening identifies tuzin as a novel vaccine candidate against visceral leishmaniasis.

    Science.gov (United States)

    Lakshmi, Bhavana Sethu; Wang, Ruobing; Madhubala, Rentala

    2014-06-24

    Leishmaniasis is a neglected tropical disease caused by Leishmania species. It is a major health concern affecting 88 countries and threatening 350 million people globally. Unfortunately, there are no vaccines and there are limitations associated with the current therapeutic regimens for leishmaniasis. The emerging cases of drug-resistance further aggravate the situation, demanding rapid drug and vaccine development. The genome sequence of Leishmania, provides access to novel genes that hold potential as chemotherapeutic targets or vaccine candidates. In this study, we selected 19 antigenic genes from about 8000 common Leishmania genes based on the Leishmania major and Leishmania infantum genome information available in the pathogen databases. Potential vaccine candidates thus identified were screened using an in vitro high throughput immunological platform developed in the laboratory. Four candidate genes coding for tuzin, flagellar glycoprotein-like protein (FGP), phospholipase A1-like protein (PLA1) and potassium voltage-gated channel protein (K VOLT) showed a predominant protective Th1 response over disease exacerbating Th2. We report the immunogenic properties and protective efficacy of one of the four antigens, tuzin, as a DNA vaccine against Leishmania donovani challenge. Our results show that administration of tuzin DNA protected BALB/c mice against L. donovani challenge and that protective immunity was associated with higher levels of IFN-γ and IL-12 production in comparison to IL-4 and IL-10. Our study presents a simple approach to rapidly identify potential vaccine candidates using the exhaustive information stored in the genome and an in vitro high-throughput immunological platform. Copyright © 2014. Published by Elsevier Ltd.

  9. Leishmania infantum Genetic Diversity and Lutzomyia longipalpis Mitochondrial Haplotypes in Brazil

    OpenAIRE

    Ribolla, Paulo Eduardo Martins; Gushi, Letícia Tsieme; Pires e Cruz, Maria do Socorro; Costa, Carlos Henrique Nery; Costa, Dorcas Lamounier; Lima Júnior, Manoel Sebastião da Costa; Dorval, Maria Elizabeth Moraes Cavalheiros; Gutierrez de Oliveira, Alessandra; da Cunha Santos, Mirella Ferreira; Fonseca Camargo-Neves, Vera Lúcia; Fortaleza, Carlos Magno Castello Branco; Alonso, Diego Peres

    2016-01-01

    Leishmania infantum is the etiological agent of visceral leishmaniasis (VL) in the Americas with domestic dogs being its major reservoir hosts. The main VL vector is the sandfly Lutzomyia longipalpis, while other Lutzomyia species may play a role in disease transmission. Although the genetic structure of L. infantum populations has been widely evaluated, only a few studies have addressed this subject coupled to the genetic structure of the respective sandfly vectors. In this study, we analyze...

  10. Nucleosides of 4-methylthio-1,2,3-triazol-5-yl-carboxylic acid derivatives

    International Nuclear Information System (INIS)

    Shingarova, I.D.; Yartseva, I.V.; Preobrazhenskaya, M.N.

    1987-01-01

    2-β-D-Ribofuranosyl-4-methylthio-5-methoxycarbonyl-1,2,3-triazole was obtained by fusing 4-methylthio-5-methoxycarbonyl-1,2,3-triazole together with tetraacyl-D-ribofuranose, followed by deacylation, and its amide and hydrazide were prepared. The structures of the new nucleosides were established by converting them into known 2-nucleosides of 1,2,3-triazol-4-yl-carboxylic acid derivatives. By comparing PMR spectra with previously reported PMR spectra for the isomeric 1- and 2-nucleosides of 1,2,3-triazol-4-yl-carboxylic acid derivatives, the synthesized nucleosides could be assigned to 2-substituted triazoles

  11. Cutaneous immune mechanisms in canine leishmaniosis due to Leishmania infantum.

    Science.gov (United States)

    Papadogiannakis, E I; Koutinas, A F

    2015-02-15

    Canine leishmaniosis (CanL) caused by the parasite Leishmania infantum is a systemic disease with variable clinical signs. The disease is endemic in the Mediterranean countries and dogs are the main domestic reservoir of the parasite. The quite complicated immune response against the parasite is crucial for the evolution of CanL infection with the skin playing a major role in its immunopathogenesis. After the inoculation of Leishmania promastigotes into the dermis by sand fly bites, complement factors, Langerhan's cells, neutrophils, fibroblasts and keratinocytes are involved in the activation of the innate arm of the skin immune system, with the macrophages and dendritic cells to play a major key role. The effective activation of cellular immunity is the cornerstone of dog's resistance against the parasite. Promastigotes reaching the dermis are engulfed, processed and transferred by APCs to draining lymph nodes to stimulate naïve T-cells for proliferation and differentiation into armed effector T-cells. Th1 cells activate the infected macrophages to kill Leishmania, whereas Th2 cells divert the immune response to humoral immunity and down regulation of cellular immunity with Th1 cell anergy. Inhibition of co-stimulatory molecules expression by infected macrophages contributes to T-cell anergy. In canine subclinical infections cutaneous lymphocytic infiltrate and parasites are absent, as opposed to dogs with clinical leishmaniosis. CD8+ cells constitute a significant population of cellular immunity in CanL since they outnumber CD4+ cells in the dermis, producing IFN-γ in sub clinically infected dogs and high levels of IL-4 in dogs with clinical leishmaniosis. Numerous B-lymphocytes have been shown to heavily infiltrate the dermis at least in exfoliative dermatitis in CanL. A mixed Th1/Th2 cytokine profile has been found in the dermis of naturally infected with L. infantum dogs. In the skin of dogs with clinical leishmaniosis, where plasma cells outnumber T

  12. Members of a large retroposon family are determinants of post-transcriptional gene expression in Leishmania.

    Directory of Open Access Journals (Sweden)

    Frédéric Bringaud

    2007-09-01

    Full Text Available Trypanosomatids are unicellular protists that include the human pathogens Leishmania spp. (leishmaniasis, Trypanosoma brucei (sleeping sickness, and Trypanosoma cruzi (Chagas disease. Analysis of their recently completed genomes confirmed the presence of non-long-terminal repeat retrotransposons, also called retroposons. Using the 79-bp signature sequence common to all trypanosomatid retroposons as bait, we identified in the Leishmania major genome two new large families of small elements--LmSIDER1 (785 copies and LmSIDER2 (1,073 copies--that fulfill all the characteristics of extinct trypanosomatid retroposons. LmSIDERs are approximately 70 times more abundant in L. major compared to T. brucei and are found almost exclusively within the 3'-untranslated regions (3'UTRs of L. major mRNAs. We provide experimental evidence that LmSIDER2 act as mRNA instability elements and that LmSIDER2-containing mRNAs are generally expressed at lower levels compared to the non-LmSIDER2 mRNAs. The considerable expansion of LmSIDERs within 3'UTRs in an organism lacking transcriptional control and their role in regulating mRNA stability indicate that Leishmania have probably recycled these short retroposons to globally modulate the expression of a number of genes. To our knowledge, this is the first example in eukaryotes of the domestication and expansion of a family of mobile elements that have evolved to fulfill a critical cellular function.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  15. 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.

  16. High throughput screens yield small molecule inhibitors of Leishmania CRK3:CYC6 cyclin-dependent kinase.

    Directory of Open Access Journals (Sweden)

    Roderick G Walker

    2011-04-01

    Full Text Available Leishmania species are parasitic protozoa that have a tightly controlled cell cycle, regulated by cyclin-dependent kinases (CDKs. Cdc2-related kinase 3 (CRK3, an essential CDK in Leishmania and functional orthologue of human CDK1, can form an active protein kinase complex with Leishmania cyclins CYCA and CYC6. Here we describe the identification and synthesis of specific small molecule inhibitors of bacterially expressed Leishmania CRK3:CYC6 using a high throughput screening assay and iterative chemistry. We also describe the biological activity of the molecules against Leishmania parasites.In order to obtain an active Leishmania CRK3:CYC6 protein kinase complex, we developed a co-expression and co-purification system for Leishmania CRK3 and CYC6 proteins. This active enzyme was used in a high throughput screening (HTS platform, utilising an IMAP fluorescence polarisation assay. We carried out two chemical library screens and identified specific inhibitors of CRK3:CYC6 that were inactive against the human cyclin-dependent kinase CDK2:CycA. Subsequently, the best inhibitors were tested against 11 other mammalian protein kinases. Twelve of the most potent hits had an azapurine core with structure activity relationship (SAR analysis identifying the functional groups on the 2 and 9 positions as essential for CRK3:CYC6 inhibition and specificity against CDK2:CycA. Iterative chemistry allowed synthesis of a number of azapurine derivatives with one, compound 17, demonstrating anti-parasitic activity against both promastigote and amastigote forms of L. major. Following the second HTS, 11 compounds with a thiazole core (active towards CRK3:CYC6 and inactive against CDK2:CycA were tested. Ten of these hits demonstrated anti-parasitic activity against promastigote L. major.The pharmacophores identified from the high throughput screens, and the derivatives synthesised, selectively target the parasite enzyme and represent compounds for future hit

  17. 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

  18. Structure-enhanced methods in the development of non-nucleoside inhibitors targeting HIV reverse transcriptase variants.

    Science.gov (United States)

    Frey, Kathleen M

    2015-01-01

    Resistance continues to emerge as a leading cause for antiretroviral treatment failure. Several mutations in HIV reverse transcriptase (RT) confer resistance to non-nucleoside inhibitors (NNRTIs), vital components of antiretroviral combination therapies. Since the majority of mutations are located in the NNRTI binding pocket, crystal structures of RT variants in complex with NNRTIs have provided ideas for new drug design strategies. This article reviews the impact of RT crystal structures on the multidisciplinary design and development of new inhibitors with improved resistance profiles.

  19. 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.

  20. Tilting the balance between RNA interference and replication eradicates Leishmania RNA virus 1 and mitigates the inflammatory response.

    Science.gov (United States)

    Brettmann, Erin A; Shaik, Jahangheer S; Zangger, Haroun; Lye, Lon-Fye; Kuhlmann, F Matthew; Akopyants, Natalia S; Oschwald, Dayna M; Owens, Katherine L; Hickerson, Suzanne M; Ronet, Catherine; Fasel, Nicolas; Beverley, Stephen M

    2016-10-25

    Many Leishmania (Viannia) parasites harbor the double-stranded RNA virus Leishmania RNA virus 1 (LRV1), which has been associated with increased disease severity in animal models and humans and with drug treatment failures in humans. Remarkably, LRV1 survives in the presence of an active RNAi pathway, which in many organisms controls RNA viruses. We found significant levels (0.4 to 2.5%) of small RNAs derived from LRV1 in both Leishmania braziliensis and Leishmania guyanensis, mapping across both strands and with properties consistent with Dicer-mediated cleavage of the dsRNA genome. LRV1 lacks cis- or trans-acting RNAi inhibitory activities, suggesting that virus retention must be maintained by a balance between RNAi activity and LRV1 replication. To tilt this balance toward elimination, we targeted LRV1 using long-hairpin/stem-loop constructs similar to those effective against chromosomal genes. LRV1 was completely eliminated, at high efficiency, accompanied by a massive overproduction of LRV1-specific siRNAs, representing as much as 87% of the total. For both L. braziliensis and L. guyanensis, RNAi-derived LRV1-negative lines were no longer able to induce a Toll-like receptor 3-dependent hyperinflammatory cytokine response in infected macrophages. We demonstrate in vitro a role for LRV1 in virulence of L. braziliensis, the Leishmania species responsible for the vast majority of mucocutaneous leishmaniasis cases. These findings establish a targeted method for elimination of LRV1, and potentially of other Leishmania viruses, which will facilitate mechanistic dissection of the role of LRV1-mediated virulence. Moreover, our data establish a third paradigm for RNAi-viral relationships in evolution: one of balance rather than elimination.

  1. Two purine nucleoside phosphorylases in Bacillus subtilis. Purification and some properties of the adenosine-specific phosphorylase

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank

    1978-01-01

    Two purine nucleoside phosphorylases (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) were purified from vegetative Bacillus subtilis cells. One enzyme, inosine-guanosine phosphorylase, showed great similarity to the homologous enzyme of Bacillus cereus. It appeared...

  2. Humoral and cellular immune responses to synthetic peptides of the Leishmania donovani kinetoplastid membrane protein-11

    DEFF Research Database (Denmark)

    Jensen, A T; Gasim, S; Ismail, A

    1998-01-01

    Native kinetoplastid membrane protein-11 (KMP-11), purified from crude extracts of Leishmania donovani parasites, activates T cells from individuals who have recovered from visceral leishmaniasis. In this work we used three 38-mer peptides spanning the amino acid sequence of the L. donovani KMP-11...... as solid-phase ligands in enzyme-linked immunosorbent assays (ELISAs) and as stimulating antigens in lymphoproliferative assays in order to evaluate humoral and cellular immune responses to well-defined sequences of the protein. Antibody reactivity against the three peptides was measured in plasma from 63......-11 peptides was detected in plasma from Sudanese patients suffering from Leishmania major infections and in plasma from Sudanese and Danish patients infected with Plasmodium falciparum. In lymphoproliferative assays, 10 of 17 PBMC isolates from donors previously infected with L. donovani showed...

  3. Evidence for Seroprevalence in Human Localized Cutaneous Leishmaniasis Caused by Leishmania donovani in Sri Lanka

    Directory of Open Access Journals (Sweden)

    Yamuna Deepani Siriwardana

    2018-01-01

    Full Text Available Visceral leishmaniasis (VL is considered as a major health threat in the Indian subcontinent. Leishmania donovani, a usually visceralizing species, causes cutaneous leishmaniasis (CL in Sri Lanka. However, visceralizing potential of the local L. donovani is not yet fully understood. This project studied the seroprevalence of local CL by using an in-house ELISA. An IgG-based ELISA using crude Leishmania antigen (Ag was developed and validated. A total of 50 laboratory confirmed cases of locally acquired CL were examined using the newly developed ELISA. According to the optimized ELISA, seroprevalence of anti-Leishmania IgG antibodies in the study group was 34.0% (n=17/50. Majority of seropositive individuals were males (n=13/17, representing 76%. Nearly half of the seropositive individuals were young adults (20–40 years, n=9/17, 53%. Higher proportions of single lesions, large lesions, and nodular lesions were associated with a seroconversion. A proportion of local L. donovani infections leading to CL have the ability to raise an antibody response in the host. This may indicate early systemic involvement as one possibility. Study of a large number of patients with adequate follow-up would be useful.

  4. Leishmania mortality in sand fly blood meal is not species-specific and does not result from direct effect of proteinases.

    Science.gov (United States)

    Pruzinova, Katerina; Sadlova, Jovana; Myskova, Jitka; Lestinova, Tereza; Janda, Jozef; Volf, Petr

    2018-01-15

    Leishmania development in sand flies is confined to the alimentary tract and is closely connected with blood meal digestion. Previously, it has been published that activities of sand fly midgut proteases are harmful to Leishmania, especially to amastigote-promastigote transition forms. However, our experiments with various Leishmania-sand fly pairs gave quite opposite results. We evaluated the effect of semi-digested midgut content on different life stages of Leishmania donovani and Leishmania major in vitro. Various morphological forms of parasites, including macrophage-derived amastigotes and transition forms, were incubated 2 h with midguts dissected at various intervals (6-72 h) post-blood meal or with commercially available proteinase, and their viability was determined using flow cytometry. In parallel, using amastigote-initiated experimental infections, we compared development of L. donovani in sand flies that are either susceptible (Phlebotomus argentipes and P. orientalis) or refractory (P. papatasi and Sergentomyia schwetzi) to this parasite. In vitro, sand fly midgut homogenates affected L. major and L. donovani in a similar way; in all sand fly species, the most significant mortality effect was observed by the end of the blood meal digestion process. Surprisingly, the most susceptible Leishmania stages were promastigotes, while mortality of transforming parasites and amastigotes was significantly lower. Parasites were also susceptible to killing by rabbit blood in combination with proteinase, but resistant to proteinase itself. In vivo, L. donovani developed late-stage infections in both natural vectors; in P. argentipes the development was much faster than in P. orientalis. On the other hand, in refractory species P. papatasi and S. schwetzi, promastigotes survived activity of digestive enzymes but were lost during defecation. We demonstrated that Leishmania transition forms are more resistant to the killing effect of semi-digested blood meal than 24

  5. An enzymatic glycosylation of nucleoside analogues using beta-galactosidase from Escherichia coli

    Czech Academy of Sciences Publication Activity Database

    Blažek, Jiří; Jansa, Petr; Baszczyňski, Ondřej; Kaiser, Martin Maxmilian; Otmar, Miroslav; Krečmerová, Marcela; Dračínský, Martin; Holý, Antonín; Králová, B.

    2012-01-01

    Roč. 20, č. 9 (2012), s. 3111-3118 ISSN 0968-0896 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : glycosylation * galactosylation * beta-galactosidase * enzymatic synthesis * nucleoside * acyclic nucleoside analogues Subject RIV: CC - Organic Chemistry Impact factor: 2.903, year: 2012

  6. Synthesis of carbocyclic homo-N-nucleosides from iridoids

    DEFF Research Database (Denmark)

    Franzyk, Henrik; Rasmussen, Jon Holbech; Mazzei, Rafaele Antonio

    1998-01-01

    Two iridoid glucosides, antirrhinoside (1) and catalpol (2), were converted into selectively protected polysubstituted cyclopentylmethanols, which were subsequently used to prepare carbocyclic homo-N-nucleosides (5, 6 and 14). A purine moiety was introduced either by the Mitsunobu reaction...... or by substitution of a primary triflate with the tetrabutylammonium salt of 6-iodopurine. The latter method was superior with regard to both ease of purification and yield. The N-9 vs. N-7 regioselectivity of the salts of different 6-substituted purine derivatives was briefly investigated....

  7. Nucleoside phosphonic acids in thymidine phosphorylase inhibition: Structure - activity relationship

    Czech Academy of Sciences Publication Activity Database

    Panova, Natalya; Kóšiová, Ivana; Petrová, Magdalena; Vaněk, Václav; Liboska, Radek; Kovačková, Soňa; Kočalka, Petr; Králíková, Šárka; Točík, Zdeněk; Páv, Ondřej; Pačes, Ondřej; Rejman, Dominik; Rosenberg, Ivan

    -, č. 52 (2008), s. 665-666 ISSN 0261-3166. [Joint Symposium of the International Roundtable on Nucleosides, Nucleotides and Nucleic Acids /18./ and the International Symposium on Nucleic Acid Chemistry /35./. Kyoto, 08.09.2008-12.09.2008] R&D Projects: GA MŠk(CZ) LC06061; GA MŠk(CZ) LC06077; GA MŠk LC512 Institutional research plan: CEZ:AV0Z40550506 Keywords : thymidine phosphorylase * inhibitors * phosphonic acids Subject RIV: CC - Organic Chemistry

  8. Purine nucleoside phosphorylase deficiency in two unrelated Saudi patients

    International Nuclear Information System (INIS)

    Alangari, Abdullah; AlHarbi, Abdullah; AlGhonaium Abdulaziz; Santisteban, Ines; Hershfield, Michael

    2009-01-01

    Purine nucleoside phosphorylase (PNP) deficiency is a rare autosomal recessive metabolic disorder that results in combined immunodeficiency, neurologic dysfunction and autoimmunity. PNP deficiency has never been reported from Saudi Arabia or in patients with an Arabic ethnic background. We report on two Saudi girls with PNP deficiency. Both showed severe lymphopenia and neurological involvement. Sequencing of the PNP gene of one girl revealed a novel missense mutation Pro146>Leu in exon 4 due to a change in the codon from CCT>CTT. Expression of PNP (146L) cDNA in E coli indicated that the mutation greatly reduced, but did not completely eliminate PNP activity. (author)

  9. Spread of Leishmania infantum in Europe with dog travelling.

    Science.gov (United States)

    Maia, Carla; Cardoso, Luís

    2015-09-30

    Leishmania infantum is the etiological agent of canine leishmaniosis (CanL) in Europe, where it is endemic in the Mediterranean region, with dogs being considered the major reservoir of the parasite for humans and other mammalian hosts. The main transmission mode of Leishmania is by the bite of infected phlebotomine sand fly insects (genus Phlebotomus), which are the only proven vectors of this zoonotic protozoan. Less common, non-vectorial transmission between dogs include infection through transfused blood products from infected donors, transplacental and venereal transmission. CanL has exhibited an expansion to new locations in Europe, mainly northwards, either by territorial contiguity, often in association with global warming that favours vectorial transmission, or by the long-distance importation of infected dogs. The increasing incidence of CanL in countries where the disease is not endemic is challenging owners, veterinarians and government authorities. Most infected dogs in these new areas have been relocated from or travelled with their owners to endemic regions, but in some cases transmission might have also been autochthonous. In the absence of prophylactic measures, the introduction of infected dogs in areas previously free of endemic CanL but which have competent sand fly vectors can result in a potential persistence of L. infantum. The spread of L. infantum in Europe is reviewed with a focus on transmission, epidemiology and geographic distribution of endemic and non-endemic CanL, infection and disease in humans and animal hosts other than dogs, together with prevention and additional control strategies. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Cross-species genetic exchange between visceral and cutaneous strains of Leishmania in the sand fly vector.

    Science.gov (United States)

    Romano, Audrey; Inbar, Ehud; Debrabant, Alain; Charmoy, Melanie; Lawyer, Phillip; Ribeiro-Gomes, Flavia; Barhoumi, Mourad; Grigg, Michael; Shaik, Jahangheer; Dobson, Deborah; Beverley, Stephen M; Sacks, David L

    2014-11-25

    Genetic exchange between Leishmania major strains during their development in the sand fly vector has been experimentally shown. To investigate the possibility of genetic exchange between different Leishmania species, a cutaneous strain of L. major and a visceral strain of Leishmania infantum, each bearing a different drug-resistant marker, were used to coinfect Lutzomyia longipalpis sand flies. Eleven double-drug-resistant progeny clones, each the product of an independent mating event, were generated and submitted to genotype and phenotype analyses. The analysis of multiple allelic markers across the genome suggested that each progeny clone inherited at least one full set of chromosomes from each parent, with loss of heterozygosity at some loci, and uniparental retention of maxicircle kinetoplast DNA. Hybrids with DNA contents of approximately 2n, 3n, and 4n were observed. In vivo studies revealed clear differences in the ability of the hybrids to produce pathology in the skin or to disseminate to and grow in the viscera, suggesting polymorphisms and differential inheritance of the gene(s) controlling these traits. The studies, to our knowledge, represent the first experimental confirmation of cross-species mating in Leishmania, opening the way toward genetic linkage analysis of important traits and providing strong evidence that genetic exchange is responsible for the generation of the mixed-species genotypes observed in natural populations.

  11. Purine nucleoside analogues inhibit the repair of radiation-induced potentially lethal damage in mammalian cells in culture

    International Nuclear Information System (INIS)

    Nakatsugawa, S.; Kumar, A.

    1982-01-01

    Three purine nucleoside analogues were found to be much more effective than pyrimidine nucleoside analogues in the screening of PLD repair inhibitors using X-irradiated Chinese hamster HA-1 cells. Among the three purine nucleoside analogues, 3'-dG was the most effective at a non-toxic concentration. (author)

  12. The crystal structure of the hexameric purine nucleoside phosphorylase from Bacillus subtilis in complex with adenosine

    International Nuclear Information System (INIS)

    Giuseppe, P.O.; Meza, A.N.; Martins, N.H.; Santos, C.R.; Murakami, M.T.

    2012-01-01

    Full text: Purine nucleoside phosphorylases (PNPs) play a key role in the purine-salvage pathway in both prokaryotes and eukaryotes. Its ribosyltransferase activity is of great biotechnological interest due to potential application in the synthesis of nucleoside analogues used in the treatment of antiviral infections and in anticancer chemotherapy. Trimeric PNPs are found mainly in vertebrates and are specific for 6-oxo-purines whereas hexameric PNPs are prevalent in prokaryotes and exhibit a broad range of substrates including 6-oxo and 6-amino purines. BsPNP233, the hexameric PNP from B. subtilis, is able to catalyze the bioconversion of ribavirin, an anti-viral drug, and is relatively thermostable, being a good target for industrial use. Here we report the crystal structures of BsPNP233 in the apo form and in complex with adenosine solved at 2.65 and 1.91 resolution, respectively. The apo and ligand-bound BsPNP233 subunits superposed with an overall r.m.s. deviation of 0.31 for all Cα atoms, which suggests that no major conformational changes occur upon substrate binding. Based on the crystal structure of BsPNP233 in complex with adenosine we have defined the active site residues implicated in binding the ribose (H4 * , R43 * , M64, R87, E178, M179, E180) and the nitrogenous base (S90, C91, G92, S202, V177, F159). These residues are highly conserved among the bacterial hexameric PNPs, suggesting they share the same mode of interaction with the substrates. This work will probably contribute to a better understanding of the molecular basis for the broad substrate specificity of hexameric PNPs and to projects aiming the rational design of PNPs for industrial purposes. (author)

  13. Nucleoside Derived Antibiotics to Fight Microbial Drug Resistance: New Utilities for an Established Class of Drugs?

    Science.gov (United States)

    Serpi, Michaela; Ferrari, Valentina; Pertusati, Fabrizio

    2016-12-08

    Novel antibiotics are urgently needed to combat the rise of infections due to drug-resistant microorganisms. Numerous natural nucleosides and their synthetically modified analogues have been reported to have moderate to good antibiotic activity against different bacterial and fungal strains. Nucleoside-based compounds target several crucial processes of bacterial and fungal cells such as nucleoside metabolism and cell wall, nucleic acid, and protein biosynthesis. Nucleoside analogues have also been shown to target many other bacterial and fungal cellular processes although these are not well characterized and may therefore represent opportunities to discover new drugs with unique mechanisms of action. In this Perspective, we demonstrate that nucleoside analogues, cornerstones of anticancer and antiviral treatments, also have great potential to be repurposed as antibiotics so that an old drug can learn new tricks.

  14. The mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase of Trypanosomatidae and the glycocomal redox balance of insect stages of Trypanosoma brucei and Leishmania spp.

    NARCIS (Netherlands)

    Guerra, D.G.; Decottignies, A.; Bakker, B.M.; Michels, P.A.M.

    2006-01-01

    The genes for the mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase were identified in Trypanosoma brucei and Leishmania major genomes. We have expressed the L. major gene in Saccharomyces cerevisiae and confirmed the subcellular localization and activity of the produced enzyme. Using

  15. Molecular characterization of Leishmania spp. in reservoir hosts in endemic foci of zoonotic cutaneous leishmaniasis in Iran.

    Science.gov (United States)

    Akhoundi, Mohammad; Mohebali, Mehdi; Asadi, Mina; Mahmodi, Mohamad Reza; Amraei, Kamyar; Mirzaei, Asad

    2013-07-01

    Zoonotic cutaneous leishmaniasis (ZCL) is an expanding disease and a public health issue in Iran. In the present study, rate of natural infection of rodent populations with Leishmania was investigated in six endemic foci including 28 villages in Golestan, Esfahan, Yazd, Fars, Khuzestan and Ilam provinces. A total of 593 rodents were captured and identified as Rhombomys opimus (n = 325), Meriones libycus (n = 171), Meriones persicus (n = 27), Tatera indica (n = 37), Nesokia indica (n = 12), Rattus rattus (n = 13) and Mus musculus (n = 8). Microscopic examinations of Giemsa-stained smears showed that 108 out of 593 (18.2%) rodents were infected with Leishmania spp., whereas infection of 186 out of 593 (31.4%) rodents with Leishmania was then confirmed by ITS1-PCR. The highest rate of infection was found in R. opimus (prevalence of 35%) and M. libycus (31%). Based on Restriction Fragment Length Polymorphism (RFLP), 145 (78%) of 186 samples detected as Leishmania DNA were identified as L. major, 8 (4%) samples as L. turanica and 33 (18%) as mixed infection (L. major and L. turanica). Samples from infected rodents were inoculated subcutaneously at tail base of BALB/c mice. In 35 of them, nodules and ulcers containing amastigotes appeared at the inoculation site. The samples prepared from infected rodents were cultured in NNN medium and only two samples werepositive. Rhombomys opimus, M. libycus, M. persicus, T. indica and N. indica were confirmed as reservoir hosts of ZCL in the studied regions. Leishmania major infection was usually accompanied L. turanica in naturally infected gerbils (R. opimus and M. libycus) in Golestan, Esfahan and Fars provinces.

  16. Molecular variation in Leishmania parasites from sandflies species of a zoonotic cutaneous leishmaniasis in northeast of Iran.

    Science.gov (United States)

    Sharbatkhori, Mitra; Spotin, Adel; Taherkhani, Heshmatollah; Roshanghalb, Mona; Parvizi, Parviz

    2014-03-01

    In the well-known zoonotic cutaneous leishmaniasis (ZCL) focus in Turkmen Sahara, border of Iran and Turkmenistan, ZCL has increased among humans in the past five years. The present study was undertaken to incriminate vectors of ZCL in the region, and to find molecular variation in Leishmania parasites. The sandflies were sampled using CDC light-traps and sticky papers. All the sandflies were identified using morphological characters of the head and abdominal terminalia. DNA was extracted from the dissected thorax and attached anterior abdomen of individual female sandfly. Leishmania detection and identification of sandflies were performed using PCR, digestion of BsuRI restriction enzyme and sequencing of ITS-rDNA gene and also by semi-nested PCR to amplify minicircle kinetoplast (k) DNA of Leishmania. Leishmania infections were detected in 26 out of 206 female sandflies. Of the infected sandflies, 18 were Phlebotomus papatasi while eight were P. caucasicus/P. mongolensis. Two infections of L. turnica were detected, one in P. papatasi and other in P. caucasicus/P. mongolensis and the rest of the sandflies were found infected with L. major. Our finding showed that L. major had low diversity with only one common haplotype (GenBank Access No. EF413075). The novel haplotypes were discovered in L. major (GenBank Access No. KF152937) and in L. turanica (GenBank Access No. EF413079) in low frequency. These Leishmania parasites are circulating to maintain infections in the P. papatasi and P. caucasicus/P. mongolensis in Turkmen Sahara.

  17. Nucleosides as a carbon source in Bacillus subtilis: characterization of the drm-pupG operon.

    Science.gov (United States)

    Schuch, R; Garibian, A; Saxild, H H; Piggot, P J; Nygaard, P

    1999-10-01

    In Bacillus subtilis, nucleosides are readily taken up from the growth medium and metabolized. The key enzymes in nucleoside catabolism are nucleoside phosphorylases, phosphopentomutase, and deoxyriboaldolase. The characterization of two closely linked loci, drm and pupG, which encode phosphopentomutase (Drm) and guanosine (inosine) phosphorylase (PupG), respectively, is reported here. When expressed in Escherichia coli mutant backgrounds, drm and pupG confer phosphopentomutase and purine-nucleoside phosphorylase activity. Northern blot and enzyme analyses showed that drm and pupG form a dicistronic operon. Both enzymes are induced when nucleosides are present in the growth medium. Using mutants deficient in nucleoside catabolism, it was demonstrated that the low-molecular-mass effectors of this induction most likely were deoxyribose 5-phosphate and ribose 5-phosphate. Both Drm and PupG activity levels were higher when succinate rather than glucose served as the carbon source, indicating that the expression of the operon is subject to catabolite repression. Primer extension analysis identified two transcription initiation signals upstream of drm; both were utilized in induced and non-induced cells. The nucleoside-catabolizing system in B. subtilis serves to utilize the base for nucleotide synthesis while the pentose moiety serves as the carbon source. When added alone, inosine barely supports growth of B. subtilis. This slow nucleoside catabolism contrasts with that of E. coli, which grows rapidly on a nucleoside as a carbon source. When inosine was added with succinate or deoxyribose, however, a significant increase in growth was observed in B. subtilis. The findings of this study therefore indicate that the B. subtilis system for nucleoside catabolism differs greatly from the well-studied system in E. coli.

  18. Further observations on clinical, histopathological, and immunological features of borderline disseminated cutaneous leishmaniasis caused by Leishmania (Leishmania amazonensis

    Directory of Open Access Journals (Sweden)

    Fernando T Silveira

    2005-08-01

    Full Text Available Leishmania (Leishmania amazonensis has for some time been considered as the causative agent of two distinct forms of American cutaneous leishmaniasis (ACL: localized cutaneous leishmaniasis (LCL, and anergic diffuse cutaneous leishmaniasis (ADCL. Recently, a new intermediate form of disease, borderline disseminated cutaneous leishmaniasis (BDCL, was introduced into the clinical spectrum of ACL caused by this parasite, and in this paper we record the clinical, histopathological, and immunological features of eight more BDCL patients from Brazilian Amazonia, who acquired the disease in the Pará state, North Brazil. Seven of them had infections of one to two years' evolution and presented with primary skin lesions and the occurrence of metastases at periods varying from six to 12 months following appearance of the first lesion. Primary skin lesions ranged from 1-3 in number, and all had the aspect of an erythematous, infiltrated plaque, variously located on the head, arms or legs. There was lymphatic dissemination of infection, with lymph node enlargement in seven of the cases, and the delayed hypersensitivity skin-test (DTH was negative in all eight patients prior to their treatment. After that, there was a conversion of DTH to positive in five cases re-examined. The major histopathological feature was a dermal mononuclear infiltration, with a predominance of heavily parasitized and vacuolated macrophages, together with lymphocytes and plasma cells. In one case, with similar histopathology, the patient had acquired his infection seven years previously and he presented with the largest number of disseminated cutaneous lesions. BDCL shows clinical and histopathological features which are different from those of both LCL and ADCL, and there is a good prognosis of cure which is generally not so in the case of frank ADCL.

  19. On Leishmania enriettii and Other Enigmatic Leishmania Species of the Neotropics

    Directory of Open Access Journals (Sweden)

    Ralph Lainson

    1997-05-01

    Full Text Available There are 20 named species of the genus Leishmania at present recognized in the New World, of which 14 are known to infect man. The present paper discusses the biological, biochemical and ecological features, where known, of six species which have not till now been found to cause human leishmaniasis; namely, Leishmania (Leishmania enriettii, L. (L. hertigi, L. (L. deanei, L. (L. aristidesi, L. (L. forattinii and L. (Viannia equatorensis. A protocol is suggested for attempts to discover the natural mammalian host(s and sandfly vector of L. (L. enriettii. Doubt is cast on the validity of the species L. herreri, described in Costa Rican sloths. Following the concensus of opinion that modern trypanosomatids derive from monogenetic intestinal flagellates of arthropods, phlebotomine sandflies are best regarded as the primary hosts of Leishmania species, with mammals acting as secondary hosts providing a source of parasites for these insects. There are probably natural barriers limiting the life-cycle of most leishmanial parasites to specific sandfly vectors

  20. 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

  1. Alkylsulfanylphenyl derivatives of cytosine and 7-deazaadenine nucleosides, nucleotides and nucleoside triphosphates. Synthesis, polymerase incorporation to DNA and electrochemical study

    Czech Academy of Sciences Publication Activity Database

    Macíčková-Cahová, Hana; Pohl, Radek; Horáková Brázdilová, Petra; Havran, Luděk; Špaček, Jan; Fojta, Miroslav; Hocek, Michal

    2011-01-01

    Roč. 17, č. 21 (2011), s. 5833-5841 ISSN 0947-6539 R&D Projects: GA MŠk(CZ) LC06035; GA MŠk LC512; GA ČR GA203/09/0317; GA AV ČR(CZ) IAA400040901 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : DNA polymerases * electrochemistry * nucleosides * nucleotides * organosulfur compounds Subject RIV: CC - Organic Chemistry Impact factor: 5.925, year: 2011

  2. Abacavir-based triple nucleoside regimens for maintenance therapy in patients with HIV.

    Science.gov (United States)

    Cruciani, Mario; Mengoli, Carlo; Serpelloni, Giovanni; Parisi, Saverio G; Malena, Marina; Bosco, Oliviero

    2013-06-05

    viral load (e.g. HIV-RNA ABC had a favourable and significant impact on lipid metabolism compared to control group. An increase in CD4 cells count from baseline was evident in all analysed studies, without significant differences between ABC and controls in individual studies. The strategy of switching to triple nucleoside regimens shows weak evidence of lower incidence of side effects and a higher incidence of virologic failure in the 3NRTI group compared to controls. Simplification with 3NRTI holds the advantages of preserving other classes of antiretroviral drugs, to lower blood lipids, and to be cost effective and simple to administer.Thus, simplification with triple nucleoside regimens AZT + 3TC + ABC should be still considered for individuals who are unable to tolerate or have contraindications to NNRTI or PI based regimens. Additional data are needed on longer-term efficacy of triple NRTI regimens, particularly on the development of antiretroviral resistance. Though studies in the current review were conducted between 2001 and 2010, the large majority of patients from studies analysed received old PI regimens (e.g., indinavir, ritonavir, nelfinavir, saquinavir) not longer recommended by International Guidelines. Since current guidelines recommend new "lipid -friendly" PI, future studies should compare regimens containing these news PIs to triple NRTI regimens. More realistically, however, there are opportunities to examine these issues in existing cohorts.

  3. 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.

    Directory of Open Access Journals (Sweden)

    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

  4. Leishmania infantum HSP70-II null mutant as candidate vaccine against leishmaniasis: a preliminary evaluation

    Directory of Open Access Journals (Sweden)

    Fresno Manuel

    2011-07-01

    Full Text Available Abstract Background Visceral leishmaniasis is the most severe form of leishmaniasis and no effective vaccine exists. The use of live attenuated vaccines is emerging as a promising vaccination strategy. Results In this study, we tested the ability of a Leishmania infantum deletion mutant, lacking both HSP70-II alleles (ΔHSP70-II, to provide protection against Leishmania infection in the L. major-BALB/c infection model. Administration of the mutant line by either intraperitoneal, intravenous or subcutaneous route invariably leads to the production of high levels of NO and the development in mice of type 1 immune responses, as determined by analysis of anti-Leishmania IgG subclasses. In addition, we have shown that ΔHSP70-II would be a safe live vaccine as immunodeficient SCID mice, and hamsters (Mesocricetus auratus, infected with mutant parasites did not develop any sign of pathology. Conclusions The results suggest that the ΔHSP70-II mutant is a promising and safe vaccine, but further studies in more appropriate animal models (hamsters and dogs are needed to appraise whether this attenuate mutant would be useful as vaccine against visceral leishmaniasis.

  5. Leishmania infantum HSP70-II null mutant as candidate vaccine against leishmaniasis: a preliminary evaluation.

    Science.gov (United States)

    Carrión, Javier; Folgueira, Cristina; Soto, Manuel; Fresno, Manuel; Requena, Jose M

    2011-07-27

    Visceral leishmaniasis is the most severe form of leishmaniasis and no effective vaccine exists. The use of live attenuated vaccines is emerging as a promising vaccination strategy. In this study, we tested the ability of a Leishmania infantum deletion mutant, lacking both HSP70-II alleles (ΔHSP70-II), to provide protection against Leishmania infection in the L. major-BALB/c infection model. Administration of the mutant line by either intraperitoneal, intravenous or subcutaneous route invariably leads to the production of high levels of NO and the development in mice of type 1 immune responses, as determined by analysis of anti-Leishmania IgG subclasses. In addition, we have shown that ΔHSP70-II would be a safe live vaccine as immunodeficient SCID mice, and hamsters (Mesocricetus auratus), infected with mutant parasites did not develop any sign of pathology. The results suggest that the ΔHSP70-II mutant is a promising and safe vaccine, but further studies in more appropriate animal models (hamsters and dogs) are needed to appraise whether this attenuate mutant would be useful as vaccine against visceral leishmaniasis.

  6. The circadian clock in immune cells controls the magnitude of Leishmania parasite infection.

    Science.gov (United States)

    Kiessling, Silke; Dubeau-Laramée, Geneviève; Ohm, Hyejee; Labrecque, Nathalie; Olivier, Martin; Cermakian, Nicolas

    2017-09-07

    The intracellular parasite Leishmania uses neutrophils and macrophages as host cells upon infection. These immune cells harbour their own intrinsic circadian clocks, known to influence many aspects of their functions. Therefore, we tested whether the host circadian clocks regulate the magnitude of Leishmania major infection in mice. The extent of parasitic infection varied over 24 h in bone marrow-derived macrophages in vitro and in two different in vivo models, footpad and peritoneal cavity infection. In vivo this was paralleled by time of day-dependent neutrophil and macrophage infiltration to the infection site and rhythmic chemokine expression. Thus, rhythmic parasitic infection observed in vivo was likely initiated by the circadian expression of chemoattractants and the subsequent rhythmic infiltration of neutrophils and macrophages. Importantly, all rhythms were abolished in clock-deficient macrophages and when mice lacking the circadian clock in immune cells were infected. Therefore we demonstrated a critical role for the circadian clocks in immune cells in modulating the magnitude of Leishmania infection. To our knowledge this is the first report showing that the circadian clock controls infection by protozoan parasites in mammals. Understanding the timed regulation of host-parasite interactions will allow developing better prophylactic and therapeutic strategies to fight off vector-borne diseases.

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

    Directory of Open Access Journals (Sweden)

    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

  8. L-Asparaginase of Leishmania donovani: Metabolic target and its role in Amphotericin B resistance

    Directory of Open Access Journals (Sweden)

    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

  9. Visualisation of Leishmania donovani fluorescent hybrids during early stage development in the sand fly vector.

    Science.gov (United States)

    Sadlova, Jovana; Yeo, Matthew; Seblova, Veronika; Lewis, Michael D; Mauricio, Isabel; Volf, Petr; Miles, Michael A

    2011-01-01

    The Leishmania protozoan parasites cause devastating human diseases. Leishmania have been considered to replicate clonally, without genetic exchange. However, an accumulation of evidence indicates that there are inter-specific and intra-specific hybrids among natural populations. The first and so far only experimental proof of genetic exchange was obtained in 2009 when double drug resistant Leishmania major hybrids were produced by co-infecting sand flies with two strains carrying different drug resistance markers. However, the location and timing of hybridisation events in sand flies has not been described. Here we have co-infected Phlebotomus perniciosus and Lutzomyia longipalpis with transgenic promastigotes of Leishmania donovani strains carrying hygromycin or neomycin resistance genes and red or green fluorescent markers. Fed females were dissected at different times post bloodmeal (PBM) and examined by fluorescent microscopy or fluorescent activated cell sorting (FACS) followed by confocal microscopy. In mixed infections strains LEM3804 and Gebre-1 reached the cardia and stomodeal valves more rapidly than strains LEM4265 and LV9. Hybrids unequivocally expressing both red and green fluorescence were seen in single flies of both vectors tested, co-infected with LEM4265 and Gebre-1. The hybrids were present as short (procyclic) promastigotes 2 days PBM in the semi-digested blood in the endoperitrophic space. Recovery of a clearly co-expressing hybrid was also achieved by FACS. However, hybrids could not sustain growth in vitro. For the first time, we observed L. donovani hybrids in the sand fly vector, 2 days PBM and described the morphological stages involved. Fluorescence microscopy in combination with FACS allows visualisation and recovery of the progeny of experimental crosses but on this occasion the hybrids were not viable in vitro. Nevertheless, genetic exchange in L. donovani has profound epidemiological significance, because it facilitates the emergence

  10. Leishmania infantum Genetic Diversity and Lutzomyia longipalpis Mitochondrial Haplotypes in Brazil.

    Science.gov (United States)

    Ribolla, Paulo Eduardo Martins; Gushi, Letícia Tsieme; Pires E Cruz, Maria do Socorro; Costa, Carlos Henrique Nery; Costa, Dorcas Lamounier; Lima Júnior, Manoel Sebastião da Costa; Dorval, Maria Elizabeth Moraes Cavalheiros; Gutierrez de Oliveira, Alessandra; da Cunha Santos, Mirella Ferreira; Fonseca Camargo-Neves, Vera Lúcia; Fortaleza, Carlos Magno Castello Branco; Alonso, Diego Peres

    2016-01-01

    Leishmania infantum is the etiological agent of visceral leishmaniasis (VL) in the Americas with domestic dogs being its major reservoir hosts. The main VL vector is the sandfly Lutzomyia longipalpis, while other Lutzomyia species may play a role in disease transmission. Although the genetic structure of L. infantum populations has been widely evaluated, only a few studies have addressed this subject coupled to the genetic structure of the respective sandfly vectors. In this study, we analyzed the population structure of L. infantum in three major VL endemic areas in Brazil and associated it with Lutzomyia longipalpis geographic structure.

  11. 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.

    Directory of Open Access Journals (Sweden)

    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.

  12. Comment on ?Regulation of immunity during visceral Leishmania infection? and further discussions about the role of antibodies in infections with Leishmania

    OpenAIRE

    Gardinassi, Luiz Gustavo; de Miranda Santos, Isabel Kinney Ferreira

    2016-01-01

    Abstract Comments on the article “Regulation of immunity during visceral Leishmania infection” published in Parasites & Vectors 2016, 9:118, and further discussions about the role of antibodies in infections with Leishmania.

  13. Comment on "Regulation of immunity during visceral Leishmania infection" and further discussions about the role of antibodies in infections with Leishmania.

    Science.gov (United States)

    Gardinassi, Luiz Gustavo; de Miranda Santos, Isabel Kinney Ferreira

    2016-07-07

    Comments on the article "Regulation of immunity during visceral Leishmania infection" published in Parasites & Vectors 2016, 9:118, and further discussions about the role of antibodies in infections with Leishmania.

  14. Differential Midgut Attachment of Leishmania (Viannia braziliensis in the Sand Flies Lutzomyia (Nyssomyia whitmani and Lutzomyia (Nyssomyia intermedia

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    Rodrigo P. Soares

    2010-01-01

    Full Text Available The interaction between Leishmania and sand flies has been demonstrated in many Old and New World species. Besides the morphological differentiation from procyclic to infective metacyclic promastigotes, the parasite undergoes biochemical transformations in its major surface lipophosphoglycan (LPG. An upregulation of β-glucose residues was previously shown in the LPG repeat units from procyclic to metacyclic phase in Leishmania (Viannia braziliensis, which has not been reported in any Leishmania species. LPG has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the Subgenus Leishmania. These adaptations were explored for the first time in a species from the Subgenus Viannia, L. (V. braziliensis with its natural vectors Lutzomyia (Nyssomyia intermedia and Lutzomyia (Nyssomyia whitmani. Using two in vitro binding techniques, phosphoglycans (PGs derived from procyclic and metacyclic parasites were able to bind to the insect midgut and inhibit L. braziliensis attachment. Interestingly, L. braziliensis procyclic parasite attachment was ∼11-fold greater in the midgut of L. whitmani than in L. intermedia. The epidemiological relevance of L. whitmani as a vector of American Cutaneous Leishmaniasis (ACL in Brazil is discussed.

  15. Enhancement of Nucleoside Production in Hirsutella sinensis Based on Biosynthetic Pathway Analysis

    Science.gov (United States)

    Liu, Zhi-Qiang; Zhang, Bo; Lin, Shan; Baker, Peter James; Chen, Mao-Sheng; Xue, Ya-Ping; Wu, Hui; Xu, Feng; Yuan, Shui-Jin; Teng, Yi; Wu, Ling-Fang

    2017-01-01

    To enhance nucleoside production in Hirsutella sinensis, the biosynthetic pathways of purine and pyrimidine nucleosides were constructed and verified. The differential expression analysis showed that purine nucleoside phosphorylase, inosine monophosphate dehydrogenase, and guanosine monophosphate synthase genes involved in purine nucleotide biosynthesis were significantly upregulated 16.56-fold, 8-fold, and 5.43-fold, respectively. Moreover, dihydroorotate dehydrogenase, uridine nucleosidase, uridine/cytidine monophosphate kinase, and inosine triphosphate pyrophosphatase genes participating in pyrimidine nucleoside biosynthesis were upregulated 4.53-fold, 10.63-fold, 4.26-fold, and 5.98-fold, respectively. To enhance the nucleoside production, precursors for synthesis of nucleosides were added based on the analysis of biosynthetic pathways. Uridine and cytidine contents, respectively, reached 5.04 mg/g and 3.54 mg/g when adding 2 mg/mL of ribose, resulting in an increase of 28.6% and 296% compared with the control, respectively. Meanwhile, uridine and cytidine contents, respectively, reached 10.83 mg/g 2.12 mg/g when adding 0.3 mg/mL of uracil, leading to an increase of 176.3% and 137.1%, respectively. This report indicated that fermentation regulation was an effective way to enhance the nucleoside production in H. sinensis based on biosynthetic pathway analysis. PMID:29333435

  16. Aqueous microwaves assisted cross-coupling reactions applied to unprotected nucleosides.

    Directory of Open Access Journals (Sweden)

    CHRISTOPHE eLEN

    2015-02-01

    Full Text Available Nucleoside analogues have attracted much attention due to their potential biological activities. Amongst all synthetic nucleosides, C5-modified pyrimidines and C7- or C8-modified purines have mostly been prepared using palladium cross-coupling reactions and then studied as antitumoral and antiviral agents. Our objective is to focus this review on the Suzuki-Miyaura and on the Heck cross-couplings of nucleosides using microwave irradiations which are an alternative technology compatible with green chemistry and sustainable development.

  17. Identification of a novel compound that inhibits osteoclastogenesis by suppressing nucleoside transporters.

    Science.gov (United States)

    Katsuyama, Shun; Sugino, Kumi; Sasazawa, Yukiko; Nakano, Yoshihiko; Aono, Harumi; Morishita, Keisuke; Kawatani, Makoto; Umezawa, Kazuo; Osada, Hiroyuki; Simizu, Siro

    2016-04-01

    We screened small-molecule compounds that inhibit osteoclast differentiation to find new anti-osteoporosis agents and found that a novel compound, SUKU-1, suppressed osteoclastogenesis. We also synthesized 38 derivatives of SUKU-1 and discovered that nine of them had inhibitory effects on osteoclastogenesis and that SUKU-33 was the most potent inhibitor. Next, we investigated the mechanisms by which SUKU-33 suppressed osteoclast differentiation. By measuring the uptake of [(3) H]-uridine in cells, we found that SUKU-33 suppressed both equilibrative nucleoside transporters and concentrative nucleoside transporters. These results suggest that SUKU-33 inhibits osteoclast differentiation by suppressing nucleoside transporters. © 2016 Federation of European Biochemical Societies.

  18. A simple chemical synthesis of sugar nucleoside diphosphates in water.

    Science.gov (United States)

    Tanaka, Hidenori; Yoshimura, Yayoi; Hindsgaul, Ole

    2013-10-08

    Chemoenzymatic oligosaccharide synthesis is attractive since it eliminates the tedious multistep protection-deprotection requirements of pure chemical synthesis. Chemoenzymatic synthesis using glycosyltransferases, however, requires not only the correct enzyme to control both regio- and stereospecificity, but also the glycosyl donor to provide the sugar that is added. This unit describes a simple synthesis of sugar-nucleoside diphosphates (sugar-NDPs), the type of glycosyl donor (e.g., UDP-Glc, UDP-Gal, ADP-Glc) required by most glycosyltransferases, by using a chemical coupling reaction in water. The preparation of sugar-NDPs by this method therefore does not require any skills in synthetic organic chemistry. Copyright © 2013 John Wiley & Sons, Inc.

  19. Electrospray ionization mass spectrometric study of thallium complexes with nucleosides.

    Science.gov (United States)

    Frańska, Magdalena

    2017-10-01

    The complexes between Tl + , K + , and nucleosides were studied by using electrospray ionization mass spectrometry. It was found that for complexes of 1:1 stoichiometry, thallium complexes with cytidine were the most abundant and thallium complexes with guanosine were the second most abundant ones. The relative abundances of cytidine-Tl + to cytidine-K + complexes depended on stoichiometry (at higher stoichiometry the potassium complexes were more abundant). In other words, the relative affinity of Tl + and K + to form cytidine complexes depends on the stoichiometry of the formed complexes. Guanosine-Tl + complexes were more abundant than guanosine-K + complexes, irrespective of stoichiometry. Both guanosine tetramer and mixed cytidine/guanosine tetramer were more abundant when they were stabilized by thallium than potassium. Therefore, Tl + may affect the K + stabilization of these tetramers.

  20. Transcriptomic signature of Leishmania infected mice macrophages: a metabolic point of view.

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    Imen Rabhi

    Full Text Available We analyzed the transcriptional signatures of mouse bone marrow-derived macrophages at different times after infection with promastigotes of the protozoan parasite Leishmania major. Ingenuity Pathway Analysis revealed that the macrophage metabolic pathways including carbohydrate and lipid metabolisms were among the most altered pathways at later time points of infection. Indeed, L. major promastiogtes induced increased mRNA levels of the glucose transporter and almost all of the genes associated with glycolysis and lactate dehydrogenase, suggesting a shift to anaerobic glycolysis. On the other hand, L. major promastigotes enhanced the expression of scavenger receptors involved in the uptake of Low-Density Lipoprotein (LDL, inhibited the expression of genes coding for proteins regulating cholesterol efflux, and induced the synthesis of triacylglycerides. These data suggested that Leishmania infection disturbs cholesterol and triglycerides homeostasis and may lead to cholesterol accumulation and foam cell formation. Using Filipin and Bodipy staining, we showed cholesterol and triglycerides accumulation in infected macrophages. Moreover, Bodipy-positive lipid droplets accumulated in close proximity to parasitophorous vacuoles, suggesting that intracellular L. major may take advantage of these organelles as high-energy substrate sources. While the effect of infection on cholesterol accumulation and lipid droplet formation was independent on parasite development, our data indicate that anaerobic glycolysis is actively induced by L. major during the establishment of infection.

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

    Science.gov (United States)

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

    2012-01-01

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

  2. New Insights Into the Transmissibility of Leishmania infantum From Dogs to Sand Flies: Experimental Vector-Transmission Reveals Persistent Parasite Depots at Bite Sites.

    Science.gov (United States)

    Aslan, Hamide; Oliveira, Fabiano; Meneses, Claudio; Castrovinci, Philip; Gomes, Regis; Teixeira, Clarissa; Derenge, Candace A; Orandle, Marlene; Gradoni, Luigi; Oliva, Gaetano; Fischer, Laurent; Valenzuela, Jesus G; Kamhawi, Shaden

    2016-06-01

    Canine leishmaniasis (CanL) is a chronic fatal disease of dogs and a major source of human infection through propagation of parasites in vectors. Here, we infected 8 beagles through multiple experimental vector transmissions with Leishmania infantum-infected Lutzomyia longipalpis. CanL clinical signs varied, although live parasites were recovered from all dog spleens. Splenic parasite burdens correlated positively with Leishmania-specific interleukin 10 levels, negatively with Leishmania-specific interferon γ and interleukin 2 levels, and negatively with Leishmania skin test reactivity. A key finding was parasite persistence for 6 months in lesions observed at the bite sites in all dogs. These recrudesced following a second transmission performed at a distal site. Notably, sand flies efficiently acquired parasites after feeding on lesions at the primary bite site. In this study, controlled vector transmissions identify a potentially unappreciated role for skin at infectious bite sites in dogs with CanL, providing a new perspective regarding the mechanism of Leishmania transmissibility to vector sand flies. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.

  3. Attenuation of nucleoside and anti-cancer nucleoside analog drug uptake in prostate cancer cells by Cimicifuga racemosa extract BNO-1055.

    Science.gov (United States)

    Dueregger, Andrea; Guggenberger, Fabian; Barthelmes, Jan; Stecher, Günther; Schuh, Markus; Intelmann, Daniel; Abel, Gudrun; Haunschild, Jutta; Klocker, Helmut; Ramoner, Reinhold; Sampson, Natalie

    2013-11-15

    This study aimed to investigate the mechanisms underlying the anti-proliferative effects of the ethanolic Cimicifuga racemosa extract BNO-1055 on prostate cells and evaluate its therapeutic potential. BNO-1055 dose-dependently attenuated cellular uptake and incorporation of thymidine and BrdU and significantly inhibited cell growth after long-time exposure. Similar results were obtained using saponin-enriched sub-fractions of BNO-1055. These inhibitory effects of BNO-1055 could be mimicked using pharmacological inhibitors and isoform-specific siRNAs targeting the equilibrative nucleoside transporters ENT1 and ENT2. Moreover, BNO-1055 attenuated the uptake of clinically relevant nucleoside analogs, e.g. the anti-cancer drugs gemcitabine and fludarabine. Consistent with inhibition of the salvage nucleoside uptake pathway BNO-1055 potentiated the cytotoxicity of the de novo nucleotide synthesis inhibitor 5-FU without significantly altering its uptake. Collectively, these data show for the first time that the anti-proliferative effects of BNO-1055 result from hindered nucleoside uptake due to impaired ENT activity and demonstrate the potential therapeutic use of BNO-1055 for modulation of nucleoside transport. Copyright © 2013 Elsevier GmbH. All rights reserved.

  4. An epidemic outbreak of canine cutaneous leishmaniasis in Colombia caused by Leishmania braziliensis and Leishmania panamensis.

    Science.gov (United States)

    Vélez, Iván D; Carrillo, Lina M; López, Liliana; Rodríguez, Erwin; Robledo, Sara M

    2012-05-01

    The largest recorded outbreak of cutaneous leishmaniasis in Colombia's history occurred during 2005-2009 in soldiers of the Colombian Army, with ~40,000 cases. This outbreak was caused by the influx of military personnel into the jungle with the mission of combat illicit crops and the guerrilla. The soldiers remain for long periods within the rainforest and are exposed to the bite of infected sand flies. During the military activities, soldiers work with dogs specially trained to detect landmines, and therefore, dogs are also exposed to the infected sand flies and show high incidence of cutaneous leishmaniasis (CL). This work describes an epidemic outbreak of canine CL caused by Leishmania braziliensis and Leishmania panamensis in Colombia, South America. The clinical features of the disease and the response to treatment with pentavalent antimonials observed in 72 guard dogs from the Colombian Army are described. A program for prevention and control of canine CL is also discussed.

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

    Science.gov (United States)

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

    2017-04-01

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

  6. 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.

  7. Insights into the Molecular Mechanism of Polymerization and Nucleoside Reverse Transcriptase Inhibitor Incorporation by Human PrimPol.

    Science.gov (United States)

    Mislak, Andrea C; Anderson, Karen S

    2016-01-01

    Human PrimPol is a newly identified DNA and RNA primase-polymerase of the archaeo-eukaryotic primase (AEP) superfamily and only the second known polymerase in the mitochondria. Mechanistic studies have shown that interactions of the primary mitochondrial DNA polymerase γ (mtDNA Pol γ) with nucleoside reverse transcriptase inhibitors (NRTIs), key components in treating HIV infection, are a major source of NRTI-associated toxicity. Understanding the interactions of host polymerases with antiviral and anticancer nucleoside analog therapies is critical for preventing life-threatening adverse events, particularly in AIDS patients who undergo lifelong treatment. Since PrimPol has only recently been discovered, the molecular mechanism of polymerization and incorporation of natural nucleotide and NRTI substrates, crucial for assessing the potential for PrimPol-mediated NRTI-associated toxicity, has not been explored. We report for the first time a transient-kinetic analysis of polymerization for each nucleotide and NRTI substrate as catalyzed by PrimPol. These studies reveal that nucleotide selectivity limits chemical catalysis while the release of the elongated DNA product is the overall rate-limiting step. Remarkably, PrimPol incorporates four of the eight FDA-approved antiviral NRTIs with a kinetic profile distinct from that of mtDNA Pol γ that may manifest in toxicity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

    Directory of Open Access Journals (Sweden)

    Anjali Kumari

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

  9. Implications of a Neotropical Origin of the Genus Leishmania

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    Noyes Harry

    1998-01-01

    Full Text Available The hypothesis of a Neotropical origin of the Leishmania/Endotrypanum clade is reviewed. The position of the L. (Sauroleishmania external to the subgenus L. (Leishmania is not consistent with the Neotropical origin of the latter subgenus. It is suggested that this may be a consequence of a faster evolutionary rate in the L. (Sauroleishmania. The implications for the classsification of the phlebotomine sandflies of the hypothesis for a Neotropical origin of the Leishmania is also considered. The classification of Galati (1995 is proposed to be most consistent with the hypothesis of a Neotropical origin of the Leishmania, whilst classifications which place the New and Old World species in separate taxa are inconsistent with this hypothesis.

  10. Leishmania RNA virus controls the severity of mucocutaneous leishmaniasis.

    Science.gov (United States)

    Ives, Annette; Ronet, Catherine; Prevel, Florence; Ruzzante, Giulia; Fuertes-Marraco, Silvia; Schutz, Frederic; Zangger, Haroun; Revaz-Breton, Melanie; Lye, Lon-Fye; Hickerson, Suzanne M; Beverley, Stephen M; Acha-Orbea, Hans; Launois, Pascal; Fasel, Nicolas; Masina, Slavica

    2011-02-11

    Mucocutaneous leishmaniasis is caused by infections with intracellular parasites of the Leishmania Viannia subgenus, including Leishmania guyanensis. The pathology develops after parasite dissemination to nasopharyngeal tissues, where destructive metastatic lesions form with chronic inflammation. Currently, the mechanisms involved in lesion development are poorly understood. Here we show that metastasizing parasites have a high Leishmania RNA virus-1 (LRV1) burden that is recognized by the host Toll-like receptor 3 (TLR3) to induce proinflammatory cytokines and chemokines. Paradoxically, these TLR3-mediated immune responses rendered mice more susceptible to infection, and the animals developed an increased footpad swelling and parasitemia. Thus, LRV1 in the metastasizing parasites subverted the host immune response to Leishmania and promoted parasite persistence.

  11. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Comparison of nucleoside concentrations in blood of fish with and without tumors

    Energy Technology Data Exchange (ETDEWEB)

    Kuehl, D.W.; Johnson, R.D. (Environmental Protection Agency, Duluth, MN (United States)); Eisenschenk, L.; Naumann, S. (Univ. of Wisconsin, Superior (United States)); Regal, R.; Barnidge, P. (Univ. of Minnesota, Duluth (United States)); McKim, J. Jr. (Oregon State Univ., Corvallis (United States))

    1991-05-01

    The objective of this study was to develop and use HPLC based analytical methodology to characterize nucleosides in blood plasma and serum from fish with and without tumors, with a goal of determining if fish blood nucleoside concentrations could similarly be used as a bioindicator of tumor development in fish. The approach was to develop analytical methodology and quality assurance criteria for the analysis of nucleosides in fish blood, and to characterize nucleoside concentrations in blood of fish for which both healthy and tumor-bearing samples were available. Data would then be used to establish parameters with which tumor-bearing fish could be distinguished from healthy fish. Blood samples used to establish the diagnostic parameters were from control rainbow trout (Oncorhynchus mykiss) and those with tumors developed after exposure to aflatoxins. A second set of blood samples was from field collected black bullheads (Ictalurus melas).

  14. Modification of Purine and Pyrimidine Nucleosides by Direct C-H Bond Activation

    Directory of Open Access Journals (Sweden)

    Yong Liang

    2015-03-01

    Full Text Available Transition metal-catalyzed modifications of the activated heterocyclic bases of nucleosides as well as DNA or RNA fragments employing traditional cross-coupling methods have been well-established in nucleic acid chemistry. This review covers advances in the area of cross-coupling reactions in which nucleosides are functionalized via direct activation of the C8-H bond in purine and the C5-H or C6-H bond in uracil bases. The review focuses on Pd/Cu-catalyzed couplings between unactivated nucleoside bases with aryl halides. It also discusses cross-dehydrogenative arylations and alkenylations as well as other reactions used for modification of nucleoside bases that avoid the use of organometallic precursors and involve direct C-H bond activation in at least one substrate. The scope and efficiency of these coupling reactions along with some mechanistic considerations are discussed.

  15. Molecular moment similarity between several nucleoside analogs of thymidine and thymidine. sil@watson.ibm.com.

    Science.gov (United States)

    Silverman, B D; Pitman, M C; Platt, D E

    1999-06-01

    Molecular moment descriptors of the shape and charge distributions of twenty five nucleoside structures have been examined. The structures include thymidine as well as the difluorotoluene nucleoside analog which has been found to pair efficiently with adenine by polymerase catalysis. The remaining twenty three structures have been chosen to be as structurally similar to thymidine and to the difluorotoluene nucleoside analog as possible. The moment descriptors which include a description of the relationship of molecular charge to shape show the difluorotoluene nucleoside to be one of the most proximate molecules to thymidine in the space of the molecular moments. The calculations, therefore, suggest that polymerase specificity might be not only a consequence of molecular steric features alone but also of the molecular electrostatic environment and its registration with molecular shape.

  16. The preparation of trisubstituted alkenyl nucleoside phosphonates under ultrasound-assisted olefin cross-metathesis.

    Science.gov (United States)

    Sari, Ozkan; Hamada, Manabu; Roy, Vincent; Nolan, Steven P; Agrofoglio, Luigi A

    2013-09-06

    Intermolecular ultrasound-assisted olefin cross-metathesis is reported. This approach allows an easy access to challenging trisubstituted alkenyl nucleoside phosphonates. Regioselective chemoenzymatic deacetylation and Mitsunobu coupling are also described.

  17. Peptides derived from nucleoside beta-amino acids form an unusual 8-helix.

    Science.gov (United States)

    Threlfall, Richard; Davies, Andrew; Howarth, Nicola M; Fisher, Julie; Cosstick, Richard

    2008-02-07

    Peptides of varying length (dimers to octamers) were prepared from nucleoside beta-amino acids and conformational studies, based on NOE observations, show that the beta-peptides form an unusual 8-helix.

  18. [Bicyclic furano[2,3-D] derivatives of pyrimidine nucleosides--synthesis and antiviral properties].

    Science.gov (United States)

    Ivanov, M A; Aleksandrova, L A

    2013-01-01

    The methods of synthesis of furano- and pyrrolo[2,3-dlpyrimidine nucleosides as well as structure activity relationship of obtained compounds towards viruses of varicella zoster, hepatitis C, bovine viral diarrhea and some others are reviewed.

  19. Cell free phosphorylation method to assess the utility of new nucleotides as nucleoside reserve transcriptase inhibitors

    CSIR Research Space (South Africa)

    Lebea, Phiyani J

    2007-06-01

    Full Text Available The acyclic nucleoside phosphonates such as cidofovir, adefovir and tenofovir have proved to be effective in vitro (cell culture systems) and in vivo (animal models, clinical studies) against a wide variety of DNA virus and retrovirus infections...

  20. Enzymatic synthesis and phosphorolysis of 4(2-thioxo- and 6(5-azapyrimidine nucleosides by E. coli nucleoside phosphorylases

    Directory of Open Access Journals (Sweden)

    Vladimir A. Stepchenko

    2016-12-01

    Full Text Available The trans-2-deoxyribosylation of 4-thiouracil (4SUra and 2-thiouracil (2SUra, as well as 6-azauracil, 6-azathymine and 6-aza-2-thiothymine was studied using dG and E. coli purine nucleoside phosphorylase (PNP for the in situ generation of 2-deoxy-α-D-ribofuranose-1-phosphate (dRib-1P followed by its coupling with the bases catalyzed by either E. coli thymidine (TP or uridine (UP phosphorylases. 4SUra revealed satisfactory substrate activity for UP and, unexpectedly, complete inertness for TP; no formation of 2’-deoxy-2-thiouridine (2SUd was observed under analogous reaction conditions in the presence of UP and TP. On the contrary, 2SU, 2SUd, 4STd and 2STd are good substrates for both UP and TP; moreover, 2SU, 4STd and 2’-deoxy-5-azacytidine (Decitabine are substrates for PNP and the phosphorolysis of the latter is reversible. Condensation of 2SUra and 5-azacytosine with dRib-1P (Ba salt catalyzed by the accordant UP and PNP in Tris∙HCl buffer gave 2SUd and 2’-deoxy-5-azacytidine in 27% and 15% yields, respectively. 6-Azauracil and 6-azathymine showed good substrate properties for both TP and UP, whereas only TP recognizes 2-thio-6-azathymine as a substrate. 5-Phenyl and 5-tert-butyl derivatives of 6-azauracil and its 2-thioxo derivative were tested as substrates for UP and TP, and only 5-phenyl- and 5-tert-butyl-6-azauracils displayed very low substrate activity. The role of structural peculiarities and electronic properties in the substrate recognition by E. coli nucleoside phosphorylases is discussed.

  1. Cloning, sequencing, and expression of the PSA genes from Leishmania infantum.

    Science.gov (United States)

    Jiménez-Ruiz, A; Boceta, C; Bonay, P; Requena, J M; Alonso, C

    1998-01-15

    We describe the molecular cloning of the PSA genes from the Leishmania infantum parasite, which show high sequence similarity with the L. major PSA-2 and L. amazonensis GP46/M2 genes. The PSA genes in L. infantum are arrayed in tandem with a repetition unit of 6 kb. A single-size class of PSA mRNA of 4 kb was detected. The characterised L. infantum PSA genes code for a protein lacking the glycosylphosphatidylinositol addition signal described in other Leishmania species due to the presence of a stop codon located upstream from the DNA sequence coding for the signal. The data obtained after immunoprecipitation of PSA indicate that the protein is present as a water-soluble form, but that also a membrane-anchored form can be detected. The amino acid sequence derived from the isolated PSA gene shows that 60% of the deduced protein is formed by 13 leucine-rich repeats, each one of which is 24 amino acids long. The analysis of the consensus sequence of the repeats revealed that the L. infantum PSA as well as the described L. major PSA-2 and L. amazonensis GP46/M2 proteins may be classified as new members of the leucine-rich repeat-containing protein superfamily. The number of leucine-rich motifs, however, varies considerably between the PSA protein from L. infantum and from the other Leishmania species. The PSA protein is a major antigen determinant during L. infantum infections since 87% of the sera from naturally infected dogs recognise the recombinant PSA purified from Escherichia coli.

  2. Ring Enlargement of Cyclic Acetals and Ketals: A Way to Seven-Membered Nucleoside Phostones

    Czech Academy of Sciences Publication Activity Database

    Páv, Ondřej; Barvík, I.; Buděšínský, Miloš; Masojídková, Milena; Rosenberg, Ivan

    2007-01-01

    Roč. 9, č. 26 (2007), s. 5469-5472 ISSN 1523-7060 R&D Projects: GA ČR GA203/05/0827; GA ČR GA202/05/0628; GA MŠk(CZ) LC06061; GA MŠk(CZ) LC06077 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleoside phostones * nucleoside derivatives * chlorodiethyl phosphite * ring enlargement Subject RIV: CC - Organic Chemistry Impact factor: 4.802, year: 2007

  3. Synthesis and anti-HIV activity of novel phenyl branched cyclopropyl nucleosides.

    Science.gov (United States)

    Wu, Ying; Hong, Joon Hee

    2005-09-01

    Novel phenyl branched cyclopropyl nucleoside analogues were designed and synthesized as potential antiviral agents. Cyclopropanation was performed via classical Simmons-Smith reaction using Zn(Et)2 and CH2I2. Coupling of the mesylates 11 and 12 with natural bases (A,C,T,U) and desilylation afforded a series of novel cyclopropyl nucleosides 21-28. The synthesized compounds were evaluated for their antiviral and antitumor activity against various viruses such as HIV, HSV-1, HSV-2 and HCMV.

  4. The promastigote surface antigen gene family of the Leishmania parasite : differential evolution by positive selection and recombination - art. no. 292

    OpenAIRE

    Devault, A.; Banuls, Anne-Laure

    2008-01-01

    Background: PSA (promastigote surface antigen) is one of the major classes of membrane proteins present at the surface of the parasitic protozoan Leishmania. While it harbours leucine rich repeats, which are suggestive of its involvement in parasite-to-host physical interactions, its exact role is largely unknown. Furthermore, the extent of diversity of this gene family, both in copy number and sequence has not been established. Results: From the newly available complete genome sequences of L...

  5. In Vitro Evaluation of a Soluble Leishmania Promastigote Surface Antigen as a Potential Vaccine Candidate against Human Leishmaniasis

    OpenAIRE

    Chamakh-Ayari, Rym; Bras-Gonçalves, Rachel; Bahi-Jaber, Narges; Petitdidier, Elodie; Markikou-Ouni, Wafa; Aoun, Karim; Moreno, Javier; Carrillo, Eugenia; Salotra, Poonam; Kaushal, Himanshu; Negi, Narender Singh; Arevalo, Jorge; Falconi-Agapito, Francesca; Privat, Angela; Cruz, Maria

    2014-01-01

    PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb)...

  6. Sequencing and Gene Expression Analysis of Leishmania tropica LACK Gene.

    Science.gov (United States)

    Hammoudeh, Nour; Kweider, Mahmoud; Abbady, Abdul-Qader; Soukkarieh, Chadi

    2014-01-01

    Leishmania Homologue of receptors for Activated C Kinase (LACK) antigen is a 36-kDa protein, which provokes a very early immune response against Leishmania infection. There are several reports on the expression of LACK through different life-cycle stages of genus Leishmania, but only a few of them have focused on L.tropica. The present study provides details of the cloning, DNA sequencing and gene expression of LACK in this parasite species. First, several local isolates of Leishmania parasites were typed in our laboratory using PCR technique to verify of Leishmania parasite species. After that, LACK gene was amplified and cloned into a vector for sequencing. Finally, the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, was evaluated by Reverse Transcription-PCR (RT-PCR) technique. The typing result confirmed that all our local isolates belong to L.tropica. LACK gene sequence was determined and high similarity was observed with the sequences of other Leishmania species. Furthermore, the expression of LACK gene in both promastigotes and amastigotes forms was confirmed. Overall, the data set the stage for future studies of the properties and immune role of LACK gene products.

  7. 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

  8. Lipases in green chemistry: acylation and alcoholysis on steroids and nucleosides.

    Science.gov (United States)

    Baldessari, Alicia; Iglesias, Luis E

    2012-01-01

    In this article, we describe the application of lipases in acylation and alcoholysis reactions on steroids and nucleosides. In the field of steroids, a variety of acetyl and fatty acid derivatives of androstanes, pregnanes, and cholestanes have been prepared through lipase-catalyzed acylation and alcoholysis reactions taking advantage of the high regio- and stereoselectivity of these enzymes. The substrates as well as the products show a high degree of biological activity as neurosteroids, hormones, and glucocorticoids. The regioselective preparation of diacylated nucleosides by means of an enzymatic alcoholysis allowed the synthesis of nucleosides prodrugs or modified nucleosides. The quantitative full deacylation and dealkoxycarbonylation of nucleosides and steroids is a mild synthetic method for the deprotection of these labile compounds. Some of the reported steroid and nucleoside products are novel, and it is not possible to obtain them satisfactorily by following traditional synthetic procedures. The advantages presented by this methodology, such as selectivity, mild reaction conditions, and low environmental impact, make the lipases an important tool in the application of the principles of Green Chemistry, offering a convenient way to prepare derivatives of natural compounds with a great potential in the pharmaceutical industry.

  9. Nature's combinatorial biosynthesis and recently engineered production of nucleoside antibiotics in Streptomyces.

    Science.gov (United States)

    Chen, Shawn; Kinney, William A; Van Lanen, Steven

    2017-04-01

    Modified nucleosides produced by Streptomyces and related actinomycetes are widely used in agriculture and medicine as antibacterial, antifungal, anticancer and antiviral agents. These specialized small-molecule metabolites are biosynthesized by complex enzymatic machineries encoded within gene clusters in the genome. The past decade has witnessed a burst of reports defining the key metabolic processes involved in the biosynthesis of several distinct families of nucleoside antibiotics. Furthermore, genome sequencing of various Streptomyces species has dramatically increased over recent years. Potential biosynthetic gene clusters for novel nucleoside antibiotics are now apparent by analysis of these genomes. Here we revisit strategies for production improvement of nucleoside antibiotics that have defined mechanisms of action, and are in clinical or agricultural use. We summarize the progress for genetically manipulating biosynthetic pathways for structural diversification of nucleoside antibiotics. Microorganism-based biosynthetic examples are provided and organized under genetic principles and metabolic engineering guidelines. We show perspectives on the future of combinatorial biosynthesis, and present a working model for discovery of novel nucleoside natural products in Streptomyces.

  10. Delayed culture of Leishmania in skin biopsies.

    Science.gov (United States)

    Dedet, J P; Pratlong, F; Pradinaud, R; Moreau, B

    1999-01-01

    Between January 1997 and October 1998, 16 skin biopsies collected from 13 patients with cutaneous leishmaniasis in French Guiana were inoculated in culture medium after travel for 3-17 days from the place of biopsy to the culture laboratory in France. Each biopsy fragment was introduced near the flame of a Bunsen burner into the transport medium (RPMI medium supplemented with 10% fetal calf serum) which was maintained at ambient temperature during postal delivery to France. In France the biopsies were ground in sterile saline before being inoculated into NNN culture tubes. The cultures were incubated at 25 degrees C and subcultured every week until the 5th week. The cultures were positive in 9 cases, remained negative in 4, and were contaminated in 3 cases. Positive results were obtained at all seasons and for 3 different Leishmania species. The study indicates that delayed culture can yield useful results from biopsies taken in field conditions.

  11. Dihydrofolate reductase: A potential drug target in trypanosomes and leishmania

    Science.gov (United States)

    Zuccotto, Fabio; Martin, Andrew C. R.; Laskowski, Roman A.; Thornton, Janet M.; Gilbert, Ian H.

    1998-05-01

    Dihydrofolate reductase has successfully been used as a drug target in the area of anti-cancer, anti-bacterial and anti-malarial chemotherapy. Little has been done to evaluate it as a drug target for treatment of the trypanosomiases and leishmaniasis. A crystal structure of Leishmania major dihydrofolate reductase has been published. In this paper, we describe the modelling of Trypanosoma cruzi and Trypanosoma brucei dihydrofolate reductases based on this crystal structure. These structures and models have been used in the comparison of protozoan, bacterial and human enzymes in order to highlight the different features that can be used in the design of selective anti-protozoan agents. Comparison has been made between residues present in the active site, the accessibility of these residues, charge distribution in the active site, and the shape and size of the active sites. Whilst there is a high degree of similarity between protozoan, human and bacterial dihydrofolate reductase active sites, there are differences that provide potential for selective drug design. In particular, we have identified a set of residues which may be important for selective drug design and identified a larger binding pocket in the protozoan than the human and bacterial enzymes.

  12. Determination of nucleosides in Cordyceps sinensis and Ganoderma lucidum by high performance liquid chromatography method

    Directory of Open Access Journals (Sweden)

    Masood Shah Khan

    2015-01-01

    Full Text Available Background: Nucleosides are supportive in the regulation and modulation of various physiological processes in body, they acts as precursors in nucleic acid synthesis, enhance immune response, help in absorption of iron and influence the metabolism of fatty acids. Cordyceps sinensis and Ganoderma lucidum are well-known for its use in traditional medicine of China, Nepal and India. They are rich in nucleosides such as adenine, adenosine, cordycepin, etc. Hence, a simple, economic and accurate high-performance liquid chromatography (HPLC analytical method was proposed for determination of adenine and adenosine for the quality control of plants. Materials and Methods: Chromatographic experiments were conducted on YL9100 HPLC system (South Korea. Reversed-phase chromatography was performed on a C18 column with methanol and dihydrogen phosphate as the mobile phase in isocratic elution method at a flow rate of 1.0 mL/min. Detection was carried out at 254 nm, which gives a sharp peak of adenine and adenosine at a retention time of 6.53 ± 0.02 min and 12.41 ± 0.02, respectively. Results and Discussion: Linear regression analysis data for the calibration plot showed a good linear relationship between response and concentration in the range of 25–200 µg/mL for adenosine and 100–800 µg/mL for adenine with regression coefficient of 0.999 and 0.996, respectively. The adenine was found 0.16% and 0.71% w/w in G. lucidum and in C. sinensis, respectively, and adenosine was found to be 0.14% w/w in G. lucidum whereas absent in C. sinensis. Conclusion: The developed HPLC method for the quantification of adenosine and adenine can be used for the quality control and standardization of crude drug and for the different herbal formulations, in which adenine and adenosine are present as major constituents. The wide linearity range, sensitivity, accuracy, and simple mobile phase imply the method is suitable for routine quantification of adenosine and adenine with

  13. Determination of nucleosides in Cordyceps sinensis and Ganoderma lucidum by high performance liquid chromatography method.

    Science.gov (United States)

    Khan, Masood Shah; Parveen, Rabea; Mishra, Kshipra; Tulsawani, Rajkumar; Ahmad, Sayeed

    2015-01-01

    Nucleosides are supportive in the regulation and modulation of various physiological processes in body, they acts as precursors in nucleic acid synthesis, enhance immune response, help in absorption of iron and influence the metabolism of fatty acids. Cordyceps sinensis and Ganoderma lucidum are well-known for its use in traditional medicine of China, Nepal and India. They are rich in nucleosides such as adenine, adenosine, cordycepin, etc. Hence, a simple, economic and accurate high-performance liquid chromatography (HPLC) analytical method was proposed for determination of adenine and adenosine for the quality control of plants. Chromatographic experiments were conducted on YL9100 HPLC system (South Korea). Reversed-phase chromatography was performed on a C18 column with methanol and dihydrogen phosphate as the mobile phase in isocratic elution method at a flow rate of 1.0 mL/min. Detection was carried out at 254 nm, which gives a sharp peak of adenine and adenosine at a retention time of 6.53 ± 0.02 min and 12.41 ± 0.02, respectively. Linear regression analysis data for the calibration plot showed a good linear relationship between response and concentration in the range of 25-200 µg/mL for adenosine and 100-800 µg/mL for adenine with regression coefficient of 0.999 and 0.996, respectively. The adenine was found 0.16% and 0.71% w/w in G. lucidum and in C. sinensis, respectively, and adenosine was found to be 0.14% w/w in G. lucidum whereas absent in C. sinensis. The developed HPLC method for the quantification of adenosine and adenine can be used for the quality control and standardization of crude drug and for the different herbal formulations, in which adenine and adenosine are present as major constituents. The wide linearity range, sensitivity, accuracy, and simple mobile phase imply the method is suitable for routine quantification of adenosine and adenine with high precision and accuracy.

  14. Case Report: First Case of Cutaneous Leishmaniasis Caused by Leishmania (Viannia) braziliensis in Suriname

    NARCIS (Netherlands)

    Hu, Ricardo V. P. F.; Kent, Alida D.; Adams, Emily R.; van der Veer, Charlotte; Sabajo, Leslie O. A.; Mans, Dennis R. A.; de Vries, Henry J. C.; Schallig, Henk D. F. H.; Fat, Rudy F. M. Lai A.

    2012-01-01

    The main causative agent of cutaneous leishmaniasis (CL) in Suriname is Leishmania (Viannia) guyanensis. This case report presents a patient infected with Leishmania (Viannia) braziliensis, a species never reported before in Suriname. This finding has clinical implications, because L. braziliensis

  15. Characterization of reactive intermediates in laser photolysis of nucleoside using of sodium salt anthraquinone-2-sulfonic acid as photosensitizer

    International Nuclear Information System (INIS)

    Ma Jianhua; Lin Weizhen; Wang Wenfeng; Han Zhenhui; Yao Side; Lin Nianyun

    1999-01-01

    The interaction of triplet state of sodium salt of anthraquinone-2-sulfonic acid (AQS) with nucleosides has been investigated in CH 3 CN using KrF(248 nm) laser flash photolysis. The transient absorption spectra and kinetics obtained from the interaction of triplet AQS and nucleoside demonstrated that the primary ionic radical pair, radical cation of nucleosides and radical anion of AQS has been detected simultaneously for the first time

  16. Evaluation of PCR procedures for detecting and quantifying Leishmania donovani DNA in large numbers of dried human blood samples from a visceral leishmaniasis focus in Northern Ethiopia.

    Science.gov (United States)

    Abbasi, Ibrahim; Aramin, Samar; Hailu, Asrat; Shiferaw, Welelta; Kassahun, Aysheshm; Belay, Shewaye; Jaffe, Charles; Warburg, Alon

    2013-03-27

    Visceral Leishmaniasis (VL) is a disseminated protozoan infection caused by Leishmania donovani parasites which affects almost half a million persons annually. Most of these are from the Indian sub-continent, East Africa and Brazil. Our study was designed to elucidate the role of symptomatic and asymptomatic Leishmania donovani infected persons in the epidemiology of VL in Northern Ethiopia. The efficacy of quantitative real-time kinetoplast DNA/PCR (qRT-kDNA PCR) for detecting Leishmania donovani in dried-blood samples was assessed in volunteers living in an endemic focus. Of 4,757 samples, 680 (14.3%) were found positive for Leishmania k-DNA but most of those (69%) had less than 10 parasites/ml of blood. Samples were re-tested using identical protocols and only 59.3% of the samples with 10 parasite/ml or less were qRT-kDNA PCR positive the second time. Furthermore, 10.8% of the PCR negative samples were positive in the second test. Most samples with higher parasitemias remained positive upon re-examination (55/59 =93%). We also compared three different methods for DNA preparation. Phenol-chloroform was more efficient than sodium hydroxide or potassium acetate. DNA sequencing of ITS1 PCR products showed that 20/22 samples were Leishmania donovani while two had ITS1 sequences homologous to Leishmania major. Although qRT-kDNA PCR is a highly sensitive test, the dependability of low positives remains questionable. It is crucial to correlate between PCR parasitemia and infectivity to sand flies. While optimal sensitivity is achieved by targeting k-DNA, it is important to validate the causative species of VL by DNA sequencing.

  17. Identification and phylogenetic relationship of Iranian strains of various Leishmania species isolated from cutaneous and visceral cases of leishmaniasis based on N-acetylglucosamine-1-phosphate transferase gene.

    Science.gov (United States)

    Hajjaran, Homa; Mohebali, Mehdi; Teimouri, Aref; Oshaghi, Mohammad Ali; Mirjalali, Hamed; Kazemi-Rad, Elham; Shiee, Mohammad Reza; Naddaf, Saied Reza

    2014-08-01

    The identity of Iranian Leishmania species has been resolved to some extent by some genetic markers. In this study, based on N-acetylglucosamine-1-phosphate transferase (nagt) gene, we further elucidated the identity and phylogeny of the prevalent species in this country. DNAs of 121 isolates belonging to cutaneous leishmaniasis (CL) patients, canine visceral leishmaniasis (CVL) cases, and Rhombomys opimus rodents were amplified by targeting a partial sequence of nagt gene. All the amplicons were analyzed with restriction fragment length polymorphism (RFLP) using Acc1 enzyme, and 49 amplicons representing different reservoir hosts were sequenced and aligned with similar sequences from GenBank database. The RFLP analysis revealed that 41 CL patients were infected Leishmania tropica and 36 with Leishmania major. Among 10 CVL isolates, 6 were identified as Leishmania infantum and 4 as L. tropica. Amongst 34 rodents' isolates, 11 and 23 isolates exhibited patterns similar to those of L. major, and L. tropica/Leishmania turanica, respectively. The sequencing results from all CL patients, CVL cases, and 4 reservoir rodents were in agreement with RFLP analysis and showed 99-100% homologies with the registered species of L. major, L. tropica, and L. infantum from Turkey, Tunisia, Iraq and Israel. Of the 7 rodent isolates exhibiting RFLP patterns similar to L. tropica/L. turanica, 3 exhibited the highest homologies (99-100%) with L. turanica and 4 with Leishmania gerbilli. The 49 nagt DNA sequences were grouped into five clusters representing L. major, L. tropica, L. infantum, L. turanica and L. gerbilli species, encompassing 19 haplotypes. No correlation was observed between intraspecies divergence and geographic distribution of haplotypes. The L. tropica haplotypes exhibited more homologies with those of L. infantum than L. major (97.2% vs. 96.9%), a probable indication to the potential ability of L. tropica to visceralize. Characterization of Iranian Leishmania isolates

  18. Formation of Mixed-Ligand Complexes of Pd2+ with Nucleoside 5'-Monophosphates and Some Metal-Ion-Binding Nucleoside Surrogates

    Directory of Open Access Journals (Sweden)

    Oleg Golubev

    2014-10-01

    Full Text Available Formation of mixed-ligand Pd2+ complexes between canonical nucleoside 5'-monophosphates and five metal-ion-binding nucleoside analogs has been studied by 1H-NMR spectroscopy to test the ability of these nucleoside surrogates to discriminate between unmodified nucleobases by Pd2+-mediated base pairing. The nucleoside analogs studied included 2,6-bis(3,5-dimethylpyrazol-1-yl-, 2,6-bis(1-methylhydrazinyl- and 6-(3,5-dimethylpyrazol-1-yl-substituted 9-(β-d-ribofuranosylpurines 1–3, and 2,4-bis(3,5-dimethylpyrazol-1-yl- and 2,4-bis(1-methylhydrazinyl-substituted 5-(β-d-ribofuranosyl-pyrimidines 4–5. Among these, the purine derivatives 1-3 bound Pd2+ much more tightly than the pyrimidine derivatives 4, 5 despite apparently similar structures of the potential coordination sites. Compounds 1 and 2 formed markedly stable mixed-ligand Pd2+ complexes with UMP and GMP, UMP binding favored by 1 and GMP by 2. With 3, formation of mixed-ligand complexes was retarded by binding of two molecules of 3 to Pd2+.

  19. Rilpivirine: a new non-nucleoside reverse transcriptase inhibitor.

    Science.gov (United States)

    Sharma, Mamta; Saravolatz, Louis D

    2013-02-01

    Rilpivirine is a new non-nucleoside reverse transcriptase inhibitor (NNRTI) that is approved for HIV-1 treatment-naive adult patients in combination with other antiretroviral agents. The recommended dose is a 25 mg tablet once daily taken orally with a meal. Due to cytochrome P450 3A4 enzyme induction or gastric pH increase, rilpivirine cannot be coadministered with a number of other drugs (anticonvulsants, rifabutin, rifampicin, rifapentine, proton pump inhibitors, systemic dexamethasone and St John's wort). Rilpivirine should be used with caution when coadministered with a drug with a known risk for torsade de pointes. Rilpivirine has a better tolerability than a comparative NNRTI, efavirenz, in clinical trials, with fewer central nervous system adverse effects, rashes, lipid abnormalities and discontinuation rates. Virological failure occurs more commonly with higher baseline viral loads (>100,000 copies/mL) and lower baseline CD4 counts (<50 cells/mm(3)). Seventeen NNRTI mutations have been associated with decreased susceptibility to rilpivirine: K101E/P, E138A/G/K/Q/R, V179L, Y181C/I/V, H221Y, F227C, M230I/L, Y188L and the combination L100I + K103N. Resistance to rilpivirine largely excludes future use of the NNRTI class.

  20. Antibacterial Nucleoside-Analog Inhibitor of Bacterial RNA Polymerase.

    Science.gov (United States)

    Maffioli, Sonia I; Zhang, Yu; Degen, David; Carzaniga, Thomas; Del Gatto, Giancarlo; Serina, Stefania; Monciardini, Paolo; Mazzetti, Carlo; Guglierame, Paola; Candiani, Gianpaolo; Chiriac, Alina Iulia; Facchetti, Giuseppe; Kaltofen, Petra; Sahl, Hans-Georg; Dehò, Gianni; Donadio, Stefano; Ebright, Richard H

    2017-06-15

    Drug-resistant bacterial pathogens pose an urgent public-health crisis. Here, we report the discovery, from microbial-extract screening, of a nucleoside-analog inhibitor that inhibits bacterial RNA polymerase (RNAP) and exhibits antibacterial activity against drug-resistant bacterial pathogens: pseudouridimycin (PUM). PUM is a natural product comprising a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 6'-amino-pseudouridine. PUM potently and selectively inhibits bacterial RNAP in vitro, inhibits bacterial growth in culture, and clears infection in a mouse model of Streptococcus pyogenes peritonitis. PUM inhibits RNAP through a binding site on RNAP (the NTP addition site) and mechanism (competition with UTP for occupancy of the NTP addition site) that differ from those of the RNAP inhibitor and current antibacterial drug rifampin (Rif). PUM exhibits additive antibacterial activity when co-administered with Rif, exhibits no cross-resistance with Rif, and exhibits a spontaneous resistance rate an order-of-magnitude lower than that of Rif. PUM is a highly promising lead for antibacterial therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Visualizing multistep elevator-like transitions of a nucleoside transporter.

    Science.gov (United States)

    Hirschi, Marscha; Johnson, Zachary Lee; Lee, Seok-Yong

    2017-05-04

    Membrane transporters move substrates across the membrane by alternating access of their binding sites between the opposite sides of the membrane. An emerging model of this process is the elevator mechanism, in which a substrate-binding transport domain moves a large distance across the membrane. This mechanism has been characterized by a transition between two states, but the conformational path that leads to the transition is not yet known, largely because the available structural information has been limited to the two end states. Here we present crystal structures of the inward-facing, intermediate, and outward-facing states of a concentrative nucleoside transporter from Neisseria wadsworthii. Notably, we determined the structures of multiple intermediate conformations, in which the transport domain is captured halfway through its elevator motion. Our structures present a trajectory of the conformational transition in the elevator model, revealing multiple intermediate steps and state-dependent conformational changes within the transport domain that are associated with the elevator-like motion.

  2. Plants used in the treatment of leishmanial ulcers due to Leishmania (Viannia braziliensis in an endemic area of Bahia, Brazil

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    Flávio França

    1996-06-01

    Full Text Available This paper records the plants used in the treatment of cutaneous leishmaniasis due to Leishmania (Viannia braziliensis (L(Vb among the rural population of a cocoa- producing coastal area of Bahia state, Brazil. An enquiry conducted among a hundred patients identified 49 plant species used to treat skin ulceration caused by this Leishmania species. The principal plants used are caju-branco (Anacardium occidentale - Anacardiaceae, used by 65% of the population, folha-fogo (Clidemia hirta - Melastomataceae 39%, alfavaca-grossa (Plectranthus amboinicus - Lamiaceae 33%, mastruz (Chenopodium ambrosioides - Chenopodiaceae 31%, erva-de-santa-maria (Solatium americanum - Solanaceae (25% and transagem (Plantago major - Plantaginaceae. 2%.Este trabalho relata as plantas usadas no tratamento da leishmaniose cutânea, causada por Leishmania (Viannia braziliensis (L(Vb, na população rural da faixa litorânea produtora de cacau do estado da Bahia, Brasil. Um inquérito realizado entre 100 pacientes, identificou 49 espécies de plantas usadas para tratar úlceras de pele causadas por esta espécie de Leishmânia. As principais plantas usadas foram o cajueiro-branco (Anacardium occidentale - Anacardiaceae usado por 65% da população, a folha-fogo (Clidemia hirta - Melastomataceae 39%, a alfavaca-grossa (Plectranthus amboinicus - Lamiaceae 33%, o mastruz (Chenopodium ambrosioides - henopodiaceae 31%, a erva-de-santa-maria (Solanum americanum - Solanaceae 25% e a transagem (Plantago major - Plantaginaceae 2%.

  3. Type I signal peptidase from Leishmania is a target of the immune response in human cutaneous and visceral leishmaniasis.

    Science.gov (United States)

    Rafati, Sima; Salmanian, Ali-Hatef; Taheri, Tahere; Masina, Slavica; Schaff, Cedric; Taslimi, Yasaman; Fasel, Nicolas

    2004-05-01

    The gene encoding type I signal peptidase (Lmjsp) has been cloned from Leishmania major. Lmjsp encodes a protein of 180 amino residues with a predicted molecular mass of 20.5 kDa. Comparison of the protein sequence with those of known type I signal peptidases indicates homology in five conserved domains A-E which are known to be important, or essential, for catalytic activity. Southern blot hybridisation analysis indicates that there is a single copy of the Lmjsp gene. A recombinant SPase protein and a synthetic peptide of the L. major signal peptidase were used to examine the presence of specific antibodies in sera from either recovered or active individuals of both cutaneous and visceral leishmaniasis. This evaluation demonstrated that sera from cutaneous and visceral forms of leishmaniasis are highly reactive to both the recombinant and synthetic signal peptidase antigens. Therefore, the Leishmania signal peptidase, albeit localised intracellularly, is a significant target of the Leishmania specific immune response and highlights its potential use for serodiagnosis of cutaneous and visceral leishmaniasis.

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

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    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.

  5. Lower levels of IgG1 in comparison with IgG2a are associated with protective immunity against Leishmania tropica infection in BALB/c mice.

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    Rostamian, Mosayeb; Sohrabi, Saeed; Kavosifard, Hanie; Niknam, Hamid M

    2017-04-01

    Leishmania (L.) tropica is the causative agent of different forms of human leishmaniasis. There is little information about the role of Leishmania-specific antibodies in the immune response against L. tropica infection. The aim of this study is to evaluate the role of Leishmania-specific antibodies and their immunoglobulin G (IgG) isotypes in L. tropica infection. L. tropica at two different doses (high dose, 10 6 parasites/mouse and low dose, 10 3 parasites/mouse) were used for infection of BALB/c mice. BALB/c mice infected with Leishmania major were used for comparison. Anti-Leishmania antibodies of the IgG1 and IgG2a isotypes were assayed by enzyme-linked immunosorbent assay. Our data showed that (1) a higher parasite dose results in higher levels of antibody. (2) L. tropica infection results in a lower IgG1 antibody response, compared with L. major infection. (3) The IgG2a/IgG1 antibody response in L. tropica infection is higher than that in L. major infection. A higher IgG2a/IgG1 ratio is associated with protective immune response in L. tropica infection. These data can help to approach the complex profile of immunity against L. tropica infection. Copyright © 2015. Published by Elsevier B.V.

  6. Circulating species of Leishmania at microclimate area of Boulemane Province, Morocco: impact of environmental and human factors.

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    Hmamouch, Asmae; El Alem, Mahmoud Mohamed; Hakkour, Maryam; Amarir, Fatima; Daghbach, Hassan; Habbari, Khalid; Fellah, Hajiba; Bekhti, Khadija; Sebti, Faiza

    2017-02-22

    Cutaneous leishmaniasis (CL) is widely distributed in Morocco where its geographical range and incidence are related to environmental factors. This study aimed to examine the impact of several factors on the distribution of CL in Boulemane Province, which is characterized by several microclimates, and to identify the Leishmania species circulating in these areas. Ordinary least squares regression (OLSR) analysis was performed to study the impact of poverty, vulnerability, population density, urbanization and bioclimatic factors on the distribution of CL in this province. Molecular characterization of parasites was performed using a previously described PCR-RFLP method targeting the ITS1 of ribosomal DNA of Leishmania. A total of 1009 cases were declared in Boulemane Province between the years 2000 and 2015 with incidences fluctuating over the years (P = 0.007). Analyzing geographical maps of the study region identified four unique microclimate areas; sub-humid, semi-arid, arid and Saharan. The geographical distribution and molecular identification of species shows that the Saharan microclimate, characterized by the presence of Leishmania major was the most affected (47.78%) followed by semi-arid area where Leishmania tropica was identified in three districts. Among several environmental factors included in the study, poverty had the greatest influence on the spatial extension of the disease in this province. The incidence of CL in Boulemane Province varies between microclimate areas, and environmental factors partly explain this variation. However, the existence of CL in the most affected districts is mainly related to poverty, population movement and human behavior. To our knowledge, this the first study utilizing molecular techniques to confirm L. tropica and L. major as the causative agents of CL in Boulemane Province. Our findings indicate that the spatial and temporal distribution of CL in Boulemane Province is strongly related to poverty and population

  7. Cryptic Leishmania infantum infection in Italian HIV infected patients

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    Rubino Raffaella

    2009-12-01

    Full Text Available Abstract Background Visceral leishmaniasis (VL is a protozoan diseases caused in Europe by Leishmania (L. infantum. Asymptomatic Leishmania infection is more frequent than clinically apparent disease. Among HIV infected patients the risk of clinical VL is increased due to immunosuppression, which can reactivate a latent infection. The aims of our study were to assess the prevalence of asymptomatic L. infantum infection in HIV infected patients and to study a possible correlation between Leishmania parasitemia and HIV infection markers. Methods One hundred and forty-five HIV infected patients were screened for the presence of anti-Leishmania antibodies and L. infantum DNA in peripheral blood. Statistical analysis was carried out by using a univariate regression analysis. Results Antibodies to L. infantum were detected in 1.4% of patients. L. infantum DNA was detected in 16.5% of patients. Significant association for PCR-Leishmania levels with plasma viral load was documented (p = 0.0001. Conclusion In our area a considerable proportion of HIV infected patients are asymptomatic carriers of L. infantum infection. A relationship between high HIV viral load and high parasitemic burden, possibly related to a higher risk of developing symptomatic disease, is suggested. PCR could be used for periodic screening of HIV patients to individuate those with higher risk of reactivation of L. infantum infection.

  8. Intergenic and external transcribed spacers of ribosomal RNA genes in lizard-infecting Leishmania: molecular structure and phylogenetic relationship to mammal-infecting Leishmania in the subgenus Leishmania (Leishmania

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    Orlando Tereza C

    2002-01-01

    Full Text Available To establish the relationships of the lizard- and mammal-infecting Leishmania, we characterized the intergenic spacer region of ribosomal RNA genes from L. tarentolae and L. hoogstraali. The organization of these regions is similar to those of other eukaryotes. The intergenic spacer region was approximately 4 kb in L. tarentolae and 5.5 kb in L. hoogstraali. The size difference was due to a greater number of 63-bp repetitive elements in the latter species. This region also contained another element, repeated twice, that had an inverted octanucleotide with the potential to form a stem-loop structure that could be involved in transcription termination or processing events. The ribosomal RNA gene localization showed a distinct pattern with one chromosomal band (2.2 Mb for L. tarentolae and two (1.5 and 1.3 Mb for L. hoogstraali. The study also showed sequence differences in the external transcribed region that could be used to distinguish lizard Leishmania from the mammalian Leishmania. The intergenic spacer region structure features found among Leishmania species indicated that lizard and mammalian Leishmania are closely related and support the inclusion of lizard-infecting species into the subgenus Sauroleishmania proposed by Saf'janova in 1982.

  9. Molecular and functional characterization of two malic enzymes from Leishmania parasites.

    Science.gov (United States)

    Giordana, Lucila; Sosa, Máximo Hernán; Leroux, Alejandro E; Mendoza, Elkin F Rodas; Petray, Patricia; Nowicki, Cristina

    2018-01-01

    Leishmania parasites cause a broad spectrum of clinical manifestations in humans and the available clinical treatments are far from satisfactory. Since these pathogens require large amounts of NADPH to maintain intracellular redox homeostasis, oxidoreductases that catalyze the production of NADPH are considered as potential drug targets against these diseases. In the sequenced genomes of most Leishmania spp. two putative malic enzymes (MEs) with an identity of about 55% have been identified. In this work, the ME from L. major (LmjF24.0770, Lmj_ME-70) and its less similar homolog from L. mexicana (LmxM.24.0761, Lmex_ME-61) were cloned and functionally characterized. Both MEs specifically catalyzed NADPH production, but only Lmex_ME-61 was activated by l-aspartate. Unlike the allosterically activated human ME, Lmex_ME-61 exhibited typical hyperbolic curves without any sign of cooperativity in the absence of l-aspartate. Moreover, Lmex_ME-61 and Lmj_ME-70 differ from higher eukaryotic homologs in that they display dimeric instead of tetrameric molecular organization. Homology modeling analysis showed that Lmex_ME-61 and Lmj_ME-70 notably differ in their surface charge distribution; this feature encompasses the coenzyme binding pockets as well. However, in both isozymes, the residues directly involved in the coenzyme binding exhibited a good degree of conservation. Besides, only Lmex_ME-61 and its closest homologs were immunodetected in cell-free extracts from L. mexicana, L. amazonensis and L. braziliensis promastigotes. Our findings provide a first glimpse into the biochemical properties of leishmanial MEs and suggest that MEs could be potentially related to the metabolic differences among the species of Leishmania parasites. Published by Elsevier B.V.

  10. Stage-specific adhesion of Leishmania promastigotes to sand fly midguts assessed using an improved comparative binding assay.

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    Raymond Wilson

    2010-09-01

    Full Text Available The binding of Leishmania promastigotes to the midgut epithelium is regarded as an essential part of the life-cycle in the sand fly vector, enabling the parasites to persist beyond the initial blood meal phase and establish the infection. However, the precise nature of the promastigote stage(s that mediate binding is not fully understood.To address this issue we have developed an in vitro gut binding assay in which two promastigote populations are labelled with different fluorescent dyes and compete for binding to dissected sand fly midguts. Binding of procyclic, nectomonad, leptomonad and metacyclic promastigotes of Leishmania infantum and L. mexicana to the midguts of blood-fed, female Lutzomyia longipalpis was investigated. The results show that procyclic and metacyclic promastigotes do not bind to the midgut epithelium in significant numbers, whereas nectomonad and leptomonad promastigotes both bind strongly and in similar numbers. The assay was then used to compare the binding of a range of different parasite species (L. infantum, L. mexicana, L. braziliensis, L. major, L. tropica to guts dissected from various sand flies (Lu. longipalpis, Phlebotomus papatasi, P. sergenti. The results of these comparisons were in many cases in line with expectations, the natural parasite binding most effectively to its natural vector, and no examples were found where a parasite was unable to bind to its natural vector. However, there were interesting exceptions: L. major and L. tropica being able to bind to Lu. longipalpis better than L. infantum; L. braziliensis was able to bind to P. papatasi as well as L. major; and significant binding of L. major to P. sergenti and L. tropica to P. papatasi was observed.The results demonstrate that Leishmania gut binding is strictly stage-dependent, is a property of those forms found in the middle phase of development (nectomonad and leptomonad forms, but is absent in the early blood meal and final stages (procyclic

  11. 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.

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

    Science.gov (United States)

    Lyra, Marcelo Rosandiski; Pimentel, Maria Inês Fernandes; Madeira, Maria de Fátima; Antonio, Liliane de Fátima; Lyra, Janine Pontes de Miranda; Fagundes, Aline; Schubach, Armando de Oliveira

    2015-01-01

    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.

  13. 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.

  14. Seasonal transmission of Leishmania (Leishmania mexicana in the state of Campeche, Yucatan Peninsula, Mexico

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    Andrade-Narvaez Fernando J

    2003-01-01

    Full Text Available In the Yucatan Peninsula, Mexico, localized cutaneous leishmaniasis (LCL caused by Leishmania (Leishmania mexicana is a typical wild zoonosis restricted to the forest, and humans are only accidentally involved. The transmission of L. (L. mexicana has been related to the patient's occupation: "chicleros"(gum collectors and agricultural workers. The objective of this study was to document L. (L. mexicana seasonally of transmission in endemic areas of LCL in the state of Campeche, Yucatan Peninsula, Mexico. The timing of incidence of LCL in humans during 1993-1994, as well as the rate and time of infection in rodents and sand flies between February 1993 and March 1995 were analyzed. Rodents and sand flies were found infected between November and March, when men carried out their field activities and are exposed. Based on results analyzed, it is concluded that L. (L. mexicana in the endemic area of LCL in the state of Campeche, Yucatan Peninsula, Mexico, presents a seasonal transmission restricted to the months of November to March. The knowledge of the timing of the transmission cycle in an endemic area of leishmaniasis is very important because intervention measures on the high-risk focus and population might be restricted.

  15. Chronic interstitial pneumonitis in dogs naturally infected with Leishmania (Leishmania chagasi: a histopathological and morphometric study

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    Gonçalves Ricardo

    2003-01-01

    Full Text Available Eighteen mongrel dogs of unknown age and naturally infected with Leishmania (Leishmania chagasi, were obtained from the City Hall of Belo Horizonte, Brazil. Four dogs were used as control. Lung samples were obtained and immediately fixed in formalin. The histopathological picture of all lung tissue sections was a chronic and diffuse interstitial pneumonitis. The thickened inter-alveolar septa were characterized by the cellular exudate (mostly macrophages, lymphocytes and plasmocytes associated with collagen deposition. Morphometric analysis showed greater septal thickness in the infected animals than in controls. In fact, the morphometric study of collagen stained with ammoniac silver confirmed a larger deposition of collagen in the infected animals. The parasitologic method was carried out during the study of the lesions on the slides. However, we did not observe any correlation between the histopathologic and morphometric data and the clinical status of the animals. We conclude that the pulmonary lesions observed in all naturally infected dogs were correlated with the disease and that the morphometric method used was satisfactory for the analysis of septal thickness and of increased collagen deposition, confirming the presence of fibrosis.

  16. Chronic interstitial pneumonitis in dogs naturally infected with Leishmania (Leishmania) chagasi: a histopathological and morphometric study.

    Science.gov (United States)

    Gonçalves, Ricardo; Tafuri, Washington Luiz; Melo, Maria Norma de; Raso, Pedro; Tafuri, Wagner Luiz

    2003-01-01

    Eighteen mongrel dogs of unknown age and naturally infected with Leishmania (Leishmania) chagasi, were obtained from the City Hall of Belo Horizonte, Brazil. Four dogs were used as control. Lung samples were obtained and immediately fixed in formalin. The histopathological picture of all lung tissue sections was a chronic and diffuse interstitial pneumonitis. The thickened inter-alveolar septa were characterized by the cellular exudate (mostly macrophages, lymphocytes and plasmocytes) associated with collagen deposition. Morphometric analysis showed greater septal thickness in the infected animals than in controls. In fact, the morphometric study of collagen stained with ammoniac silver confirmed a larger deposition of collagen in the infected animals. The parasitologic method was carried out during the study of the lesions on the slides. However, we did not observe any correlation between the histopathologic and morphometric data and the clinical status of the animals. We conclude that the pulmonary lesions observed in all naturally infected dogs were correlated with the disease and that the morphometric method used was satisfactory for the analysis of septal thickness and of increased collagen deposition, confirming the presence of fibrosis.

  17. [Western blot, ELISA and indirect immunofluorescence test evaluation of Leishmania (Leishmania) infantum-infected dogs].

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    Vargas-Duarte, Jimmy J; López-Páez, Myriam C; Escovar-Castro, Jesús E; Fernández-Manrique, José

    2009-08-01

    Evaluating canine visceral leishmaniasis diagnostic test performance in Colombia and adapting the Western blot test in naturally and experimentally infected dogs. Sera were obtained from 10 experimentally L. Infantum-infected dogs, 5 naturally infected dogs, 16 healthy dogs, 26 Babesia canis, Erhlichia canis, Dirofilaria immitis, Trypanosoma cruzi and Leishmania (Viannia) spp infected dogs, 40 dogs from non-endemic areas and 150 from endemic areas. Sera were tested for L. infantum infection using immunofluorescent antibody (IFAT), ELISA and Western blot (WB) tests. Positives results were obtained for 73 % of known infected dogs by the IFAT test and false positives were obtained for 2.5 % of non-infected dogs using WB. ELISA was not efficient for diagnosis. 24 antigenic fractions were recognised in tested sera using WB; however, 29, 34, 50, 69, 75, 86, 99 and 123 kDa bands were recognised in sera from dogs from non-endemic areas, healthy dogs and Trypanosoma cruzi, Erhlichia canis, Dirofilaria immitis and Babesia canis infected dogs. The 13 kDa fraction proved potentially useful for diagnosing canine visceral leishmaniasis. The separate use of parasitological and serological test could lead to misdiagnosis of Leishmania infection; using both kinds of technique simultaneously is thus highly recommended.

  18. What has proteomics taught us about Leishmania development?

    Science.gov (United States)

    Tsigankov, Polina; Gherardini, Pier Federico; Helmer-Citterich, Manuela; Zilberstein, Dan

    2012-08-01

    Leishmania are obligatory intracellular parasitic protozoa that cycle between sand fly mid-gut and phagolysosomes of mammalian macrophages. They have developed genetically programmed changes in gene and protein expression that enable rapid optimization of cell function according to vector and host environments. During the last two decades, host-free systems that mimic intra-lysosomal environments have been devised in which promastigotes differentiate into amastigotes axenically. These cultures have facilitated detailed investigation of the molecular mechanisms underlying Leishmania development inside its host. Axenic promastigotes and amastigotes have been subjected to transcriptome and proteomic analyses. Development had appeared somewhat variable but was revealed by proteomics to be strictly coordinated and regulated. Here we summarize the current understanding of Leishmania promastigote to amastigote differentiation, highlighting the data generated by proteomics.

  19. Subversion of host cell signalling by the protozoan parasite Leishmania.

    Science.gov (United States)

    Gregory, D J; Olivier, M

    2005-01-01

    The protozoa Leishmania spp. are obligate intracellular parasites that inhabit the macrophages of their host. Since macrophages are specialized for the identification and destruction of invading pathogens, both directly and by triggering an innate immune response, Leishmania have evolved a number of mechanisms for suppressing some critical macrophage activities. In this review, we discuss how various species of Leishmania distort the host macrophage's own signalling pathways to repress the expression of various cytokines and microbicidal molecules (nitric oxide and reactive oxygen species), and antigen presentation. In particular, we describe how MAP Kinase and JAK/STAT cascades are repressed, and intracellular Ca2+ and the activities of protein tyrosine phosphatases, in particular SHP-1, are elevated.

  20. 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.

  1. Leishmania (infantum chagasi in canine urinary sediment

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    Ivete Lopes de Mendonça

    Full Text Available Canine visceral leishmaniasis (CVL is difficult to diagnosis, mainly due to the presence of asymptomatic animals, the diversity of clinical symptoms and the difficulty in obtaining diagnostic evidence of high sensitivity and specificity. The purpose of this study was to diagnose CVL in urinary sediment of 70 dogs of different breeds, sexes and ages from the veterinary hospital of the Federal University of Piauí and Zoonosis Control Center of Teresina, Brazil. The serological tests were TR DPP® for CVL and enzyme-linked immunosorbent assay (ELISA for CVL, parasitological exams of bone marrow and lymph nodes and urine sediment cultures. Leishmania was detected in the bone marrow and/or lymph node of 61.0% of the animals (43/70, and urine sediment culture was positive in 9.30% (4/43 of these animals. In the serological exams, 70.0% (49/70 were reactive using the DPP and 78.2% (55/70 were reactive using ELISA. The goal of this study was to diagnose the presence of L. (infantum chagasi in a culture of urinary sediment.

  2. 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.

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

    Science.gov (United States)

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

    2017-09-01

    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 understanding its tropism in in vivo conditions. Clinical samples (lesion aspirations and bone marrow) obtained from CL/VL patients were investigated using parasitological (smear/NNN) and DNA-based techniques. For species identification, a real time ITS1-PCR was performed using isolates and results were confirmed by hsp70 PCR-N/sequencing and cpb gene PCR/sequencing in order to reveal Leishmania donovani and Leishmania infantum discrimination. Clinical materials from CL and VL patients were also inoculated into two experimental groups (Group CL and Group VL) of Balb/C mice intraperitoneally for creating clinical picture of Turkish L. donovani strains. After 45days, the samples from visible sores of the skin were taken, and spleens and livers were removed. Measurements of the internal organs were done and touch preparations were prepared for checking the presence of amastigotes. The strains were isolated from all patients and amastigotes were seen in all smears of the patients, and then isolates were immediately stored in liquid nitrogen. In real time ITS1-PCR, the melting temperatures of all samples were out of range of L. infantum, L. tropica and L. major. Sequencing of hsp70 PCR-N showed that all isolates highly identical to previously submitted L. donovani sequences in GenBank, and cpb gene sequencing showed five isolates had longer cpbF allele, whereas one isolate contained a mixed sequence of both cpbF and cpbE. All mice in both experimental groups became infected. Compared to controls, the length and width of both liver and spleen were significantly elevated (pTurkey. Animal models using clinical samples were successfully established and important clinical

  4. The Polymerase chain reaction as a tool of molecular diagnosis of Leishmania infection in the Sudan

    International Nuclear Information System (INIS)

    Hashim, Amna Osman Yousif

    1997-06-01

    Leishmaniasis, manifesting on it's different clinical forms is endemic in different regions of the Sudan. diagnosis of the disease in the Sudan is usually done using simple methods such as microscopical examination of slit smirs, Histological sections and cultures. Serological diagnosis using enzymes linked immunosorbent assay (ELISA)- and direct agglutination test (DAT) are sometimes used as more sensitive tool has thrown light on the epidemiology of the disease in the Sudan. This study was conducted on 126 subjects to identify the parasites-causing the different clinical manifestations, to determine the genetic diversity of different isolates of Lieshmania- and to detect parasites in the peripheral blood of subjects from highly endemic foci. The study population consisted of 7 with suspected VL, 12 with suspected ML, 14 with suspected PKDL, 2 with sporotrichoid CL and 89 healthy games wardens and army soldiers from highly endemic foci. Parasites were cultured in biphasic medium and subcultured in liquid medium until mass production was stabilized. Extraction of DNA was done using three methods which were phenol/ chloroform/ isoamylalcohol, K buffer and proteinase K as well as lysing of the parasite with distilled water. The KDNA was amplified using species namely AJSI and DeB8. The products were analysed on 1.5% agarose gel-and were visualized and photographed with U.V. transilluminator and camera. Characteristic bands of 700 and 800 b.p corresponding to the full length of mini circle of L.major and L.donovani respectively were obtained on amplification of KDNA from patients with VL and CL. In some cases lower bands of 400 and 500 b.p PKDL and multiple bands for sporotrichoid CL.Leishmania DNA was detected from the conjucativa of the eye of a patient with PKDL. The genetic diversity of Leishmania parasite was determined by digesting PCR products from PKDL, sporotrichoids CL and VL patients. Different patterns were produced for each digesting product. This result

  5. Persistence of phlebotomine Leishmania vectors in urban sites of Catania (Sicily, Italy).

    Science.gov (United States)

    Lisi, Oscar; D'Urso, Vera; Vaccalluzzo, Valerio; Bongiorno, Gioia; Khoury, Cristina; Severini, Francesco; Di Muccio, Trentina; Gramiccia, Marina; Gradoni, Luigi; Maroli, Michele

    2014-12-09

    Pioneering research on "Mediterranean Kala-Azar" carried out by Adler and Theodor early in the past century (~1930s) had identified Catania city (Sicily) as a major focus of the disease nowadays known as zoonotic visceral leishmaniasis (VL). Despite the fact that disease in both humans and dogs has continued to be highly prevalent in the Catania province up to the present times, research on Leishmania vectors in this urban focus dates back to that distant period. This study aimed to evaluate the persistence and current composition of the sand fly fauna in urban environments of Catania in recent years, 2006 and 2013. In 2006 fifty-one suitable collecting sites were identified within 44 sub-units of a grid drawn to include the urban Catania area. In 2013 the survey was restricted to four of the most productive and representative sites resulting from the 2006 survey. In both periods 3 collections per month were performed using standard sticky traps set for 3 days in wall holes/cavities along public roads, from the end of April through December. 43/51 sites (84.3%) were found positive for sand flies. The 2006 collections accounted for a total of 4341 specimens including six species. Among competent Leishmania vector species, P. perniciosus was the most prevalent (36.5%) being identified in all sand fly-positive sites, with significant abundance in those of the old city centre. Other species of interest were P. sergenti (2.5%) and P. neglectus (1.5%). The 2013 survey produced 1130 sand flies, of which 39.5% were P. perniciosus, 1.6% P. sergenti and 0.7% P. neglectus. A search for Leishmania DNA in a small sample of 72 P. perniciosus females revealed 11% infection prevalence. Our findings from an old urban focus of leishmaniasis demonstrate that phlebotomine sand flies have adapted fairly well to the drastic environmental changes that have occurred in cities of the Western world in the past century and still represent a potential risk for Leishmania transmission.

  6. Synthesis of nucleoside and nucleotide conjugates of bile acids, and polymerase construction of bile acid-functionalized DNA

    Czech Academy of Sciences Publication Activity Database

    Ikonen, Satu; Macíčková-Cahová, Hana; Pohl, Radek; Šanda, Miloslav; Hocek, Michal

    2010-01-01

    Roč. 8, č. 5 (2010), s. 1194-1201 ISSN 1477-0520 R&D Projects: GA MŠk LC512; GA ČR GA203/09/0317 Institutional research plan: CEZ:AV0Z40550506 Keywords : steroids * nucleosides * nucleoside triphosphates * DNA polymerase Subject RIV: CC - Organic Chemistry Impact factor: 3.451, year: 2010

  7. Structural and Enzymatic Characterization of a Nucleoside Diphosphate Sugar Hydrolase from Bdellovibrio bacteriovorus.

    Directory of Open Access Journals (Sweden)

    Andres H de la Peña

    Full Text Available Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 -- a Nudix hydrolase from Bdellovibrio bacteriovorus-that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively. Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-β-α NDPSase fold differentiates NDPSases from ADPRases.

  8. Highly regioselective synthesis of undecylenic acid esters of purine nucleosides catalyzed by Candida antarctica lipase B.

    Science.gov (United States)

    Gao, Wen-Li; Li, Ning; Zong, Min-Hua

    2011-11-01

    Regioselective undecylenoylation of purine nucleosides as potential dual prodrugs was achieved by Candida antarctica lipase B using adenosine as a model reactant. The optimum organic solvent, molar ratio of vinyl ester to nucleoside, enzyme dosage, reaction temperature and molecular sieve amount were anhydrous THF, 5:1, 20 U/ml, 45°C and 75 mg/ml, respectively. Under the optimum conditions, the initial reaction rate, yield and 5'-regioselectivity were 1.1 mM/h, 90% and >99%, respectively. The enzymatic acylation of various nucleosides furnished the desired 5'-ester derivatives with the yields of 60-95% and 5'-regioselectivities of >99%. In addition, the lipase displayed excellent operational stability in THF, and retained 96% of its initial activity after reused for five batches.

  9. Structure of purine nucleoside phosphorylase (DeoD) from Bacillus anthracis

    International Nuclear Information System (INIS)

    Grenha, Rosa; Levdikov, Vladimir M.; Fogg, Mark J.; Blagova, Elena V.; Brannigan, James A.; Wilkinson, Anthony J.; Wilson, Keith S.

    2005-01-01

    The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis was solved by X-ray crystallography using molecular replacement and refined at a resolution of 2.24 Å. Protein structures from the causative agent of anthrax (Bacillus anthracis) are being determined as part of a structural genomics programme. Amongst initial candidates for crystallographic analysis are enzymes involved in nucleotide biosynthesis, since these are recognized as potential targets in antibacterial therapy. Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway. The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis has been solved by molecular replacement at 2.24 Å resolution and refined to an R factor of 18.4%. This is the first report of a DeoD structure from a Gram-positive bacterium

  10. Further support for a palaearctic origin of Leishmania

    Directory of Open Access Journals (Sweden)

    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.

  11. CD8+ T cells in Leishmania infections: friends or foes?

    Directory of Open Access Journals (Sweden)

    Simona eStager

    2012-01-01

    Full Text Available Host protection against several intracellular pathogens requires the induction of CD8+ T cell responses. CD8+ T cells are potent effector cells that can produce high amounts of pro-inflammatory cytokines and kill infected target cells efficiently. However, a protective role for CD8+ T cells during Leishmania infections is still controversial and largely depends on the infection model. In this review, we discuss the role of CD8+ T cells during various types Leishmania infections, following vaccination, and as potential immunotherapeutic targets.

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

    Science.gov (United States)

    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 exa