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 ...

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

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

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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.

Studies on Stibanate unresponsive isolates of Leishmania donovani

Indian Academy of Sciences (India)

Unknown

503. Keywords. Drug resistance; Leishmania donovani; sodium stibogluconate; visceral leishmaniasis ... cases to less than 5% in treated ones (Zuckerman and. Lainson 1977). Later on .... Effect of Stibanate on in vitro growth of Ag83 promastigotes. (A) Virulent .... In rural areas where health care facilities are not adequate ...

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

Czech Academy of Sciences Publication Activity Database

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

2015-01-01

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

Natural infection of the opossum Didelphis albiventris (Marsupialia, Didelphidae with Leishmania donovani, in Brazil

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Ítalo A. Sherlock

1984-12-01

Full Text Available An opossum, Didelphis albiventris, from Jacobina, bahia State, was found naturally infected with Leishmania donovani, being the first non-canid wild mammal to be detected with agent of kala-azar in the New World.Um gambá, Didelphis albiventris, de Jacobina, Bahia, foi encontrado com infecção natural pela Leishmania donovani, sendo o primeiro mamífero silvestre não-canídeo a ser achado com o agente do calazar nas Américas.

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

NARCIS (Netherlands)

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

2017-01-01

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

The lignan niranthin poisons Leishmania donovani topoisomerase IB and favours a Th1 immune response in mice

Science.gov (United States)

Chowdhury, Sayan; Mukherjee, Tulika; Mukhopadhyay, Rupkatha; Mukherjee, Budhaditya; Sengupta, Souvik; Chattopadhyay, Sharmila; Jaisankar, Parasuraman; Roy, Syamal; Majumder, Hemanta K

2012-01-01

Niranthin, a lignan isolated from the aerial parts of the plant Phyllanthus amarus, exhibits a wide spectrum of pharmacological activities. In the present study, we have shown for the first time that niranthin is a potent anti-leishmanial agent. The compound induces topoisomerase I-mediated DNA–protein adduct formation inside Leishmania cells and triggers apoptosis by activation of cellular nucleases. We also show that niranthin inhibits the relaxation activity of heterodimeric type IB topoisomerase of L. donovani and acts as a non-competitive inhibitor interacting with both subunits of the enzyme. Niranthin interacts with DNA–protein binary complexes and thus stabilizes the ‘cleavable complex’ formation and subsequently inhibits the religation of cleaved strand. The compound inhibits the proliferation of Leishmania amastigotes in infected cultured murine macrophages with limited cytotoxicity to the host cells and is effective against antimony-resistant Leishmania parasites by modulating upregulated P-glycoprotein on host macrophages. Importantly, besides its in vitro efficacy, niranthin treatment leads to a switch from a Th2- to a Th1-type immune response in infected BALB/c mice. The immune response causes production of nitric oxide, which results in almost complete clearance of the liver and splenic parasite burden after intraperitoneal or intramuscular administration of the drug. These findings can be exploited to develop niranthin as a new drug candidate against drug-resistant leishmaniasis. PMID:23027614

Kinetoplastid membrane protein-11 is present in promastigotes and amastigotes of Leishmania amazonensis and its surface expression increases during metacyclogenesis

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Denise CS Matos

2010-05-01

Full Text Available Kinetoplastid membrane protein-11 (KMP-11, a protein present in all kinetoplastid protozoa, is considered a potential candidate for a leishmaniasis vaccine. A suitable leishmaniasis vaccine candidate molecule must be expressed in amastigotes, the infective stage for mammals. However, the expression of KMP-11 in Leishmania amastigotes has been a subject of controversy. We evaluated the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, of Leishmania amazonensis by immunoblotting, flow cytometry and immunocytochemistry, using a monoclonal antibody against KMP-11. We found that KMP-11 is present in promastigotes and amastigotes. In both stages, the protein was found in association with membrane structures (at the cell surface, flagellar pocket and intracellular vesicles. More importantly, its surface expression is higher in amastigotes than in promastigotes and increases during metacyclogenesis. The increased expression of KMP-11 in metacyclic promastigotes, and especially in amastigotes, indicates a role for this molecule in the parasite relationship with the mammalian host. The presence of this molecule in amastigotes is consistent with the previously demonstrated immunoprotective capacity of vaccine prototypes based on the KMP-11-coding gene and the presence of humoral and cellular immune responses to KMP-11 in Leishmania-infected humans and animals.

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

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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

Interferon-¿ and interleukin-4 in human Leishmania donovani infections

DEFF Research Database (Denmark)

Kemp, M; Kurtzhals, J A; Kharazmi, A

1993-01-01

Clinical and immunological similarities between Leishmania donovani infections in humans and L. major infections in mice suggest that some of the pathophysiological mechanisms are the same in the two conditions. Both infections can result either in a fatal systemic disease or in a self-limiting i......Clinical and immunological similarities between Leishmania donovani infections in humans and L. major infections in mice suggest that some of the pathophysiological mechanisms are the same in the two conditions. Both infections can result either in a fatal systemic disease or in a self......-limiting infection with few and mild symptoms. In the murine model the outcome of the infection is critically related to the cytokines produced by T lymphocytes activated by leishmanial antigens. Activation of the IFN-gamma producing Th1 subset of CD4 positive T cells results in cure and survival, whereas activation...... of the IL-4 secreting Th2 subset results in a progressive disease with fatal outcome. A similar Th1/Th2 dichotomy in the cytokine response to L. donovani may exist in humans, and may have influence on the outcome of infection. In murine leishmaniasis the levels of IL-4 and IFN-gamma at the time of infection...

Insulin-Like Growth Factor-I Induces Arginase Activity in Leishmania amazonensis Amastigote-Infected Macrophages through a Cytokine-Independent Mechanism

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Celia Maria Vieira Vendrame

2014-01-01

Full Text Available Leishmania (Leishmania amazonensis exhibits peculiarities in its interactions with hosts. Because amastigotes are the primary form associated with the progression of infection, we studied the effect of insulin-like growth factor (IGF-I on interactions between L. (L. amazonensis amastigotes and macrophages. Upon stimulation of infected macrophages with IGF-I, we observed decreased nitric oxide production but increased arginase expression and activity, which lead to increased parasitism. However, stimulation of amastigote-infected macrophages with IGF-I did not result in altered cytokine levels compared to unstimulated controls. Because IGF-I is present in tissue fluids and also within macrophages, we examined the possible effect of this factor on phosphatidylserine (PS exposure on amastigotes, seen previously in tissue-derived amastigotes leading to increased parasitism. Stimulation with IGF-I induced PS exposure on amastigotes but not on promastigotes. Using a PS-liposome instead of amastigotes, we observed that the PS-liposome but not the control phosphatidylcholine-liposome led to increased arginase activity in macrophages, and this process was not blocked by anti-TGF-β antibodies. Our results suggest that in L. (L. amazonensis amastigote-infected macrophages, IGF-I induces arginase activity directly in amastigotes and in macrophages through the induction of PS exposure on amastigotes in the latter, which could lead to the alternative activation of macrophages through cytokine-independent mechanisms.

Epidemiology of Leishmania donovani infection in high-transmission foci in Nepal

DEFF Research Database (Denmark)

Rijal, Suman; Uranw, Surendra; Chappuis, François

2010-01-01

OBJECTIVE: Nepal reports a visceral leishmaniasis (VL) incidence of 5 per 10 000 per year on the basis of notification by health facilities, but little community-based epidemiological information exists. We report data on prevalence rates of Leishmania donovani infection in ten communities in East...

IgG and IgG subclasses antibody responses to rK39 in Leishmania donovani infections

International Nuclear Information System (INIS)

Daifalla, N.S.; El Hassan, A.M.

1998-01-01

Leishmania donovani infection cause a wide spectrum of human diseases ranging from self-healing subclinical infections to severe visceral leishmaniasis, post kal-azar dermal leishmaiasis, and mucosal leishmaiasis. The infection associated with high levels of anti-leishmania antibodies which offer a potential parameter for the serological diagnosis of L. donovani infection replacing the invasive parasitological methods. rK39, a cloned antigen of L. chagasis was reported to have high levels of anti-leishmania antibodies in Sudanese and American visceral leishmaniasis patients. In an assessment of rK39-ELISA in detecting L. donovani infection we found that the antigen detected visceral leishmaniasis, post kala-azar dermal leishmaniasis, and mucosal leismaniasis with the sensitives of 96.6%, 95.91% and 90.91% respectively. The test has the specificity of 96.7%. Further investigation of 25 visceral leishmaniasis patients showed elevated anti-rK39 antibody responses of IgG subclasses with IgG1 and IgG3 significantly higher than IgG4. igG3 showed the highest sensitivity (84.00%) whereas IgG1 showed the highest sensitivity (100%). The dynamics of the serological reactivity to rK39 in l.donovani infections will be discussed in relation to exposure, infection, cure and relapse.(Author)

Genetic polymorphism within the Leishmania donovani complex: correlation with geographic origin

Czech Academy of Sciences Publication Activity Database

Zemanová, Eva; Jirků, Milan; Mauricio, I. L.; Miles, M. A.; Lukeš, Julius

2004-01-01

Roč. 70, č. 6 (2004), s. 613-617 ISSN 0002-9637 Grant - others:European Community(XE) QLK2-CT-2001-01810 Institutional research plan: CEZ:AV0Z6022909 Keywords : genetic polymorphism * Leishmania donovani * RAPD Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.013, year: 2004

Evidence for Seroprevalence in Human Localized Cutaneous Leishmaniasis Caused by Leishmania donovani in Sri Lanka

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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.

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

DEFF Research Database (Denmark)

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

1994-01-01

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

The epidemiology of Leishmania donovani infection in high transmission foci in India

DEFF Research Database (Denmark)

Singh, Shri P; Picado, Albert; Boelaert, Marleen

2010-01-01

OBJECTIVE: Visceral Leishmaniasis (VL) is highly prevalent in Bihar, India. India and its neighbours aim at eliminating VL, but several knowledge gaps in the epidemiology of VL may hamper that effort. The prevalence of asymptomatic infections with Leishmania donovani and their role in transmission...

Reprogramming neutral lipid metabolism in mouse dendritic leucocytes hosting live Leishmania amazonensis amastigotes.

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Hervé Lecoeur

Full Text Available BACKGROUND: After loading with live Leishmania (L amazonensis amastigotes, mouse myeloid dendritic leucocytes/DLs are known to undergo reprogramming of their immune functions. In the study reported here, we investigated whether the presence of live L. amazonensis amastigotes in mouse bone marrow-derived DLs is able to trigger re-programming of DL lipid, and particularly neutral lipid metabolism. METHODOLOGY/PRINCIPAL FINDINGS: Affymetrix-based transcriptional profiles were determined in C57BL/6 and DBA/2 mouse bone marrow-derived DLs that had been sorted from cultures exposed or not to live L. amazonensis amastigotes. This showed that live amastigote-hosting DLs exhibited a coordinated increase in: (i long-chain fatty acids (LCFA and cholesterol uptake/transport, (ii LCFA and cholesterol (re-esterification to triacyl-sn-glycerol (TAG and cholesteryl esters (CE, respectively. As these neutral lipids are known to make up the lipid body (LB core, oleic acid was added to DL cultures and LB accumulation was compared in live amastigote-hosting versus amastigote-free DLs by epi-fluorescence and transmission electron microscopy. This showed that LBs were both significantly larger and more numerous in live amastigote-hosting mouse dendritic leucocytes. Moreover, many of the larger LB showed intimate contact with the membrane of the parasitophorous vacuoles hosting the live L. amazonensis amastigotes. CONCLUSIONS/SIGNIFICANCE: As leucocyte LBs are known to be more than simple neutral lipid repositories, we set about addressing two related questions. Could LBs provide lipids to live amastigotes hosted within the DL parasitophorous vacuole and also deliver? Could LBs impact either directly or indirectly on the persistence of L. amazonensis amastigotes in rodent skin?

Identification of a differentially expressed mRNA in axenic Leishmania panamensis amastigotes

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José Arturo Gutiérrez

2001-08-01

Full Text Available Differential display technique was applied in order to identify transcripts which are present in axenic amastigotes but not in promastigotes of the Leishmania panamensis parasites. One of them was cloned and the sequence reveals an open reading frame of 364 amino acids (aprox. 40 kDa. The deduced protein is homologous to the serine/threonine protein kinases and specially to the mitogen activates protein kinases from eukaryotic species. Southern blot analysis suggest that this transcript, named lpmkh, is present in the genome of the parasite as a single copy gene. These results could imply that lpmkh could be involved in the differentiation process or the preservation of amastigotes in axenic conditions.

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

International Nuclear Information System (INIS)

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

Leishmania donovani: an in vitro study of antimony-resistant amphotericin B-sensitive isolates

DEFF Research Database (Denmark)

Sharief, Abdalla Hassan; Gasim Khalil, Eltahir Awad; Theander, Thor G

2006-01-01

Drug sensitivity of clinically antimony-unresponsive Leishmania donovani isolates from Eastern Sudan was evaluated in an in vitro culture system against sodium stibogluconate (Pentostam) and Amphotericin B. Eight isolates, six from antimony-resistant and two from clinically responsive patients were...

Identification of Leishmania donovani Topoisomerase 1 inhibitors via intuitive scaffold hopping and bioisosteric modification of known Top 1 inhibitors

Science.gov (United States)

Mamidala, Rajinikanth; Majumdar, Papiya; Jha, Kunal Kumar; Bathula, Chandramohan; Agarwal, Rahul; Chary, M. Thirumala; Mazumdar, H. K.; Munshi, Parthapratim; Sen, Subhabrata

2016-05-01

A library of arylidenefuropyridinediones was discovered as potent inhibitors of Leishmania donovani Topoisomerase 1 (LdTop1) where the active molecules displayed considerable inhibition with single digit micromolar EC50 values. This molecular library was designed via intuitive scaffold hopping and bioisosteric modification of known topoisomerase 1 inhibitors such as camptothecin, edotecarin and etc. The design was rationalized by molecular docking analysis of the compound prototype with human topoisomerase 1 (HTop1) and Leishmania donovani topoisomerase 1(LdTop1). The most active compound 4 displayed no cytotoxicity against normal mammalian COS7 cell line (~100 fold less inhibition at the EC50). Similar to camptothecin, 4 interacted with free LdTop1 as observed in the preincubation DNA relaxation inhibition experiment. It also displayed anti-protozoal activity against Leishmania donovani promastigote. Crystal structure investigation of 4 and its molecular modelling with LdTop1 revealed putative binding sites in the enzyme that could be harnessed to generate molecules with better potency.

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

Science.gov (United States)

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

2015-01-01

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

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

Science.gov (United States)

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

2009-08-01

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

2-Alkynoic fatty acids inhibit topoisomerase IB from Leishmania donovani.

Science.gov (United States)

Carballeira, Néstor M; Cartagena, Michelle; Sanabria, David; Tasdemir, Deniz; Prada, Christopher F; Reguera, Rosa M; Balaña-Fouce, Rafael

2012-10-01

2-Alkynoic fatty acids display antimycobacterial, antifungal, and pesticidal activities but their antiprotozoal activity has received little attention. In this work we synthesized the 2-octadecynoic acid (2-ODA), 2-hexadecynoic acid (2-HDA), and 2-tetradecynoic acid (2-TDA) and show that 2-ODA is the best inhibitor of the Leishmania donovani DNA topoisomerase IB enzyme (LdTopIB) with an EC(50)=5.3±0.7μM. The potency of LdTopIB inhibition follows the trend 2-ODA>2-HDA>2-TDA, indicating that the effectiveness of inhibition depends on the fatty acid carbon chain length. All of the studied 2-alkynoic fatty acids were less potent inhibitors of the human topoisomerase IB enzyme (hTopIB) as compared to LdTopIB. 2-ODA also displayed in vitro activity against Leishmania donovani (IC(50)=11.0μM), but it was less effective against other protozoa, Trypanosoma cruzi (IC(50)=48.1μM) and Trypanosoma brucei rhodesiense (IC(50)=64.5μM). The antiprotozoal activity of the 2-alkynoic fatty acids, in general, followed the trend 2-ODA>2-HDA>2-TDA. The experimental information gathered so far indicates that 2-ODA is a promising antileishmanial compound. Copyright © 2012 Elsevier Ltd. All rights reserved.

Transcriptional signatures of BALB/c mouse macrophages housing multiplying Leishmania amazonensis amastigotes

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Lang Thierry

2009-03-01

Full Text Available Abstract Background Mammal macrophages (MΦ display a wide range of functions which contribute to surveying and maintaining tissue integrity. One such function is phagocytosis, a process known to be subverted by parasites like Leishmania (L. Indeed, the intracellular development of L. amazonensis amastigote relies on the biogenesis and dynamic remodelling of a phagolysosome, termed the parasitophorous vacuole, primarily within dermal MΦ. Results Using BALB/c mouse bone marrow-derived MΦ loaded or not with amastigotes, we analyzed the transcriptional signatures of MΦ 24 h later, when the amastigote population was growing. Total RNA from MΦ cultures were processed and hybridized onto Affymetrix Mouse430_2 GeneChips®, and some transcripts were also analyzed by Real-Time quantitative PCR (RTQPCR. A total of 1,248 probe-sets showed significant differential expression. Comparable fold-change values were obtained between the Affymetrix technology and the RTQPCR method. Ingenuity Pathway Analysis software® pinpointed the up-regulation of the sterol biosynthesis pathway (p-value = 1.31e-02 involving several genes (1.95 to 4.30 fold change values, and the modulation of various genes involved in polyamine synthesis and in pro/counter-inflammatory signalling. Conclusion Our findings suggest that the amastigote growth relies on early coordinated gene expression of the MΦ lipid and polyamine pathways. Moreover, these MΦ hosting multiplying L. amazonensis amastigotes display a transcriptional profile biased towards parasite-and host tissue-protective processes.

Latent infection with Leishmania donovani in highly endemic villages in Bihar, India.

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Epco Hasker

Full Text Available Asymptomatic persons infected with the parasites causing visceral leishmaniasis (VL usually outnumber clinically apparent cases by a ratio of 4-10 to 1. We describe patterns of markers of Leishmania donovani infection and clinical VL in relation to age in Bihar, India.We selected eleven villages highly endemic for Leishmania donovani. During a 1-year interval we conducted two house to house surveys during which we collected blood samples on filter paper from all consenting individuals aged 2 years and above. Samples were tested for anti-leishmania serology by Direct Agglutination Test (DAT and rK39 ELISA. Data collected during the surveys included information on episodes of clinical VL among study participants.We enrolled 13,163 persons; 6.2% were reactive to DAT and 5.9% to rK39. Agreement between the tests was weak (kappa = 0.30. Among those who were negative on both tests at baseline, 3.6% had converted to sero-positive on either of the two tests one year later. Proportions of sero-positives and sero-converters increased steadily with age. Clinical VL occurred mainly among children and young adults (median age 19 years.Although infection with L. donovani is assumed to be permanent, serological markers revert to negative. Most VL cases occur at younger ages, yet we observed a steady increase with age in the frequency of sero-positivity and sero-conversion. Our findings can be explained by a boosting effect upon repeated exposure to the parasite or by intermittent release of parasites in infected subjects from safe target cells. A certain proportion of sero-negative subjects could have been infected but below the threshold of antibody abundance for our serologic testing.

Comparison of Bloodmeal Digestion and the Peritrophic Matrix in Four Sand Fly Species Differing in Susceptibility to Leishmania donovani.

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Pruzinova, Katerina; Sadlova, Jovana; Seblova, Veronika; Homola, Miroslav; Votypka, Jan; Volf, Petr

2015-01-01

The early stage of Leishmania development in sand flies is closely connected with bloodmeal digestion. Here we compared various parameters of bloodmeal digestion in sand flies that are either susceptible (Phlebotomus argentipes and P. orientalis) or refractory (P. papatasi and Sergentomyia schwetzi) to Leishmania donovani, to study the effects on vector competence. The volume of the bloodmeal ingested, time of defecation of bloodmeal remnants, timing of formation and degradation of the peritrophic matrix (PM) and dynamics of proteolytic activities were compared in four sand fly species. Both proven vectors of L. donovani showed lower trypsin activity and slower PM formation than refractory species. Interestingly, the two natural L. donovani vectors strikingly differed from each other in secretion of the PM and midgut proteases, with P. argentipes possessing fast bloodmeal digestion with a very high peak of chymotrypsin activity and rapid degradation of the PM. Experimental infections of P. argentipes did not reveal any differences in vector competence in comparison with previously studied P. orientalis; even the very low initial dose (2×103 promastigotes/ml) led to fully developed late-stage infections with colonization of the stomodeal valve in about 40% of females. We hypothesise that the period between the breakdown of the PM and defecation of the bloodmeal remnants, i.e. the time frame when Leishmania attach to the midgut in order to prevent defecation, could be one of crucial parameters responsible for the establishment of Leishmania in the sand fly midgut. In both natural L. donovani vectors this period was significantly longer than in S. schwetzi. Both vectors are equally susceptible to L. donovani; as average bloodmeal volumes taken by females of P. argentipes and P. orientalis were 0.63 μl and 0.59 μl, respectively, an infective dose corresponding to 1-2 parasites was enough to initiate mature infections.

rK39 enzyme-linked immunosorbent assay for diagnosis of Leishmania donovani infection

NARCIS (Netherlands)

Zijlstra, E. E.; Daifalla, N. S.; Kager, P. A.; Khalil, E. A.; El-Hassan, A. M.; Reed, S. G.; Ghalib, H. W.

1998-01-01

The rK39 enzyme-linked immunosorbent assay (ELISA) was compared with the direct agglutination test (DAT) for Leishmania donovani infection in the Sudan. rK39 ELISA proved more sensitive than DAT in diagnosis of kala-azar (93 and 80%, respectively); both tests may remain positive up to 24 months

Serodiagnosis of Leishmania donovani infections: assessment of enzyme-linked immunosorbent assays using recombinant L. donovani gene B protein (GBP) and a peptide sequence of L. donovani GBP

DEFF Research Database (Denmark)

Jensen, A T; Gasim, S; Moller, T

1999-01-01

The repetitive sequence of Leishmania major gene B protein (GBP) has previously been shown to be a useful tool in the diagnosis of cutaneous leishmaniasis (CL). Here, we have assessed enzyme-linked immunosorbent assays (ELISAs) using recombinant L. donovani GBP (rGBP) and a peptide sequence of L...... for malaria but free of leishmaniasis was negative in both assays....

Humoral and cellular immune responses to glucose regulated protein 78 - a novel Leishmania donovani antigen

DEFF Research Database (Denmark)

Jensen, Anja T R; Ismail, Ahmed; Gaafar, Ameera

2002-01-01

The recently cloned glucose regulated protein 78 (GRP78) of Leishmania donovani has been suggested as a new and promising Leishmania vaccine candidate. We assessed antibody and T-cell reactivity to GRP78 in an enzyme-linked immunosorbent assay (ELISA) and in lymphoproliferative assays. Serological...... with a positive leishmanin skin test showed antibody reactivity to recombinant GRP78 (rGRP78). In lymphoproliferative assays, 9 of 13 isolates of peripheral blood mononuclear cells (PBMC) from individuals previously infected with L. donovani and one of three individuals previously infected with L. major showed...... in an area endemic for malaria but free of leishmaniasis and plasma from healthy Danes was negative in the assay. GRP78 antibody was detected in 10% and 5% of plasma samples from Sudanese and Ghanaian malaria patients, respectively, whereas 35% of plasma samples from otherwise healthy Sudanese individuals...

Technetium-99m labeling anti-amastigote polyclonal antibodies of Leishmania amazonensis

International Nuclear Information System (INIS)

Araujo, J.G.V.C.; Toledo, V.P.C.P.; Guimaraes, T.M.P.D.; Bernardo-Filho, M.; Simal, C.J.R.; Mota, L.G.; Diniz, S.O.F.; Cardoso, V.N.

2002-01-01

Anti-amastigote polyclonal antibody (IgG) was incubated with solutions of stannous chloride and sodium borohidride. After that, 3.7 MBq of technetium-99m ( 99m Tc) was added. A labeling yield of the antibody about 84% was obtained. After filtration of 99m Tc-IgG, the radiochemical purity increased from 84 to 95%. The labeling of IgG with 99m Tc did not modify the immunoreactivity of the antibody, since it was able to identify in vitro and in vivo the specific antigen of Leishmania amazonensis

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

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

International Nuclear Information System (INIS)

Singh, Sushma; Jhingran, Anupam; Sharma, Ankur; Simonian, Alina R.; Soininen, Pasi; Vepsalainen, Jouko; Khomutov, Alex R.; Madhubala, Rentala

2008-01-01

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

Leishmania donovani argininosuccinate synthase is an active enzyme associated with parasite pathogenesis.

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Ines Lakhal-Naouar

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

Genomic and transcriptomic alterations in Leishmania donovani lines experimentally resistant to antileishmanial drugs.

Science.gov (United States)

Rastrojo, Alberto; García-Hernández, Raquel; Vargas, Paola; Camacho, Esther; Corvo, Laura; Imamura, Hideo; Dujardin, Jean-Claude; Castanys, Santiago; Aguado, Begoña; Gamarro, Francisco; Requena, Jose M

2018-04-13

Leishmaniasis is a serious medical issue in many countries around the World, but it remains largely neglected in terms of research investment for developing new control and treatment measures. No vaccines exist for human use, and the chemotherapeutic agents currently used are scanty. Furthermore, for some drugs, resistance and treatment failure are increasing to alarming levels. The aim of this work was to identify genomic and trancriptomic alterations associated with experimental resistance against the common drugs used against VL: trivalent antimony (Sb III , S line), amphotericin B (AmB, A line), miltefosine (MIL, M line) and paromomycin (PMM, P line). A total of 1006 differentially expressed transcripts were identified in the S line, 379 in the A line, 146 in the M line, and 129 in the P line. Also, changes in ploidy of chromosomes and amplification/deletion of particular regions were observed in the resistant lines regarding the parental one. A series of genes were identified as possible drivers of the resistance phenotype and were validated in both promastigotes and amastigotes from Leishmania donovani, Leishmania infantum and Leishmania major species. Remarkably, a deletion of the gene LinJ.36.2510 (coding for 24-sterol methyltransferase, SMT) was found to be associated with AmB-resistance in the A line. In the P line, a dramatic overexpression of the transcripts LinJ.27.T1940 and LinJ.27.T1950 that results from a massive amplification of the collinear genes was suggested as one of the mechanisms of PMM resistance. This conclusion was reinforced after transfection experiments in which significant PMM-resistance was generated in WT parasites over-expressing either gene LinJ.27.1940 (coding for a D-lactate dehydrogenase-like protein, D-LDH) or gene LinJ.27.1950 (coding for an aminotransferase of branched-chain amino acids, BCAT). This work allowed to identify new drivers, like SMT, the deletion of which being associated with resistance to AmB, and the tandem D

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

Background: Protozoan parasites, such as Leishmania, still pose an enormous public health problem in many countries throughout the world. Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies. Several innovative approaches are under...... 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...... 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...

Increased Transmissibility of Leishmania donovani From the Mammalian Host to Vector Sand Flies After Multiple Exposures to Sand Fly Bites.

Science.gov (United States)

Valverde, Joanna G; Paun, Andrea; Inbar, Ehud; Romano, Audrey; Lewis, Michael; Ghosh, Kashinath; Sacks, David

2017-04-15

Patients with active visceral leishmaniasis are important reservoirs in the anthroponotic transmission cycle of Leishmania donovani. The role of the blood or skin as a source of infection to sand flies remains unclear, and the possible effect of multiple exposures to fly bites on transmissibility has not been addressed. L. donovani-infected hamsters underwent xenodiagnoses with Lutzomyia longipalpis on the same or different sites on the abdomen on 2 consecutive days or by artificial feeding on the skin or blood. The transmission of L. donovani from sick hamsters to flies was surprisingly low (mean, 24% of fed flies). New flies fed on the same site acquired significantly more infections (mean, 61%; P Leishmania donovani. Using the hamster model of visceral disease, we demonstrate that prior exposure to bites of uninfected sand flies potentiates their ability to transmit infection to the vector. Published by Oxford University Press for the Infectious Diseases Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.

l-Arginine Uptake by Cationic Amino Acid Transporter Promotes Intra-Macrophage Survival of Leishmania donovani by Enhancing Arginase-Mediated Polyamine Synthesis

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Abhishek Mandal

2017-07-01

Full Text Available The survival of intracellular protozoan parasite, Leishmania donovani, the causative agent of Indian visceral leishmaniasis (VL, depends on the activation status of macrophages. l-Arginine, a semi-essential amino acid plays a crucial regulatory role for activation of macrophages. However, the role of l-arginine transport in VL still remains elusive. In this study, we demonstrated that intra-macrophage survival of L. donovani depends on the availability of extracellular l-arginine. Infection of THP-1-derived macrophage/human monocyte-derived macrophage (hMDM with Leishmania, resulted in upregulation of l-arginine transport. While investigating the involvement of the transporters, we observed that Leishmania survival was greatly impaired when the transporters were blocked either using inhibitor or siRNA-mediated downregulation. CAT-2 was found to be the main isoform associated with l-arginine transport in L. donovani-infected macrophages. l-arginine availability and its transport regulated the host arginase in Leishmania infection. Arginase and inducible nitric oxide synthase (iNOS expression were reciprocally regulated when assayed using specific inhibitors and siRNA-mediated downregulation. Interestingly, induction of iNOS expression and nitric oxide production were observed in case of inhibition of arginase in infected macrophages. Furthermore, inhibition of l-arginine transport as well as arginase resulted in decreased polyamine production, limiting parasite survival inside macrophages. l-arginine availability and transport regulated Th1/Th2 cytokine levels in case of Leishmania infection. Upregulation of l-arginine transport, induction of host arginase, and enhanced polyamine production were correlated with increased level of IL-10 and decreased level of IL-12 and TNF-α in L. donovani-infected macrophages. Our findings provide clear evidence for targeting the metabolism of l-arginine and l-arginine-metabolizing enzymes as an important

Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes.

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Rita Marcia Cardoso de Paiva

2015-12-01

Full Text Available Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several Leishmania species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of Trypanosoma cruzi and Leishmania spp. Here, we showed that the genome of L. braziliensis contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in L. braziliensis amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in L. braziliensis, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after in vitro infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole.

Point mutations in a nucleoside transporter gene from Leishmania donovani confer drug resistance and alter substrate selectivity

OpenAIRE

Vasudevan, Gayatri; Ullman, Buddy; Landfear, Scott M.

2001-01-01

Leishmania parasites lack a purine biosynthetic pathway and depend on surface nucleoside and nucleobase transporters to provide them with host purines. Leishmania donovani possess two closely related genes that encode high affinity adenosine-pyrimidine nucleoside transporters LdNT1.1 and LdNT1.2 and that transport the toxic adenosine analog tubercidin in addition to the natural substrates. In this study, we have characterized a drug-resistant clonal mutant of L. do...

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

Science.gov (United States)

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

2016-03-01

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

Role of efflux pumps and intracellular thiols in natural antimony resistant isolates of Leishmania donovani.

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Smita Rai

Full Text Available BACKGROUND: In view of the recent upsurge in the phenomenon of therapeutic failure, drug resistance in Leishmania, developed under natural field conditions, has become a great concern yet little understood. Accordingly, the study of determinants of antimony resistance is urgently warranted. Efflux transporters have been reported in Leishmania but their role in clinical resistance is still unknown. The present study was designed to elucidate the mechanism of natural antimony resistance in L. donovani field isolates by analyzing the functionality of efflux pump(s and expression profiles of known genes involved in transport and thiol based redox metabolism. METHODOLOGY/PRINCIPAL FINDINGS: We selected 7 clinical isolates (2 sensitive and 5 resistant in addition to laboratory sensitive reference and SbIII resistant mutant strains for the present study. Functional characterization using flow cytometry identified efflux pumps that transported substrates of both P-gp and MRPA and were inhibited by the calmodulin antagonist trifluoperazine. For the first time, verapamil sensitive efflux pumps for rhodamine 123 were observed in L. donovani that were differentially active in resistant isolates. RT-PCR confirmed the over-expression of MRPA in isolates with high resistance index only. Resistant isolates also exhibited consistent down regulation of AQP1 and elevated intracellular thiol levels which were accompanied with increased expression of ODC and TR genes. Interestingly, γ-GCS is not implicated in clinical resistance in L. donovani isolates. CONCLUSIONS/SIGNIFICANCE: Here we demonstrate for the first time, the role of P-gp type plasma membrane efflux transporter(s in antimony resistance in L. donovani field isolates. Further, decreased levels of AQP1 and elevated thiols levels have emerged as biomarkers for clinical resistance.

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

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Sreenivas Gannavaram

2011-01-01

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

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.

Vaccine development against Leishmania donovani

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Amrita eDas

2012-05-01

Full Text Available Visceral leishmaniasis (VL caused by Leishmania donovani and Leishmania infantum/ chagasi represents the second most challenging infectious disease worldwide, affecting nearly 500,000 people and 60,000 deaths annually. Zoonotic VL (ZVL caused by L. infantum is re-emergent canid zoonoses which represents a complex epidemiological cycle in New world where domestic dogs serve as reservoir host responsible for potentially fatal human infection where dog culling is the only control measure for eliminating reservoir host. Lifelong immunity in human against reinfection has motivated several attempts in developing prophylactic vaccines against the disease but very few have progressed beyond experimental stage. Absence of any licensed vaccine along with high toxicity and increasing resistance to the current chemotherapeutic drugs has further complicated the situation in endemic regions of the world. Advances in vaccinology, including recombinant proteins, novel antigen-delivery systems/adjuvants, heterologous prime-boost regimens and strategies for intracellular antigen presentation, have contributed to recent advances in vaccine development against VL. Attempts to develop an effective vaccine for use in domestic dogs in areas of canine VL should be pursued for preventing human infection. Studies in animal models and human patients have revealed the pathogenic mechanisms of disease progression and features of protective immunity. This review will summarize the accumulated knowledge about pathogenesis, immune response and prerequisites for protective immunity against human VL. Authors will discuss promising vaccine targets, their developmental status and future prospects in a quest for rational vaccine development against VL. In addition, several challenges such as safety issues, a renewed and coordinated commitment to basic research, preclinical studies and trial design will be addressed to overcome the problems faced in developing effective vaccines

Genetic Manipulation of Leishmania donovani to Explore the Involvement of Argininosuccinate Synthase in Oxidative Stress Management

Science.gov (United States)

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

2016-01-01

Reactive oxygen and nitrogen species (ROS and RNS) produced by the phagocytic cells are the most common arsenals used to kill the intracellular pathogens. However, Leishmania, an intracellular pathogen, has evolved mechanisms to survive by counterbalancing the toxic oxygen metabolites produced during infection. Polyamines, the major contributor in this anti-oxidant machinery, are largely dependent on the availability of L-arginine in the intracellular milieu. Argininosuccinate synthase (ASS) plays an important role as the rate-limiting step required for converting L-citrulline to argininosuccinate to provide arginine for an assortment of metabolic processes. Leishmania produce an active ASS enzyme, yet it has an incomplete urea cycle as it lacks an argininosuccinate lyase (ASL). There is no evidence for endogenous synthesis of L-arginine in Leishmania, which suggests that these parasites salvage L-arginine from extracellular milieu and makes the biological function of ASS and the production of argininosuccinate in Leishmania unclear. Our previous quantitative proteomic analysis of Leishmania promastigotes treated with sub-lethal doses of ROS, RNS, or a combination of both, led to the identification of several differentially expressed proteins which included ASS. To assess the involvement of ASS in stress management, a mutant cell line with greatly reduced ASS activity was created by a double-targeted gene replacement strategy in L. donovani promastigote. Interestingly, LdASS is encoded by three copies of allele, but Western blot analysis showed the third allele did not appear to express ASS. The free thiol levels in the mutant LdASS-/-/+ cell line were decreased. Furthermore, the cell viability in L-arginine depleted medium was greatly attenuated on exposure to different stress environments and was adversely impacted in its ability to infect mice. These findings suggest that ASS is important for Leishmania donovani to counterbalance the stressed environments

Genetic Validation of Leishmania donovani Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity

OpenAIRE

Sanya Chadha; N. Arjunreddy Mallampudi; Debendra K. Mohapatra; Rentala Madhubala; Ira J. Blader; Greg Matlashewski; Frederick Buckner

2017-01-01

ABSTRACT Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis. Increasing resistance and severe side effects of existing drugs have led to the need to identify new chemotherapeutic targets. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and are required for protein synthesis. aaRSs are known drug targets for bacterial and fungal pathogens. Here, we have characterized and evaluated the essentiality of L.?donovani lysyl-tRNA synthetase (LdLysRS). Two different codin...

In Vitro Susceptibilities of Wild and Drug Resistant Leishmania donovani Amastigote Stages to Andrographolide Nanoparticle: Role of Vitamin E Derivative TPGS for Nanoparticle Efficacy

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Mondal, Subhasish; Roy, Partha; Das, Suvadra; Halder, Asim; Mukherjee, Arup; Bera, Tanmoy

2013-01-01

Visceral leishmaniasis (VL) is a chronic protozoan infection in humans associated with significant global morbidity and mortality. There is an urgent need to develop drugs and strategy that will improve therapeutic response for effective clinical treatment of drug resistant VL. To address this need, andrographolide (AG) nanoparticles were designed with P-gp efflux inhibitor vitamin E TPGS (D-α-tocopheryl polyethyleneglycol 1000 succinate) for sensitivity against drug resistant Leishmania strains. AG loaded PLGA (50∶50) nanoparticles (AGnps) stabilized by vitamin E TPGS were prepared for delivery into macrophage cells infested with sensitive and drug resistant amastigotes of Leishmania parasites. Physico-chemical characterization of AGnps by photon correlation spectroscopy exhibited an average particle size of 179.6 nm, polydispersity index of 0.245 and zeta potential of −37.6 mV. Atomic force microscopy and transmission electron microscopy visualization revealed spherical nanoparticles with smooth surfaces. AGnps displayed sustained AG release up to 288 hours as well as minimal particle aggregation and drug loss even after three months study period. Antileishmanial activity as revealed from selectivity index in wild-type strain was found to be significant for AGnp with TPGS in about one-tenth of the dosage of the free AG and one-third of the dosage of the AGnp without TPGS. Similar observations were also found in case of in vitro generated drug resistant and field isolated resistant strains of Leishmania. Cytotoxicity of AGnp with and without TPGS was significantly less than standard antileishmanial chemotherapeutics like amphotericin B, paromomycin or sodium stibogluconate. Macrophage uptake of AGnps was almost complete within one hour as evident from fluorescent microscopy studies. Thus, based on these observations, it can be concluded that the low-selectivity of AG in in vitro generated drug resistant and field isolated resistant strains was improved in

In vitro susceptibilities of wild and drug resistant leishmania donovani amastigote stages to andrographolide nanoparticle: role of vitamin E derivative TPGS for nanoparticle efficacy.

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Subhasish Mondal

Full Text Available Visceral leishmaniasis (VL is a chronic protozoan infection in humans associated with significant global morbidity and mortality. There is an urgent need to develop drugs and strategy that will improve therapeutic response for effective clinical treatment of drug resistant VL. To address this need, andrographolide (AG nanoparticles were designed with P-gp efflux inhibitor vitamin E TPGS (D-α-tocopheryl polyethyleneglycol 1000 succinate for sensitivity against drug resistant Leishmania strains. AG loaded PLGA (50∶50 nanoparticles (AGnps stabilized by vitamin E TPGS were prepared for delivery into macrophage cells infested with sensitive and drug resistant amastigotes of Leishmania parasites. Physico-chemical characterization of AGnps by photon correlation spectroscopy exhibited an average particle size of 179.6 nm, polydispersity index of 0.245 and zeta potential of -37.6 mV. Atomic force microscopy and transmission electron microscopy visualization revealed spherical nanoparticles with smooth surfaces. AGnps displayed sustained AG release up to 288 hours as well as minimal particle aggregation and drug loss even after three months study period. Antileishmanial activity as revealed from selectivity index in wild-type strain was found to be significant for AGnp with TPGS in about one-tenth of the dosage of the free AG and one-third of the dosage of the AGnp without TPGS. Similar observations were also found in case of in vitro generated drug resistant and field isolated resistant strains of Leishmania. Cytotoxicity of AGnp with and without TPGS was significantly less than standard antileishmanial chemotherapeutics like amphotericin B, paromomycin or sodium stibogluconate. Macrophage uptake of AGnps was almost complete within one hour as evident from fluorescent microscopy studies. Thus, based on these observations, it can be concluded that the low-selectivity of AG in in vitro generated drug resistant and field isolated resistant strains was

Role of the domestic dog as a reservoir host of Leishmania donovani in eastern Sudan

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Schallig Henk DFH

2009-06-01

Full Text Available Abstract Background The study aims to determine the role of domestic dogs in transmission of visceral leishmaniasis in eastern Sudan. A cross-sectional survey was conducted in 10 villages along the River Rahad in eastern Sudan to elucidate the role of domestic dogs (Canis familiaris, Linnaeus, 1758 as a reservoir host of Leishmania donovani. In this study, 87 dogs were screened for infection by Leishmania donovani. Blood and lymph node samples were taken from 87 and 33 dogs respectively and subsequently screened by the Polymerase Chain Reaction (PCR and Direct Agglutination Test (DAT test. Additional lymph node smears were processed for microscopy and parasite culture. Host preference of the visceral leishmaniasis (VL vector in the area, Phlebotomus orientalis, and other sandflies for the Nile rat (Arvicanthis niloticus, É. Geoffrey, 1803, the genet (Genetta genetta, Linnaeus, 1758, the mongoose (Herpeistes ichneumon, Linnaeus, 1758, and the domestic dog were determined by counting numbers of sand flies attracted to CDC traps that were baited by these animals. Results DAT on blood samples detected anti-Leishmania antibodies in 6 samples (6.9%. Two out of 87 (2.3% blood samples tested were PCR positive, giving an amplification product of 560 bp. The two positive samples by PCR were also positive by DAT. However, none of the 33 lymph nodes aspirates were Leishmania positive when screened by microscopy, culture and genus-specific PCR. The dog-baited trap significantly attracted the highest number of P. orientalis and sand fly species (P Conclusion It is concluded that the results obtained from host attraction studies indicate that dog is more attractive for P. orientalis than Egyptian mongoose, common genet and Nile rat.

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.

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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.

Genetic Validation of Leishmania donovani Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity.

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Chadha, Sanya; Mallampudi, N Arjunreddy; Mohapatra, Debendra K; Madhubala, Rentala

2017-01-01

Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis. Increasing resistance and severe side effects of existing drugs have led to the need to identify new chemotherapeutic targets. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and are required for protein synthesis. aaRSs are known drug targets for bacterial and fungal pathogens. Here, we have characterized and evaluated the essentiality of L. donovani lysyl-tRNA synthetase ( Ld LysRS). Two different coding sequences for lysyl-tRNA synthetases are annotated in the Leishmania genome database. Ld LysRS-1 (LdBPK_150270.1), located on chromosome 15, is closer to apicomplexans and eukaryotes, whereas Ld LysRS-2 (LdBPK_300130.1), present on chromosome 30, is closer to bacteria. In the present study, we have characterized Ld LysRS-1. Recombinant Ld LysRS-1 displayed aminoacylation activity, and the protein localized to the cytosol. The Ld LysRS-1 heterozygous mutants had a restrictive growth phenotype and attenuated infectivity. Ld LysRS-1 appears to be an essential gene, as a chromosomal knockout of Ld LysRS-1 could be generated when the gene was provided on a rescuing plasmid. Cladosporin, a fungal secondary metabolite and a known inhibitor of LysRS, was more potent against promastigotes (50% inhibitory concentration [IC 50 ], 4.19 µM) and intracellular amastigotes (IC 50 , 1.09 µM) than were isomers of cladosporin (3-epi-isocladosporin and isocladosporin). These compounds exhibited low toxicity to mammalian cells. The specificity of inhibition of parasite growth caused by these inhibitors was further assessed using Ld LysRS-1 heterozygous mutant strains and rescue mutant promastigotes. These inhibitors inhibited the aminoacylation activity of recombinant Ld LysRS. Our data provide a framework for the development of a new class of drugs against this parasite. IMPORTANCE Aminoacyl-tRNA synthetases are housekeeping enzymes essential for protein translation, providing charged tRNAs for

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

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

2016-03-01

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

Characterization of Leishmania isolates from Nepalese patients with visceral leishmaniasis.

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Pandey, Kishor; Yanagi, Testuo; Pandey, Basu Dev; Mallik, Arun Kumar; Sherchand, Jeevan Bahadur; Kanbara, Hiroji

2007-05-01

In Nepal, visceral leishmaniasis (VL) is endemic in 13 districts of the central and eastern regions. A total of 166 bone-marrow aspirates were obtained from patients with suspected VL. Ninety-seven were identified as positive by microscopy, and 29 of those were successfully isolated and cultured. We characterized these isolates by molecular analysis and by their ability to infect mice. PCR-restriction fragment length polymorphism analysis of the mini-exon and the cysteine proteinase b gene showed that all isolates were Leishmania donovani, and the restriction pattern of the Nepalese isolates corresponded to the standard Indian strain of L. donovani but differed from that of the Kenyan strain. The single-strand conformation polymorphism analysis of ribosomal internal transcribed spacer showed no genetic heterogeneity within Nepalese isolates. Intraperitoneal inoculation with the promastigotes of all isolates resulted in amastigote proliferation in the spleen of 20 nude mice, of which ten isolates were highly infective, and ten were moderately infective, including one BALB/c mouse. Of the 20 amastigotes isolated from the spleen of nude mice, only the ten highly infective isolates infected BALB/c mice, of which, two isolates were considered to have low infectivity, three isolates were considered to be moderately infective, and five isolates were considered to be highly infective.

Comparative study of structural models of Leishmania donovani and human GDP-mannose pyrophosphorylases.

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Daligaux, Pierre; Bernadat, Guillaume; Tran, Linh; Cavé, Christian; Loiseau, Philippe M; Pomel, Sébastien; Ha-Duong, Tâp

2016-01-01

Leishmania is the parasite responsible for the neglected disease leishmaniasis. Its virulence and survival require biosynthesis of glycoconjugates, whose guanosine diphospho-d-mannose pyrophosphorylase (GDP-MP) is a key player. However, experimentally resolved structures of this enzyme are still lacking. We herein propose structural models of the GDP-MP from human and Leishmania donovani. Based on a multiple sequences alignment, the models were built with MODELLER and then carefully refined with all atom molecular dynamics simulations in explicit solvent. Their quality was evaluated against several standard criteria, including their ability to bind GDP-mannose assessed by redocking calculations. Special attention was given in this study to interactions of the catalytic site residues with the enzyme substrate and competitive inhibitors, opening the perspective of medicinal chemistry developments. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Homology modeling of Leishmania donovani enolase and its molecular interaction with novel inhibitors

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Jay Prakash Mahato

2017-01-01

Full Text Available Introduction: The treatment of Indian tropical disease such as kala-azar is likely to be troublesome to the clinicians as AmpB- and miltefosine-resistant Leishmania donovani has been reported. The rationale behind designed a novel inhibitors of model of L. donovani enolase and performing a binding study with its inhibitors to gain details of the interaction between protein residues and ligand molecules. Methods and Materials: The L. donovani enolase model consists of two typical domains. The N-terminal one contains three-stranded antiparallel β-sheets, followed by six α-helices. The C-terminal domain composes of eleven-stranded mixed α/β-barrel with connectivity. The first α-helix within the C-terminal domain, H7, and the second β-strand, S7, of the barrel domain was arranged in an antiparallel fashion compared to all other α-helices and β-strands. The root-mean-square deviation between predicted model and template is 0.4 Å. The overall conformation of L. donovani enolase model is similar to those of Trypanosoma cruzi enolase and Streptococcus pneumoniae enolase crystal structures. Result: The key amino acid residues within the docking complex model involved in the interaction between model enolase structure and ligand molecule are Lys70, Asn165, Ala168, Asp17, and Asn213. Conclusion: Our theoretical prediction may lead to the establishment of prophylactic and therapeutic approaches for the treatment of kala-azar. This biomedical informatics analysis will help us to combat future kala-azar.

Tentativa de transmissão da Leishmania donovani pela picada do Lutzomyia longipalpis entre cães

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Ítalo A. Sherlock

1972-02-01

Full Text Available Os Autores apresentam dados sôbre tentativas de transmissão experimental da Leishmania donovani pela picada de Lutzomyia longipalpis entre cães. Dois cães jovens sadios foram picados respectivamente por dois e sete flebótomos ricamente infectados e não adquiriram leishmaniose.

Gluconeogenesis in Leishmania mexicana

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Rodriguez-Contreras, Dayana; Hamilton, Nicklas

2014-01-01

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

Glycosphingolipid antigens from Leishmania (L. amazonensis amastigotes: Binding of anti-glycosphingolipid monoclonal antibodies in vitro and in vivo

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A.H. Straus

1997-03-01

Full Text Available Specific glycosphingolipid antigens of Leishmania (L. amazonensis amastigotes reactive with the monoclonal antibodies (MoAbs ST-3, ST-4 and ST-5 were isolated, and their structure was partially elucidated by negative ion fast atom bombardment mass spectrometry. The glycan moieties of five antigens presented linear sequences of hexoses and N-acetylhexosamines ranging from four to six sugar residues, and the ceramide moieties were found to be composed by a sphingosine d18:1 and fatty acids 24:1 or 16:0. Affinities of the three monoclonal antibodies to amastigote glycosphingolipid antigens were also analyzed by ELISA. MoAb ST-3 reacted equally well with all glycosphingolipid antigens tested, whereas ST-4 and ST-5 presented higher affinities to glycosphingolipids with longer carbohydrate chains, with five or more sugar units (slow migrating bands on HPTLC. Macrophages isolated from footpad lesions of BALB/c mice infected with Leishmania (L. amazonensis were incubated with MoAb ST-3 and, by indirect immunofluorescence, labeling was only detected on the parasite, whereas no fluorescence was observed on the surface of the infected macrophages, indicating that these glycosphingolipid antigens are not acquired from the host cell but synthesized by the amastigote. Intravenous administration of 125I-labeled ST-3 antibody to infected BALB/c mice showed that MoAb ST-3 accumulated significantly in the footpad lesions in comparison to blood and other tissues

Genetic Manipulation of Leishmania donovani to Explore the Involvement of Argininosuccinate Synthase in Oxidative Stress Management.

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Abul Hasan Sardar

2016-03-01

Full Text Available Reactive oxygen and nitrogen species (ROS and RNS produced by the phagocytic cells are the most common arsenals used to kill the intracellular pathogens. However, Leishmania, an intracellular pathogen, has evolved mechanisms to survive by counterbalancing the toxic oxygen metabolites produced during infection. Polyamines, the major contributor in this anti-oxidant machinery, are largely dependent on the availability of L-arginine in the intracellular milieu. Argininosuccinate synthase (ASS plays an important role as the rate-limiting step required for converting L-citrulline to argininosuccinate to provide arginine for an assortment of metabolic processes. Leishmania produce an active ASS enzyme, yet it has an incomplete urea cycle as it lacks an argininosuccinate lyase (ASL. There is no evidence for endogenous synthesis of L-arginine in Leishmania, which suggests that these parasites salvage L-arginine from extracellular milieu and makes the biological function of ASS and the production of argininosuccinate in Leishmania unclear. Our previous quantitative proteomic analysis of Leishmania promastigotes treated with sub-lethal doses of ROS, RNS, or a combination of both, led to the identification of several differentially expressed proteins which included ASS. To assess the involvement of ASS in stress management, a mutant cell line with greatly reduced ASS activity was created by a double-targeted gene replacement strategy in L. donovani promastigote. Interestingly, LdASS is encoded by three copies of allele, but Western blot analysis showed the third allele did not appear to express ASS. The free thiol levels in the mutant LdASS-/-/+ cell line were decreased. Furthermore, the cell viability in L-arginine depleted medium was greatly attenuated on exposure to different stress environments and was adversely impacted in its ability to infect mice. These findings suggest that ASS is important for Leishmania donovani to counterbalance the stressed

The mitochondrial SIR2 related protein 2 (SIR2RP2 impacts Leishmania donovani growth and infectivity.

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Nimisha Mittal

2017-05-01

Full Text Available Leishmania donovani, a protozoan parasite is the major causative agent of visceral leishmaniasis. Increased toxicity and resistance to the existing repertoire of drugs has been reported. Hence, an urgent need exists for identifying newer drugs and drug targets. Previous reports have shown sirtuins (Silent Information Regulator from kinetoplastids as promising drug targets. Leishmania species code for three SIR2 (Silent Information Regulator related proteins. Here, we for the first time report the functional characterization of SIR2 related protein 2 (SIR2RP2 of L. donovani.Recombinant L. donovani SIR2RP2 was expressed in E. coli and purified. The enzymatic functions of SIR2RP2 were determined. The subcellular localization of LdSIR2RP2 was done by constructing C-terminal GFP-tagged full-length LdSIR2RP2. Deletion mutants of LdSIR2RP2 were generated in Leishmania by double targeted gene replacement methodology. These null mutants were tested for their proliferation, virulence, cell cycle defects, mitochondrial functioning and sensitivity to known SIR2 inhibitors.Our data suggests that LdSIR2RP2 possesses NAD+-dependent ADP-ribosyltransferase activity. However, NAD+-dependent deacetylase and desuccinylase activities were not detected. The protein localises to the mitochondrion of the promastigotes. Gene deletion studies showed that ΔLdSIR2RP2 null mutants had restrictive growth phenotype associated with accumulation of cells in the G2/M phase and compromised mitochondrial functioning. The null mutants had attenuated infectivity. Deletion of LdSIR2RP2 resulted in increased sensitivity of the parasites to the known SIR2 inhibitors. The sirtuin inhibitors inhibited the ADP-ribosyltransferase activity of recombinant LdSIR2RP2. In conclusion, sirtuins could be used as potential new drug targets for visceral leishmaniasis.

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

DEFF Research Database (Denmark)

Chen, M; Zhai, L; Christensen, S B

2001-01-01

Our previous studies have shown that chalcones exhibit potent antileishmanial and antimalarial activities in vitro and in vivo. Preliminary studies showed that these compounds destroyed the ultrastructure of Leishmania parasite mitochondria and inhibited the respiration and the activity...... of mitochondrial dehydrogenases of Leishmania parasites. The present study was designed to further investigate the mechanism of action of chalcones, focusing on the parasite respiratory chain. The data show that licochalcone A inhibited the activity of fumarate reductase (FRD) in the permeabilized Leishmania major...... promastigote and in the parasite mitochondria, and it also inhibited solubilized FRD and a purified FRD from L. donovani. Two other chalcones, 2,4-dimethoxy-4'-allyloxychalcone (24m4ac) and 2,4-dimethoxy-4'-butoxychalcone (24mbc), also exhibited inhibitory effects on the activity of solubilized FRD in L. major...

Effect of BMAP-28 antimicrobial peptides on Leishmania major promastigote and amastigote growth: role of leishmanolysin in parasite survival.

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Miriam A Lynn

Full Text Available Protozoan parasites, such as Leishmania, still pose an enormous public health problem in many countries throughout the world. Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies. Several innovative approaches are under investigation, including the utilization of host defence peptides (HDPs as emerging anti-parasitic therapies. HDPs are characterised by their small size, amphipathic nature and cationicity, which induce permeabilization of cell membranes, whilst modulating the immune response of the host. Recently, members 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.An MTS viability assay was utilized to show the potent antiparasitic activity of BMAP-28 and its protease resistant isomers against L. major promastigotes in vitro. Cell membrane permeability assays, caspase 3/7, Tunel assays and morphologic studies suggested that this was a late stage apoptotic cell death 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.Interestingly, D-BMAP-28 appears to be the most potent antiparasitic of the three isomers against wild type L. major promastigotes and amastigotes. These exciting results suggest that BMAP-28 and its protease resistant isomers have significant therapeutic potential as novel anti-leishmanials.

Bases experimentales para la identificacion de Leishmania spp. de America por morfometria de amastigotos Experimental basis for the identification of Leishmania spp. of America by morphometry of amastigotes

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Haideé Urdaneta

1982-12-01

Full Text Available Estudios morfométricos sobre los amastigotos de dieciocho poblaciones de cuatro aislados pertenecientes a dos especies venezolanas de Leishmania (L. braziliensis y L. garnhami indican que la posición del einetoplasto nose modifica en modo estadísticamente significativo cuando los parásitos son sometidos a pasajes por hamsteres y ratones,cultivos en medio NNN o por infección en un vector (Lu. townsendi. La posición del cinetoplasto, medido como la distancia entre el extremo posterior del amastigoto al cinetoplasto, dividido entre la distancia del organoide al extremo anterior de la célula, permite diferenciar a L. braziliensis de L. mexicana y L. garnhami. Los otros parámetros morfométricos no son tan confiables.Morphometric studies on amastigotes of eigtheen populations from four isolates of two species of Venezuelan Leishmania (L. braziliensis and L. garnhami indicate that the position of the kinetoplast is not significantly modified when parasites are submitted to pasages into hamsters, and culture in NNN medium or infection in a vector (Lu. townsendi. The location of the kinetoplast measured as the distance from the posterior end of the amastigote to the kinetoplast divided by the distance of the same organoid to the anterior end of the amastigote allows to differenciate L. braziliensis from L. mexicana and L. garnhami. Other morphometric parameters are not so confiable.

Paromomycin affects translation and vesicle-mediated trafficking as revealed by proteomics of paromomycin -susceptible -resistant Leishmania donovani.

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Bhavna Chawla

Full Text Available Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis (VL and is responsible for significant mortality and morbidity. Increasing resistance towards antimonial drugs poses a great challenge in chemotherapy of VL. Paromomycin is an aminoglycosidic antibiotic and is one of the drugs currently being used in the chemotherapy of cutaneous and visceral leishmaniasis. To understand the mode of action of this antibiotic at the molecular level, we have investigated the global proteome differences between the wild type AG83 strain and a paromomycin resistant (PRr strain of L. donovani. Stable isotope labeling of amino acids in cell culture (SILAC followed by quantitative mass spectrometry of the wild type AG83 strain and the paromomycin resistant (PRr strain identified a total of 226 proteins at ≥ 95% confidence. Data analysis revealed upregulation of 29 proteins and down-regulation of 21 proteins in the PRr strain. Comparative proteomic analysis of the wild type and the paromomycin resistant strains showed upregulation of the ribosomal proteins in the resistant strain indicating role in translation. Elevated levels of glycolytic enzymes and stress proteins were also observed in the PRr strain. Most importantly, we observed upregulation of proteins that may have a role in intracellular survival and vesicular trafficking in the PRr strain. Furthermore, ultra-structural analysis by electron microscopy demonstrated increased number of vesicular vacuoles in PRr strain when compared to the wild-type strain. Drug affinity pull-down assay followed by mass spectrometery identified proteins in L. donovani wild type strain that were specifically and covalently bound to paromomycin. These results provide the first comprehensive insight into the mode of action and underlying mechanism of resistance to paromomycin in Leishmania donovani.

Towards multilocus sequence typing of the Leishmania donovani complex: Resolving genotypes and haplotypes for five polymorphic metabolic enzymes (ASAT, GPI, NH1, NH2, PGD)

Czech Academy of Sciences Publication Activity Database

Mauricio, I. L.; Yeo, M.; Baghaei, M.; Doto, D.; Pratlong, F.; Zemanová, Eva; Dedet, J.-P.; Lukeš, Julius; Miles, M. A.

2006-01-01

Roč. 36, č. 7 (2006), s. 757-769 ISSN 0020-7519 Grant - others:European Comission(EU) QLK2-CT-2001-01810 Institutional research plan: CEZ:AV0Z60220518 Keywords : Leishmania donovani * Leishmania infantum * multilocus sequence typing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.337, year: 2006

Leishmania donovani resides in modified early endosomes by upregulating Rab5a expression via the downregulation of miR-494

Science.gov (United States)

Verma, Jitender Kumar; Rastogi, Ruchir

2017-01-01

Several intracellular pathogens arrest the phagosome maturation in the host cells to avoid transport to lysosomes. In contrast, the Leishmania containing parasitophorous vacuole (PV) is shown to recruit lysosomal markers and thus Leishmania is postulated to be residing in the phagolysosomes in macrophages. Here, we report that Leishmania donovani specifically upregulates the expression of Rab5a by degrading c-Jun via their metalloprotease gp63 to downregulate the expression of miR-494 in THP-1 differentiated human macrophages. Our results also show that miR-494 negatively regulates the expression of Rab5a in cells. Subsequently, L. donovani recruits and retains Rab5a and EEA1 on PV to reside in early endosomes and inhibits transport to lysosomes in human macrophages. Similarly, we have also observed that Leishmania PV also recruits Rab5a by upregulating its expression in human PBMC differentiated macrophages. However, the parasite modulates the endosome by recruiting Lamp1 and inactive pro-CathepsinD on PV via the overexpression of Rab5a in infected cells. Furthermore, siRNA knockdown of Rab5a or overexpression of miR-494 in human macrophages significantly inhibits the survival of the parasites. These results provide the first mechanistic insights of parasite-mediated remodeling of endo-lysosomal trafficking to reside in a specialized early endocytic compartment. PMID:28650977

Targeting Leishmania amazonensis amastigotes through macrophage internalisation of a hydroxymethylnitrofurazone nanostructured polymeric system.

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Monteiro, Lis Marie; Löbenberg, Raimar; Ferreira, Elizabeth Igne; Cotrim, Paulo Cesar; Kanashiro, Edite; Rocha, Mussya; Chung, Man Chin; Bou-Chacra, Nadia

2017-07-01

Dextran-coated poly (n-butyl cyanoacrylate) nanoparticles (PBCA-NPs) were prepared and were evaluated for enhanced delivery of a promising anti-Leishmania drug candidate, hydroxymethylnitrofurazone (NFOH), to phagocytic cells. Currently available chemotherapy for leishmaniasis, such as pentavalent antimonials, presents low safety and efficacy. Furthermore, widespread drug resistance in leishmaniasis is rapidly emerging. To overcome these drawbacks, the use of nanosized delivery systems can reduce systemic drug toxicity and increase the drug concentration in infected macrophages, therefore improving treatment of leishmaniasis. PBCA-NPs containing NFOH (PBCA-NFOH-NPs) were prepared by an anionic emulsion polymerisation method. The z-average and polydispersity index (PDI) were determined by photon correlation spectroscopy, the zeta potential by microelectrophoresis and the entrapment efficiency by HPLC. Cytotoxicity was determined using macrophages from BALB/c mice. Efficacy tests were performed using Leishmania amazonensis promastigotes and amastigotes. The z-average of PBCA-NFOH-NPs was 151.5 ± 61.97 nm, with a PDI of 0.104 ± 0.01, a zeta potential of -10.1 ± 6.49 mV and an entrapment efficiency of 64.47 ± 0.43%. Efficacy in amastigotes revealed IC 50 values of 0.33 µM and 31.2 µM for the nanostructured and free NFOH, respectively (95-fold increase). The cytotoxicity study indicated low toxicity of the PBCA-NFOH-NPs to macrophages. The selectivity index was 370.6, which is 49-fold higher than free NFOH (7.6). Such findings indicated that improved efficacy could be due to NP internalisation following site-specific drug delivery and reactivation of immune protective reactions by the NP components. Thus, PBCA-NFOH-NPs have the potential to significantly improve the treatment of leishmaniasis, with reduced systemic side effects. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Natural hybrid of Leishmania infantum/L. donovani: development in Phlebotomus tobbi, P. perniciosus and Lutzomyia longipalpis and comparison with non-hybrid strains differing in tissue tropism.

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Seblova, Veronika; Myskova, Jitka; Hlavacova, Jana; Votypka, Jan; Antoniou, Maria; Volf, Petr

2015-11-25

Infection caused by parasites from L. donovani complex can manifest as a serious visceral disease or a self-healing milder cutaneous form. The different tropism and pathology in humans is caused by the interaction between parasites, host and vector determinants but the mechanisms are not well understood. In Cukurova region in Turkey we previously identified a major focus of cutaneous leishmaniasis caused by L. donovani/infantum hybrids (CUK strain) and isolated this parasite from the locally abundant sand fly, Phlebotomus tobbi. Here, we present the first experimental study with P. tobbi. We tested the susceptibility of this species to various Leishmania under laboratory conditions, characterized glycoproteins in the P. tobbi midgut putatively involved in parasite-vector interaction and compared the development of the CUK strain in the sand fly with one other dermotropic and three viscerotropic strains belonging to the L. donovani complex. Females of laboratory reared P. tobbi, P. perniciosus and Lutzomyia longipalpis were infected using membrane feeding on rabbit blood containing promastigotes of various Leishmania species with different tropisms. The individual guts were checked microscopically for presence and localization of Leishmania parasites; the number of parasites was assessed more precisely by qPCR. In addition, glycosylation of midgut proteins of P. tobbi was studied by lectin blotting of midgut lysate with lectins specific for terminal sugars of N-type and O-type glycans. High infection rates, heavy parasite loads and late-stage infection with colonization of the stomodeal valve were observed in P. tobbi infected by Leishmania major or L. infantum CUK hybrid. In parallel, lectin blotting revealed the presence of O-glycosylated proteins in the P. tobbi midgut. In P. perniciosus and L. longipalpis all five Leishmania strains tested developed well. In both vectors, significantly higher parasite numbers were detected by qPCR for dermotropic L. donovani

Deciphering the interplay between cysteine synthase and thiol cascade proteins in modulating Amphotericin B resistance and survival of Leishmania donovani under oxidative stress

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Kuljit Singh

2017-08-01

Full Text Available Leishmania donovani is the causative organism of the neglected human disease known as visceral leishmaniasis which is often fatal, if left untreated. The cysteine biosynthesis pathway of Leishmania may serve as a potential drug target because it is different from human host and regulates downstream components of redox metabolism of the parasites; essential for their survival, pathogenicity and drug resistance. However, despite the apparent dependency of redox metabolism of cysteine biosynthesis pathway, the role of L. donovani cysteine synthase (LdCS in drug resistance and redox homeostasis has been unexplored. Herein, we report that over-expression of LdCS in Amphotericin B (Amp B sensitive strain (S1-OE modulates resistance towards oxidative stress and drug pressure. We observed that antioxidant enzyme activities were up-regulated in S1-OE parasites and these parasites alleviate intracellular reactive oxygen species (ROS efficiently by maintaining the reduced thiol pool. In contrast to S1-OE parasites, Amp B sensitive strain (S1 showed higher levels of ROS which was positively correlated with the protein carbonylation levels and negatively correlated with cell viability. Moreover, further investigations showed that LdCS over-expression also augments the ROS-primed induction of LdCS-GFP as well as endogenous LdCS and thiol pathway proteins (LdTryS, LdTryR and LdcTXN in L. donovani parasites; which probably aids in stress tolerance and drug resistance. In addition, the expression of LdCS was found to be up-regulated in Amp B resistant isolates and during infective stationary stages of growth and consistent with these observations, our ex vivo infectivity studies confirmed that LdCS over-expression enhances the infectivity of L. donovani parasites. Our results reveal a novel crosstalk between LdCS and thiol metabolic pathway proteins and demonstrate the crucial role of LdCS in drug resistance and redox homeostasis of Leishmania. Keywords

Seasonal variation in the prevalence of sand flies infected with Leishmania donovani.

Science.gov (United States)

Tiwary, Puja; Kumar, Dinesh; Mishra, Mukesh; Singh, Rudra Pratap; Rai, Madhukar; Sundar, Shyam

2013-01-01

Visceral Leishmaniasis (VL) is a life threatening neglected infectious disease in the Indian subcontinent, transmitted by the bite of female sand flies. Estimation of the infectivity in the vector population, collected in different seasons, may be useful to better understanding the transmission dynamics of VL as well as to plan vector control measures. We collected sand flies from highly endemic regions of Bihar state, India for one year over three seasons. The species of the sand flies were confirmed by species-specific PCR-RFLP. Leishmania donovani infection was investigated in 1397 female Phlebotomus argentipes using PCR, targeting the Leishmania specific minicircle of the kDNA region. Further, the parasitic load in the infected sand flies was measured using quantitative PCR. Though sand flies were most abundant in the rainy season, the highest rate of infection was detected in the winter season with 2.84% sand flies infected followed by the summer and rainy seasons respectively. This study can help in vector elimination programmes and to reduce disease transmission.

The Leishmania donovani complex: Genotypes of five metabolic enzymes (ICD, ME, MPI, G6PDH and FH), new targets for multilocus sequence typing

Czech Academy of Sciences Publication Activity Database

Zemanová, Eva; Jirků, Milan; Mauricio, I. L.; Horák, Aleš; Miles, M. A.; Lukeš, Julius

2007-01-01

Roč. 37, č. 2 (2007), s. 149-160 ISSN 0020-7519 R&D Projects: GA MŠk 2B06129 Grant - others:EU(EU) QLK2-CT-2001-01810 Institutional research plan: CEZ:AV0Z60220518 Source of funding: R - rámcový projekt EK Keywords : Leishmania donovani complex * zymodeme * multilocus sequence typing * Leishmania * phylogenetic network Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.392, year: 2007

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

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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

Leishmania donovani infection induces anemia in hamsters by differentially altering erythropoiesis in bone marrow and spleen.

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William P Lafuse

Full Text Available Leishmania donovani is a parasite that causes visceral leishmaniasis by infecting and replicating in macrophages of the bone marrow, spleen, and liver. Severe anemia and leucopenia is associated with the disease. Although immune defense mechanisms against the parasite have been studied, we have a limited understanding of how L. donovani alters hematopoiesis. In this study, we used Syrian golden hamsters to investigate effects of L. donovani infection on erythropoiesis. Infection resulted in severe anemia and leucopenia by 8 weeks post-infection. Anemia was associated with increased levels of serum erythropoietin, which indicates the hamsters respond to the anemia by producing erythropoietin. We found that infection also increased numbers of BFU-E and CFU-E progenitor populations in the spleen and bone marrow and differentially altered erythroid gene expression in these organs. In the bone marrow, the mRNA expression of erythroid differentiation genes (α-globin, β-globin, ALAS2 were inhibited by 50%, but mRNA levels of erythroid receptor (c-kit, EpoR and transcription factors (GATA1, GATA2, FOG1 were not affected by the infection. This suggests that infection has a negative effect on differentiation of erythroblasts. In the spleen, erythroid gene expression was enhanced by infection, indicating that the anemia activates a stress erythropoiesis response in the spleen. Analysis of cytokine mRNA levels in spleen and bone marrow found that IFN-γ mRNA is highly increased by L. donovani infection. Expression of the IFN-γ inducible cytokine, TNF-related apoptosis-inducing ligand (TRAIL, was also up-regulated. Since TRAIL induces erythroblasts apoptosis, apoptosis of bone marrow erythroblasts from infected hamsters was examined by flow cytometry. Percentage of erythroblasts that were apoptotic was significantly increased by L. donovani infection. Together, our results suggest that L. donovani infection inhibits erythropoiesis in the bone marrow by

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...

Seroepidemiology and molecular diversity of Leishmania donovani complex in Georgia.

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Babuadze, Giorgi; Farlow, Jason; de Koning, Harry P; Carrillo, Eugenia; Chakhunashvili, Giorgi; Murskvaladze, Mari; Kekelidze, Merab; Karseladze, Irakli; Kokaia, Nora; Kalandadze, Irine; Tsereteli, David; Markhvashvili, Ivane; Sidamonidze, Ketevan; Chanturia, Gvantsa; Adeishvili, Ekaterine; Imnadze, Paata

2016-05-13

Leishmaniasis includes multiple clinical syndromes, most notably visceral, cutaneous, and mucosal forms. Visceral leishmaniasis (VL), also known as kala-azar, is a potentially fatal disease endemic to large parts of Africa and Asia, and in South-Eastern Europe (Greece, Turkey, Georgia). Visceral leishmaniasis is a parasitic zoonosis caused by species of the L. donovani complex. In the classical epidemiological model the main reservoir for VL are canines. The study included a cohort of 513 individuals of both genders (190 males and 323 females) from the ages of 1 to 70 years that were screened in ten villages across two districts in Kakheti using the Kalazar Detect™ rK39 rapid diagnostic test. The phylogenetic diversity patterns of local strains, based on the rDNA internal transcribed spacer (ITS) sequences, were assessed for samples obtained from patients with suspected L. donovani infection, from canine reservoirs and from Phlebotomus sand flies obtained from different geographical areas of Georgia and from Azerbaijan. Out of a total of 600 domestic dog blood samples 95 (15.8 %) were positive by rK39 rapid diagnostic tests. For symptomatic domestic dogs, the testing of conjunctival swabs or bone marrow aspirates revealed a higher VL incidence in Kvareli District (Kvareli; 19.4 %, n = 329) compared with that observed for Sagarejo District (Sagarejo; 11.4 %, n = 271). A total of 231 sand flies of both genders were collected during the 2-month period; of the 114 females, 1.75 % were PCR positive for the presence of Leishmania spp. VL infection rates remain high in both canines and humans in Georgia, with disease in several known natural foci. The genetic relationships derived from rDNA internal transcribed spacer (ITS) sequence comparisons identified genetic subgroups, revealing preliminary insights into the genetic structure of L. donovani complex members currently circulating in the South Caucasus and demonstrates the utility of ITS-based genotyping

Solute carrier protein family 11 member 1 (Slc11a1) activation efficiently inhibits Leishmania donovani survival in host macrophages.

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Singh, Nisha; Gedda, Mallikarjuna Rao; Tiwari, Neeraj; Singh, Suya P; Bajpai, Surabhi; Singh, Rakesh K

2017-09-01

Visceral leishmaniasis (kala-azar), a life threatening disease caused by L. donovani , is a latent threat to more than 147 million people living in disease endemic South East Asia region of the Indian subcontinent. The therapeutic option to control leishmanial infections are very limited, and at present comprise only two drugs, an antifungal amphotericin B and an antitumor miltefosine, which are also highly vulnerable for parasitic resistance. Therefore, identification and development of alternate control measures is an exigent requirement to control leishmanial infections. In this study, we report that functionally induced expression of solute carrier protein family 11 member 1 ( Slc11a1), a transmembrane divalent cationic transporter recruited on the surface of phagolysosomes after phagocytosis of parasites, effectively inhibits Leishmania donovani growth in host macrophages. Further, the increased Slc11a1 functionality also resulted in increased production of NOx, TNF-α and IL-12 by activated macrophages. The findings of this study signify the importance of interplay between Slc11a1 expression and macrophages activation that can be effectively used to control of Leishmania growth and survival.

Safety and skin delayed-type hypersensitivity response in vervet monkeys immunized with Leishmania donovani sonicate antigen delivered with adjuvants

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Joshua M. Mutiso

2012-02-01

Full Text Available In this study, we report on the safety and skin delayed-type hypersensitivity (DTH, responses of the Leishmania donovani whole cell sonicate antigen delivered in conjunction with alum-BCG (AlBCG, Montanide ISA 720 (MISA or Monophosphoryl lipid A (MPLA in groups of vervet monkeys. Following three intradermal injections of the inoculums on days 0, 28 and 42, safety and DTH responses were assessed. Preliminary tumor necrosis factor alpha (TNF-α and interferon gamma (IFN-γ levels were also measured and these were compared with DTH. Only those animals immunized with alum-BCG reacted adversely to the inoculum by producing ulcerative erythematous skin indurations. Non-parametric analysis of variance followed by a post-test showed significantly higher DTH responses in the MISA+Ag group compared with other immunized groups (p < 0.001. The MPLA+Ag group indicated significantly lower DTH responses to the sonicate antigen compared with the AlBCG+Ag group. There was a significant correlation between the DTH and cytokine responses (p < 0.0001. Based on this study we conclude that Leishmania donovani sonicate antigen containing MISA 720 is safe and is associated with a strong DTH reaction following immunization.

Phytochemical screening of the exudate of Aloe otallensis and its effect on Leishmania donovani

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Zerihun Tesfaye Nigusse

2016-06-01

Full Text Available Objective: To evaluate the antileishmanial activity of methanolic extract of Aloe otallensis (A. otallensis on the promastigote stage of Leishmania donovani (L. donovani as compared to standard drugs and to screen its phytochemical constituents. Methods: Phytochemical screening was done by using the method mentioned by Evans and Trease on methanolic extract of the exudates of Aloe otallensis leaves. The extract was also evaluated for in vitro antileishmanial activity against L. donavani which is found from the Parasitology Unit of Black Lion Hospital. The result was compared to standard drugs of sodium stibogluconate, milfostin and paramomycin. Results: The extract has a good antileishmanial activity with an IC50 of 0.1230 μg/mL on L. donovani (AM 563. The experimental data showed that relatively it had better activity than paramomycin and milfostin but less activity than sodium stibogluconate. The data analyses were done by GraphPad Prism version 5 software after it was read by ELISA reader at the wave length of 650 nm. The phytochemical screening of the exudates of A. otallensis showed the presence of phenol, alkaloid and saponin. Conclusions: The methanol extract of the exudates of A. otallensis has a good antileishmaniasis activity and this may be attributed to phenol, alkaloid and saponin present in the plant. But it needs further analysis for the conformation of which constituent presents in high concentration to know which one has the strongest effect.

Molecular Preadaptation to Antimony Resistance in Leishmania donovani on the Indian Subcontinent.

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Dumetz, F; Cuypers, B; Imamura, H; Zander, D; D'Haenens, E; Maes, I; Domagalska, M A; Clos, J; Dujardin, J-C; De Muylder, G

2018-04-25

Antimonials (Sb) were used for decades for chemotherapy of visceral leishmaniasis (VL). Now abandoned in the Indian subcontinent (ISC) because of Leishmania donovani resistance, this drug offers a unique model for understanding drug resistance dynamics. In a previous phylogenomic study, we found two distinct populations of L. donovani : the core group (CG) in the Gangetic plains and ISC1 in the Nepalese highlands. Sb resistance was only encountered within the CG, and a series of potential markers were identified. Here, we analyzed the development of resistance to trivalent antimonials (Sb III ) upon experimental selection in ISC1 and CG strains. We observed that (i) baseline Sb III susceptibility of parasites was higher in ISC1 than in the CG, (ii) time to Sb III resistance was higher for ISC1 parasites than for CG strains, and (iii) untargeted genomic and metabolomic analyses revealed molecular changes along the selection process: these were more numerous in ISC1 than in the CG. Altogether these observations led to the hypothesis that CG parasites are preadapted to Sb III resistance. This hypothesis was experimentally confirmed by showing that only wild-type CG strains could survive a direct exposure to the maximal concentration of Sb III The main driver of this preadaptation was shown to be MRPA , a gene involved in Sb III sequestration and amplified in an intrachromosomal amplicon in all CG strains characterized so far. This amplicon emerged around 1850 in the CG, well before the implementation of antimonials for VL chemotherapy, and we discuss here several hypotheses of selective pressure that could have accompanied its emergence. IMPORTANCE The "antibiotic resistance crisis" is a major challenge for scientists and medical professionals. This steady rise in drug-resistant pathogens also extends to parasitic diseases, with antimony being the first anti- Leishmania drug that fell in the Indian subcontinent (ISC). Leishmaniasis is a major but neglected infectious

Transfer of innate resistance and susceptibility to Leishmania donovani infection in mouse radiation bone marrow chimaeras

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Crocker, P.R.; Blackwell, J.M.; Bradley, D.J. (London School of Hygiene and Tropical Medicine (UK))

1984-07-01

Reciprocal radiation bone marrow chimaeras were made between H-2-compatible strains of mice innately resistant or susceptible to visceral leishmaniasis. In initial experiments, susceptibility but not resistance to Leishmania donovani could be transferred with donor bone marrow into irradiated recipients. In subsequent experiments it was possible to transfer both resistance and susceptibility. This was achieved either by selecting more radiosensitive mouse strains as susceptible recipients, or alternatively by increasing the irradiation dose for the susceptible recipients used in the initial experiments. Using the higher irradiation dose, successful transfer of resistance and susceptibility between congenic mice carrying the Lshsup(r) and Lshsup(s) alleles on the more radioresistant B10 genetic background provided firm evidence that the results obtained in this study were specifically related to expression of the Lsh gene. It is concluded that Lsh gene-controlled resistance and susceptibility to L. donovani is determined by bone marrow-derived cells. The cell type(s) involved is likely to be of the macrophage lineage.

Transfer of innate resistance and susceptibility to Leishmania donovani infection in mouse radiation bone marrow chimaeras

International Nuclear Information System (INIS)

Crocker, P.R.; Blackwell, J.M.; Bradley, D.J.

1984-01-01

Reciprocal radiation bone marrow chimaeras mere made between H-2-compatible strains of mice innately resistant or susceptible to visceral leishmaniasis. In initial experiments, susceptibility but not resistance to Leishmania donovani could be transferred with donor bone marrow into irradiated recipients. In subsequent experiments it was possible to transfer both resistance and susceptibility. This was achieved either by selecting more radiosensitive mouse strains as susceptible recipients, or alternatively by increasing the irradiation dose for the susceptible recipients used in the initial experiments. Using the higher irradiation dose, successful transfer of resistance and susceptibility between congenic mice carrying the Lshsup(r) and Lshsup(s) alleles on the more radioresistant B10 genetic background provided firm evidence that the results obtained in this study were specifically related to expression of the Lsh gene. It is concluded that Lsh gene-controlled resistance and susceptibility to L. donovani is determined by bone marrow-derived cells. The cell type(s) involved is likely to be of the macrophage lineage. (author)

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

Science.gov (United States)

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

2017-01-01

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

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

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

2017-01-01

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

Antigenicity and protective efficacy of a Leishmania amastigote-specific protein, member of the super-oxygenase family, against visceral leishmaniasis.

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Vivian T Martins

Full Text Available The present study aimed to evaluate a hypothetical Leishmania amastigote-specific protein (LiHyp1, previously identified by an immunoproteomic approach performed in Leishmania infantum, which showed homology to the super-oxygenase gene family, attempting to select a new candidate antigen for specific serodiagnosis, as well as to compose a vaccine against VL.The LiHyp1 DNA sequence was cloned; the recombinant protein (rLiHyp1 was purified and evaluated for its antigenicity and immunogenicity. The rLiHyp1 protein was recognized by antibodies from sera of asymptomatic and symptomatic animals with canine visceral leishmaniasis (CVL, but presented no cross-reactivity with sera of dogs vaccinated with Leish-Tec, a Brazilian commercial vaccine; with Chagas' disease or healthy animals. In addition, the immunogenicity and protective efficacy of rLiHyp1 plus saponin was evaluated in BALB/c mice challenged subcutaneously with virulent L. infantum promastigotes. rLiHyp1 plus saponin vaccinated mice showed a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with the recombinant protein. Immunized and infected mice, as compared to the control groups (saline and saponin, showed significant reductions in the number of parasites found in the liver, spleen, bone marrow, and in the paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, produced mainly by CD4 T cells. In these mice, a decrease in the parasite-mediated IL-4 and IL-10 response could also be observed.The present study showed that this Leishmania oxygenase amastigote-specific protein can be used for a more sensitive and specific serodiagnosis of asymptomatic and symptomatic CVL and, when combined with a Th1-type adjuvant, can also be employ as a candidate antigen to develop vaccines against VL.

Zinc depletion promotes apoptosis-like death in drug-sensitive and antimony-resistance Leishmania donovani.

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Saini, Shalini; Bharati, Kavita; Shaha, Chandrima; Mukhopadhyay, Chinmay K

2017-09-05

Micronutrients are essential for survival and growth for all the organisms including pathogens. In this manuscript, we report that zinc (Zn) chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethylenediamine (TPEN) affects growth and viability of intracellular pathogen Leishmania donovani (LD) by a concentration and time dependent manner. Simultaneous addition of zinc salt reverses the effect of TPEN. Further experiments provide evidence of apoptosis-like death of the parasite due to Zn-depletion. TPEN treatment enhances caspase-like activity suggesting increase in apoptosis-like events in LD. Specific inhibitors of cathepsin B and Endoclease G block TPEN-induced leishmanial death. Evidences show involvement of reactive oxygen species (ROS) potentially of extra-mitochondrial origin in TPEN-induced LD death. Pentavalent antimonials remained the prime source of treatment against leishmaniasis for several decades; however, antimony-resistant Leishmania is now common source of the disease. We also reveal that Zn-depletion can promote apoptosis-like death in antimony-resistant parasites. In summary, we present a new finding about the role of zinc in the survival of drug sensitive and antimony-resistant LD.

F1 Domain of the Leishmania (Leishmania donovani Nucleoside Hydrolase Promotes a Th1 Response in Leishmania (Leishmania infantum Cured Patients and in Asymptomatic Individuals Living in an Endemic Area of Leishmaniasis

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Eugenia Carrillo

2017-07-01

Full Text Available The Leishmania (Leishmania donovani nucleoside hydrolase NH36 is the main antigen of the Leishmune® vaccine and one of the promising candidates for vaccination against visceral leishmaniasis. The antigenicity of the N-terminal (F1, the central (F2, or the C-terminal recombinant domain (F3 of NH36 was evaluated using peripheral blood mononuclear cells (PBMC from individuals infected with L. (L. infantum from an endemic area of visceral leishmaniasis of Spain. Both NH36 and F1 domains significantly increased the PBMC proliferation stimulation index of cured patients and infected asymptomatic individuals compared to healthy controls. Moreover, F1 induced a 19% higher proliferative response than NH36 in asymptomatic exposed subjects. In addition, in patients cured from visceral leishmaniasis, proliferation in response to NH36 and F1 was accompanied by a significant increase of IFN-γ and TNF-α secretion, which was 42–43% higher, in response to F1 than to NH36. The interleukin 17 (IL-17 secretion was stronger in asymptomatic subjects, in response to F1, as well as in cured cutaneous leishmaniasis after NH36 stimulation. While no IL-10 secretion was determined by F1, a granzyme B increase was detected in supernatants from cured patients after stimulation with either NH36 or F1. These data demonstrate that F1 is the domain of NH36 that induces a recall cellular response in individuals with acquired resistance to the infection by L. (L. infantum. In addition, F1 and NH36 discriminated the IgG3 humoral response in patients with active visceral leishmaniasis due to L. (L. donovani (Ethiopia and L. (L. infantum (Spain from that of endemic and non-endemic area controls. NH36 showed higher reactivity with sera from L. (L. donovani-infected individuals, indicating species specificity. We conclude that the F1 domain, previously characterized as an inducer of the Th1 and Th17 responses in cured/exposed patients infected with L. (L. infantum chagasi, may

Identification of a secreted casein kinase 1 in Leishmania donovani: effect of protein over expression on parasite growth and virulence.

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Mary Dan-Goor

Full Text Available Casein kinase 1 (CK1 plays an important role in eukaryotic signaling pathways, and their substrates include key regulatory proteins involved in cell differentiation, proliferation and chromosome segregation. The Leishmania genome encodes six potential CK1 isoforms, of which five have orthologs in other trypanosomatidae. Leishmania donovani CK1 isoform 4 (Ldck1.4, orthologous to LmjF27.1780 is unique to Leishmania and contains a putative secretion signal peptide. The full-length gene and three shorter constructs were cloned and expressed in E. coli as His-tag proteins. Only the full-length 62.3 kDa protein showed protein kinase activity indicating that the N-terminal and C-terminal domains are essential for protein activity. LdCK1.4-FLAG was stably over expressed in L. donovani, and shown by immunofluorescence to be localized primarily in the cytosol. Western blotting using anti-FLAG and anti-CK1.4 antibodies showed that this CK1 isoform is expressed and secreted by promastigotes. Over expression of LdCK1.4 had a significant effect on promastigote growth in culture with these parasites growing to higher cell densities than the control parasites (wild-type or Ld:luciferase, P<0.001. Analysis by flow cytometry showed a higher percentage, ∼4-5-fold, of virulent metacyclic promastigotes on day 3 among the LdCK1.4 parasites. Finally, parasites over expressing LdCK1.4 gave significantly higher infections of mouse peritoneal macrophages compared to wild-type parasites, 28.6% versus 6.3%, respectively (p = 0.0005. These results suggest that LdCK1.4 plays an important role in parasite survival and virulence. Further studies are needed to validate CK1.4 as a therapeutic target in Leishmania.

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

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

2017-02-01

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

Safety and skin delayed-type hypersensitivity response in vervet monkeys immunized with Leishmania donovani sonicate antigen delivered with adjuvants

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Mutiso,Joshua M.; Macharia,John C.; Taracha,Evans; Wafula,Kellern; Rikoi,Hitler; Gicheru,Michael M.

2012-01-01

In this study, we report on the safety and skin delayed-type hypersensitivity (DTH), responses of the Leishmania donovani whole cell sonicate antigen delivered in conjunction with alum-BCG (AlBCG), Montanide ISA 720 (MISA) or Monophosphoryl lipid A (MPLA) in groups of vervet monkeys. Following three intradermal injections of the inoculums on days 0, 28 and 42, safety and DTH responses were assessed. Preliminary tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) levels ...

IL-17A promotes susceptibility during experimental visceral leishmaniasis caused by Leishmania donovani.

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Terrazas, Cesar; Varikuti, Sanjay; Kimble, Jennifer; Moretti, Ellen; Boyaka, Prosper N; Satoskar, Abhay R

2016-03-01

Leishmania donovani is an intracellular parasite that infects professional phagocytes and causes visceral leishmaniasis (VL). The immune response during VL has been extensively studied in the context of T-helper (Th)1 and Th2 responses. Immunity against this parasite is dependent on IFN-γ production and subsequent macrophage activation, and the Th2 response promotes granuloma formation. The cytokine IL-17A is associated with neutrophilic inflammation. Depletion of neutrophils during experimental VL results in enhanced parasitic loads. Furthermore, although patients resistant to VL showed enhanced levels of IL-17A in circulation, little is known about the role of IL-17A during VL infection. Here, we used IL-17A-deficient mice and IL-17A reporter mice to address the role of IL-17A during VL. IL-17A(-/-) mice were highly resistant to VL infection, showing decreased parasites in the liver and spleen. This unexpected phenotype was associated with enhanced IFN-γ production by T cells and decreased accumulation of neutrophils and monocytes, resulting in reduced number of granulomas. We also found γδ T and Th17 cells as the main IL-17A(+) cells during VL infection. Our data reveal an unexpected role of IL-17A rendering susceptibility against L. donovani by regulating the IFN-γ response and promoting detrimental inflammation. © FASEB.

Leishmania hijacking of the macrophage intracellular compartments.

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Liévin-Le Moal, Vanessa; Loiseau, Philippe M

2016-02-01

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

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

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

2016-08-01

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

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

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

2016-01-01

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

Mapping the antigenicity of the parasites in Leishmania donovani infection by proteome serology.

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Michael Forgber

Full Text Available BACKGROUND: Leishmaniasis defines a cluster of protozoal diseases with diverse clinical manifestations. The visceral form caused by Leishmania donovani is the most severe. So far, no vaccines exist for visceral leishmaniasis despite indications of naturally developing immunity, and sensitive immunodiagnostics are still at early stages of development. METHODOLOGY/PRINCIPLE FINDINGS: Establishing a proteome-serological methodology, we mapped the antigenicity of the parasites and the specificities of the immune responses in human leishmaniasis. Using 2-dimensional Western blot analyses with sera and parasites isolated from patients in India, we detected immune responses with widely divergent specificities for up to 330 different leishmanial antigens. 68 antigens were assigned to proteins in silver- and fluorochrome-stained gels. The antigenicity of these proteins did not correlate with the expression levels of the proteins. Although some antigens are shared among different parasite isolates, there are extensive differences and no immunodominant antigens, but indications of antigenic drift in the parasites. Six antigens were identified by mass spectrometry. CONCLUSIONS/SIGNIFICANCE: Proteomics-based dissection of the serospecificities of leishmaniasis patients provides a comprehensive inventory of the complexity and interindividual heterogeneity of the host-responses to and variations in the antigenicity of the Leishmania parasites. This information can be instrumental in the development of vaccines and new immune monitoring and diagnostic devices.

Immunotherapy against visceral leishmaniasis with the nucleoside hydrolase-DNA vaccine of Leishmania donovani.

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Gamboa-León, R; Paraguai de Souza, E; Borja-Cabrera, G P; Santos, F N; Myashiro, L M; Pinheiro, R O; Dumonteil, E; Palatnik-de-Sousa, C B

2006-05-29

The nucleoside hydrolase (NH36) of Leishmania (L.) donovani is a vital enzyme which releases purines or pyrimidines of foreign DNA to be used in the synthesis of parasite DNA. As a bivalent DNA vaccine, the VR1012-NH36 was immunoprotective against visceral and cutaneous murine leishmaniasis. In this work we tested the immunotherapy against Leishmania (L.) chagasi infection, using two doses of 100 or 20 microg VR1012-NH36 vaccine (i.m. route), and, as a possible immunomodulator, aqueous garlic extract (8 mg/kg/day by the i.p. route), which was effective in immunotherapy of cutaneous murine leishmaniasis. Liver parasitic load was significantly reduced following treatment with 100 microg (91%) and 20 microg (77%) of the DNA vaccine, and by 20 microg DNA vaccine and garlic extract (76%) (p=0.023). Survival was 33% for saline controls, 100% for the 100 microg vaccine, and 83 and 67% for the 20 microg vaccine with and without garlic extract addition, respectively. Garlic treatment alone did not reduce parasite load (p>0.05), but increased survival (100%). The NH36-DNA vaccine was highly effective as a new tool for the therapy and control of visceral leishmaniasis, while the mild protective effect of garlic might be related to an unspecific enhancement of IFN-gamma secretion.

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

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

2012-07-01

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

Presence of Leishmania amastigotes in peritoneal fluid of a dog with leishmaniasis from Alagoas, Northeast Brazil Presença de formas amastigotas de Leishmania em fluido peritoneal de cão com leishmaniose proveniente de Alagoas, nordeste do Brasil

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Filipe Dantas-Torres

2006-08-01

Full Text Available The goal of this short communication is to report the uncommon presence of intracellular amastigotes of Leishmania in peritoneal fluid of a dog with leishmaniasis from Alagoas State, Brazil. Physical examination of an adult male rottweiler suspected to be suffering of leishmaniasis revealed severe loss of weight, ascitis, splenomegaly, moderately enlarged lymph nodes, onychogryphosis, generalized alopecia, skin ulcers on the posterior limbs, and conjunctivitis. Samples of bone marrow, popliteal lymph node, skin ulcer, and peritoneal fluid were collected and smears of each sample were prepared and stained with hematoxylin and eosin. Numerous amastigotes were detected in bone marrow, popliteal lymph node, and skin ulcer smears. Smears of peritoneal fluid revealed the unusual presence of several free and intracellular amastigotes of Leishmania. Future studies are needed to determine whether the cytology of ascitic fluid represents a useful tool for diagnosis Leishmania infection in ascitic dogs, particularly in those living in areas where canine leishmaniasis is enzootic.O objetivo desta comunicação é descrever a presença incomum de formas amastigotas de Leishmania em fluido peritoneal de um cão com leishmaniose proveniente do Estado de Alagoas, nordeste do Brasil. O exame físico de um cão macho adulto da raça rottweiler, apresentando suspeita de leishmaniose, revelou perda de peso severa, esplenomegalia, linfonodos moderadamente aumentados, ascite, onicogrifose, alopecia generalizada, conjuntivite e presença de lesões cutâneas ulceradas localizadas nos membros posteriores. Foram coletadas amostras de medula óssea, linfonodo poplíteo, fluido peritoneal e úlcera cutânea. A partir das amostras, foram elaborados esfregaços, os quais foram corados pela hematoxilina e eosina. Inúmeras formas amastigotas foram detectadas na medula óssea, linfonodo poplíteo e úlcera cutânea. Esfregaços de fluido peritoneal revelaram a presença, n

Over-expression of 60s ribosomal L23a is associated with cellular proliferation in SAG resistant clinical isolates of Leishmania donovani.

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Sanchita Das

Full Text Available Sodium antimony gluconate (SAG unresponsiveness of Leishmania donovani (Ld had effectively compromised the chemotherapeutic potential of SAG. 60s ribosomal L23a (60sRL23a, identified as one of the over-expressed protein in different resistant strains of L.donovani as observed with differential proteomics studies indicates towards its possible involvement in SAG resistance in L.donovani. In the present study 60sRL23a has been characterized for its probable association with SAG resistance mechanism.The expression profile of 60s ribosomal L23a (60sRL23a was checked in different SAG resistant as well as sensitive strains of L.donovani clinical isolates by real-time PCR and western blotting and was found to be up-regulated in resistant strains. Ld60sRL23a was cloned, expressed in E.coli system and purified for raising antibody in swiss mice and was observed to have cytosolic localization in L.donovani. 60sRL23a was further over-expressed in sensitive strain of L.donovani to check its sensitivity profile against SAG (Sb V and III and was found to be altered towards the resistant mode.This study reports for the first time that the over expression of 60sRL23a in SAG sensitive parasite decreases the sensitivity of the parasite towards SAG, miltefosine and paramomycin. Growth curve of the tranfectants further indicated the proliferative potential of 60sRL23a assisting the parasite survival and reaffirming the extra ribosomal role of 60sRL23a. The study thus indicates towards the role of the protein in lowering and redistributing the drug pressure by increased proliferation of parasites and warrants further longitudinal study to understand the underlying mechanism.

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

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Rodriguez-Contreras, Dayana; Hamilton, Nicklas

2014-11-21

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

Understanding the transmission dynamics of Leishmania donovani to provide robust evidence for interventions to eliminate visceral leishmaniasis in Bihar, India the LCNTDR Collection : Advances in scientific research for NTD control

NARCIS (Netherlands)

M.M. Cameron (Mary M.); A. Acosta-Serrano (Alvaro); C. Bern (Caryn); M. Boelaert (Marleen); M. Den Boer (Margriet); S. Burza (Sakib); L.A.C. Chapman (Lloyd A. C.); A. Chaskopoulou (Alexandra); M. Coleman (Michael); O. Courtenay (Orin); S. Croft (Simon); P.K. Das (P.); E. Dilger (Erin); G. Foster (Geraldine); R. Garlapati (Rajesh); L. Haines (Lee); A. Harris (Angela); J. Hemingway (Janet); T.D. Hollingsworth (T. Déirdre); S. Jervis (Sarah); G.F. Medley (Graham F.); M. Miles (Michael); M. Paine (Mark); A. Picado (Albert); R. Poché (Richard); P. Ready (Paul); M. Rogers (Matthew); M. Rowland (Mark); S. Sundar (Shyam); S.J. de Vlas (Sake); D. Weetman (David)

2016-01-01

textabstractVisceral Leishmaniasis (VL) is a neglected vector-borne disease. In India, it is transmitted to humans by Leishmania donovani-infected Phlebotomus argentipes sand flies. In 2005, VL was targeted for elimination by the governments of India, Nepal and Bangladesh by 2015. The elimination

Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of Visceral Leishmaniasis patients and the monitoring of their response to treatment

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Ghosh, Prakash; Khan, Md. Anik Ashfaq; Duthie, Malcolm S.; Vallur, Aarthy C.; Picone, Alessandro; Howard, Randall F.; Reed, Steven G.

2017-01-01

Sustained elimination of Visceral Leishmaniasis (VL) requires the reduction and control of parasite reservoirs to minimize the transmission of Leishmania donovani infection. A simple, reproducible and definitive diagnostic procedure is therefore indispensable for the early and accurate detection of parasites in VL, Relapsed VL (RVL) and Post Kala-azar Dermal Leishmaniasis (PKDL) patients, all of whom are potential reservoirs of Leishmania parasites. To overcome the limitations of current diagnostic approaches, a novel quantitative real-time polymerase chain reaction (qPCR) method based on Taqman chemistry was devised for the detection and quantification of L. donovani in blood and skin. The diagnostic efficacy was evaluated using archived peripheral blood buffy coat DNA from 40 VL, 40 PKDL, 10 RVL, 20 cured VL, and 40 cured PKDL along with 10 tuberculosis (TB) cases and 80 healthy endemic controls. Results were compared to those obtained using a Leishmania-specific nested PCR (Ln-PCR). The real time PCR assay was 100% (95% CI, 91.19–100%) sensitive in detecting parasite genomes in VL and RVL samples and 85.0% (95% CI, 70.16–94.29%) sensitive for PKDL samples. In contrast, the sensitivity of Ln-PCR was 77.5% (95% CI, 61.55–89.16%) for VL samples, 100% (95%CI, 69.15–100%) for RVL samples, and 52.5% (95% CI, 36.13–68.49%) for PKDL samples. There was significant discordance between the two methods with the overall sensitivity of the qPCR assay being considerably higher than Ln-PCR. None of the assay detected L. donovani DNA in buffy coats from cured VL cases, and reduced infectious burdens were demonstrated in cured PKDL cases who remained positive in 7.5% (3/40) and 2.5% (1/40) cases by real-time PCR and Ln-PCR, respectively. Both assays were 100% (95% CI, 95.98–100) specific with no positive signals in either endemic healthy control or TB samples. The real time PCR assay we developed offers a molecular tool for accurate detection of circulating L

Metabolic Reprogramming During Purine Stress in the Protozoan Pathogen Leishmania donovani

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Martin, Jessica L.; Yates, Phillip A.; Soysa, Radika; Alfaro, Joshua F.; Yang, Feng; Burnum-Johnson, Kristin E.; Petyuk, Vladislav A.; Weitz, Karl K.; Camp, David G.; Smith, Richard D.; Wilmarth, Phillip A.; David, Larry L.; Ramasamy, Gowthaman; Myler, Peter J.; Carter, Nicola S.

2014-02-27

The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over 3 months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6-48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and/or post-translational mechanisms.

Integrating ribosomal promoter vectors that offer a choice of constitutive expression profiles in Leishmania donovani.

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Soysa, Radika; Tran, Khoa D; Ullman, Buddy; Yates, Phillip A

2015-12-01

We have designed a novel series of integrating ribosomal RNA promoter vectors with five incrementally different constitutive expression profiles, covering a 250-fold range. Differential expression was achieved by placing different combinations of synthetic or leishmanial DNA sequences upstream and downstream of the transgene coding sequence in order to modulate pre-mRNA processing efficiency and mRNA stability, respectively. All of the vectors have extensive multiple cloning sites, and versions are available for producing N- or C- terminal GFP fusions at each of the possible relative expression levels. In addition, the modular configuration of the vectors allows drug resistance cassettes and other components to be readily exchanged. In toto, these vectors should be useful additions to the toolkit available for molecular and genetic studies of Leishmania donovani. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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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.

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

The effect of Leishmania major and L. donovani surface protease gp63 on surface markers on human T cells was studied using fluorescence-activated flow cytometry. Purified gp63 (63,000 m.w. glycoprotein) at concentrations above 10 micrograms/ml completely inhibited binding of six different anti-CD4......-expression of CD4, reaching 50% of the initial level after 72 h of incubation in medium. Preincubation of cells with live promastigotes showed an inhibitory effect on CD4 comparable to that seen with purified gp63. The binding of Abs directed against other surface markers present on human T-cells--CD2, CD3, CD5......, CD8, CD11A, CD25, CD45RO, CD45RA, CD58, TCR-alpha, TCR-gamma, and HLA DQ--was not inhibited by gp63. These data suggest that gp63, both in its purified form and in the form anchored to the parasite membrane, cleaves CD4 on human T cells. The cleavage of CD4 by the protease might play a role...

Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.

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Lynette Beattie

2010-03-01

Full Text Available Kupffer cells (KCs represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens. Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8(+ T cells. Here, using a combination of approaches including whole mount and thin section confocal microscopy, adoptive cell transfer and intra-vital 2-photon microscopy, we demonstrate that KCs represent the only detectable population of mononuclear phagocytes within granulomas induced by Leishmania donovani infection that are capable of presenting parasite-derived peptide to effector CD8(+ T cells. This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

Cholesterol Corrects Altered Conformation of MHC-II Protein in Leishmania donovani Infected Macrophages: Implication in Therapy

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Chakrabarti, Saikat; Roy, Syamal

2016-01-01

Background Previously we reported that Kala-azar patients show progressive decrease in serum cholesterol as a function of splenic parasite burden. Splenic macrophages (MΦ) of Leishmania donovani (LD) infected mice show decrease in membrane cholesterol, while LD infected macrophages (I-MΦ) show defective T cell stimulating ability that could be corrected by liposomal delivery of cholesterol. T helper cells recognize peptide antigen in the context of class II MHC molecule. It is known that the conformation of a large number of membrane proteins is dependent on membrane cholesterol. In this investigation we tried to understand the influence of decreased membrane cholesterol in I-MΦ on the conformation of MHC-II protein and peptide-MHC-II stability, and its bearing on the antigen specific T-cell activation. Methodology/Principal Findings MΦ of CBA/j mice were infected with Leishmania donovani (I-MΦ). Two different anti-Aκ mAbs were used to monitor the status of MHC-II protein under parasitized condition. One of them (11.5–2) was conformation specific, whereas the other one (10.2.16) was not. Under parasitized condition, the binding of 11.5–2 decreased significantly with respect to the normal counterpart, whereas that of 10.2.16 remained unaltered. The binding of 11.5–2 was restored to normal upon liposomal delivery of cholesterol in I-MΦ. By molecular dynamics (MD) simulation studies we found that there was considerable conformational fluctuation in the transmembrane domain of the MHC-II protein in the presence of membrane cholesterol than in its absence, which possibly influenced the distal peptide binding groove. This was evident from the faster dissociation of the cognate peptide from peptide-MHC complex under parasitized condition, which could be corrected by liposomal delivery of cholesterol in I-MΦ. Conclusion The decrease in membrane cholesterol in I-MΦ may lead to altered conformation of MHC II, and this may contribute to a faster dissociation of

Cross-protective immunity to Leishmania amazonensis is mediated by CD4+ and CD8+-epitopes of Leishmania donovani Nucleoside Hydrolase terminal domains

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Dirlei eNico

2014-05-01

Full Text Available The Nucleoside hydrolase of Leishmania donovani (NH36 is a phylogenetic marker of high homology among Leishmania parasites. In mice and dog vaccination NH36 induces a CD4+ T cell-driven protective response against Leishmania chagasi infection directed against its C-terminal domain (F3. The C-terminal and N-terminal domain vaccines also decreased the footpad lesion caused by Leishmania amazonensis. We studied the basis of the crossed immune response using recombinant generated peptides covering the whole NH36 sequence and saponin for mice prophylaxis against L. amazonensis. The F1 (amino acids 1-103 and F3 peptide (amino acids 199-314 vaccines enhanced the IgG and IgG2a anti-NH36 antibodies to similar levels. The F3 vaccine induced the strongest DTH response, the highest proportions of NH36-specific CD4+ and CD8+ T cells after challenge and the highest expression of IFN-γ and TNF-α. The F1 vaccine, on the other hand, induced a weaker but significant DTH response and a mild enhancement of IFN-γ and TNF-α levels. The in vivo depletion with anti-CD4 or CD8 monoclonal antibodies disclosed that cross-protection against L. amazonensis infection was mediated by a CD4+ T cell response directed against the C-terminal domain (75% of reduction of the size of footpad lesion followed by a CD8+ T cell response against the N-terminal domain of NH36 (57% of reduction of footpad lesions. Both vaccines were capable of inducing long-term cross-immunity. The amino acid sequence of NH36 showed 93% identity to the sequence of the NH A34480 of L. amazonensis which also showed the presence of completely conserved predicted epitopes for CD4+ and CD8+ T cells in F1 domain, and of CD4+ epitopes differing in a single amino acid, in F1 and F3 domains. The identification of the C-terminal and N-terminal domains as the targets of the immune response to NH36 in the model of L. amazonesis infection represents a basis for the rationale development of a bivalent vaccine

First Molecular Characterization of Leishmania Species Causing Visceral Leishmaniasis among Children in Yemen.

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Mahdy, Mohammed A K; Al-Mekhlafi, Abdulsalam M; Abdul-Ghani, Rashad; Saif-Ali, Reyadh; Al-Mekhlafi, Hesham M; Al-Eryani, Samira M; Lim, Yvonne A L; Mahmud, Rohela

2016-01-01

Visceral leishmaniasis (VL) is a debilitating, often fatal disease caused by Leishmania donovani complex; however, it is a neglected tropical disease. L. donovani complex comprises two closely related species, L. donovani that is mostly anthroponotic and L. infantum that is zoonotic. Differentiation between these two species is critical due to the differences in their epidemiology and pathology. However, they cannot be differentiated morphologically, and their speciation using isoenzyme-based methods poses a difficult task and may be unreliable. Molecular characterization is now the most reliable method to differentiate between them and to determine their phylogenetic relationships. The present study aims to characterize Leishmania species isolated from bone marrows of Yemeni pediatric patients using sequence analysis of the ribosomal internal transcribed spacer-1 (ITS1) gene. Out of 41 isolates from Giemsa-stained bone marrow smears, 25 isolates were successfully amplified by nested polymerase chain reaction and sequenced in both directions. Phylogenetic analysis using neighbor joining method placed all study isolates in one cluster with L. donovani complex (99% bootstrap). The analysis of ITS1 for microsatellite repeat numbers identified L. infantum in 11 isolates and L. donovani in 14 isolates. These data suggest the possibility of both anthroponotic and zoonotic transmission of VL-causing Leishmania species in Yemen. Exploring the possible animal reservoir hosts is therefore needed for effective control to be achieved.

Cloning, characterization and sub-cellular localization of gamma subunit of T-complex protein-1 (chaperonin) from Leishmania donovani

Energy Technology Data Exchange (ETDEWEB)

Bhaskar,; Kumari, Neeti [Division of Biochemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, PO Box 173, Lucknow (India); Goyal, Neena, E-mail: neenacdri@yahoo.com [Division of Biochemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, PO Box 173, Lucknow (India)

2012-12-07

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

Cloning, characterization and sub-cellular localization of gamma subunit of T-complex protein-1 (chaperonin) from Leishmania donovani

International Nuclear Information System (INIS)

Bhaskar,; Kumari, Neeti; Goyal, Neena

2012-01-01

Highlights: ► The study presents cloning and characterization of TCP1γ gene from L. donovani. ► TCP1γ is a subunit of T-complex protein-1 (TCP1), a chaperonin class of protein. ► LdTCPγ exhibited differential expression in different stages of promastigotes. ► LdTCPγ co-localized with actin, a cytoskeleton protein. ► The data suggests that this gene may have a role in differentiation/biogenesis. ► First report on this chapronin in Leishmania. -- Abstract: T-complex protein-1 (TCP1) complex, a chaperonin class of protein, ubiquitous in all genera of life, is involved in intracellular assembly and folding of various proteins. The gamma subunit of TCP1 complex (TCP1γ), plays a pivotal role in the folding and assembly of cytoskeleton protein(s) as an individual or complexed with other subunits. Here, we report for the first time cloning, characterization and expression of the TCP1γ of Leishmania donovani (LdTCP1γ), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1γ revealed the presence of all the characteristic features of TCP1γ. However, leishmanial TCP1γ represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1γ exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1γ as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1γ was studied in log phase promastigotes by employing indirect immunofluorescence microscopy. The protein was present not only in cytoplasm but it was also localized in nucleus, peri-nuclear region, flagella, flagellar pocket and apical region. Co-localization of LdTCP1γ with actin suggests that, this gene may have a role in maintaining the structural dynamics of cytoskeleton of parasite.

Novel features of a PIWI-like protein homolog in the parasitic protozoan Leishmania.

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Prasad K Padmanabhan

Full Text Available In contrast to nearly all eukaryotes, the Old World Leishmania species L. infantum and L. major lack the bona fide RNAi machinery genes. Interestingly, both Leishmania genomes code for an atypical Argonaute-like protein that possesses a PIWI domain but lacks the PAZ domain found in Argonautes from RNAi proficient organisms. Using sub-cellular fractionation and confocal fluorescence microscopy, we show that unlike other eukaryotes, the PIWI-like protein is mainly localized in the single mitochondrion in Leishmania. To predict PIWI function, we generated a knockout mutant for the PIWI gene in both L. infantum (Lin and L. major species by double-targeted gene replacement. Depletion of PIWI has no effect on the viability of insect promastigote forms but leads to an important growth defect of the mammalian amastigote lifestage in vitro and significantly delays disease pathology in mice, consistent with a higher expression of the PIWI transcript in amastigotes. Moreover, amastigotes lacking PIWI display a higher sensitivity to apoptosis inducing agents than wild type parasites, suggesting that PIWI may be a sensor for apoptotic stimuli. Furthermore, a whole-genome DNA microarray analysis revealed that loss of LinPIWI in Leishmania amastigotes affects mostly the expression of specific subsets of developmentally regulated genes. Several transcripts encoding surface and membrane-bound proteins were found downregulated in the LinPIWI((-/- mutant whereas all histone transcripts were upregulated in the null mutant, supporting the possibility that PIWI plays a direct or indirect role in the stability of these transcripts. Although our data suggest that PIWI is not involved in the biogenesis or the stability of small noncoding RNAs, additional studies are required to gain further insights into the role of this protein on RNA regulation and amastigote development in Leishmania.

AMP-acetyl CoA synthetase from Leishmania donovani: identification and functional analysis of 'PX4GK' motif.

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Soumya, Neelagiri; Kumar, I Sravan; Shivaprasad, S; Gorakh, Landage Nitin; Dinesh, Neeradi; Swamy, Kayala Kambagiri; Singh, Sushma

2015-04-01

An adenosine monophosphate forming acetyl CoA synthetase (AceCS) which is the key enzyme involved in the conversion of acetate to acetyl CoA has been identified from Leishmania donovani for the first time. Sequence analysis of L. donovani AceCS (LdAceCS) revealed the presence of a 'PX4GK' motif which is highly conserved throughout organisms with higher sequence identity (96%) to lower sequence identity (38%). A ∼ 77 kDa heterologous protein with C-terminal 6X His-tag was expressed in Escherichia coli. Expression of LdAceCS in promastigotes was confirmed by western blot and RT-PCR analysis. Immunolocalization studies revealed that it is a cytosolic protein. We also report the kinetic characterization of recombinant LdAceCS with acetate, adenosine 5'-triphosphate, coenzyme A and propionate as substrates. Site directed mutagenesis of residues in conserved PX4GK motif of LdAceCS was performed to gain insight into its potential role in substrate binding, catalysis and its role in maintaining structural integrity of the protein. P646A, G651A and K652R exhibited more than 90% loss in activity signifying its indispensible role in the enzyme activity. Substitution of other residues in this motif resulted in altered substrate specificity and catalysis. However, none of them had any role in modulation of the secondary structure of the protein except G651A mutant. Copyright © 2015 Elsevier B.V. All rights reserved.

Novel Arsenic Nanoparticles Are More Effective and Less Toxic than As (III) to Inhibit Extracellular and Intracellular Proliferation of Leishmania donovani

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Chakraborty, Sudipta; Bhar, Kaushik; Saha, Sandip; Chakrabarti, Rajarshi; Pal, Anjali

2014-01-01

Visceral leishmaniasis, a vector-borne tropical disease that is threatening about 350 million people worldwide, is caused by the protozoan parasite Leishmania donovani. Metalloids like arsenic and antimony have been used to treat diseases like leishmaniasis caused by the kinetoplastid parasites. Arsenic (III) at a relatively higher concentration (30 μg/mL) has been shown to have antileishmanial activity, but this concentration is reported to be toxic in several experimental mammalian systems. Nanosized metal (0) particles have been shown to be more effective than their higher oxidation state forms. There is no information so far regarding arsenic nanoparticles (As-NPs) as an antileishmanial agent. We have tested the antileishmanial properties of the As-NPs, developed for the first time in our laboratory. As-NPs inhibited the in vitro growth, oxygen consumption, infectivity, and intramacrophage proliferation of L. donovani parasites at a concentration which is about several fold lower than that of As (III). Moreover, this antileishmanial activity has comparatively less cytotoxic effect on the mouse macrophage cell line. It is evident from our findings that As-NPs have more potential than As (III) to be used as an antileishmanial agent. PMID:25614827

Estudio ultraestructural de la fagocitosis de promastigotes y amastigotes de Leishmania mexicana por la línea de células dendríticas FSDC

OpenAIRE

Ladys Sarmiento; Martha Ayala; Sandra Peña; Gerzaín Rodríguez; Zelandia Fermín; Felix J. Tapia

2006-01-01

Introducción. Las células dendríticas están presentes en la mayoría de los tejidos, ellas capturany presentan antígenos para activar a los linfocitos T. Objetivo. Se describe ultraestructuralmente la fagocitosis de Leishmania mexicana por lalínea de células dendríticas FSDC, una línea de células de Langerhans obtenida de la epidermisfetal de ratón, e inmortalizada por la transducción retroviral del oncogen v-myc. Materiales y métodos. Se obtuvieron amastigotes de la lesión de ratones ...

From genomes to vaccines: Leishmania as a model.

Science.gov (United States)

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

2002-01-01

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

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

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

2015-01-01

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

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

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Jarina Pena DaMata

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

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

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M Mohebali

2011-06-01

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

Screening For Inhibitors Of Essential Leishmania Glucose Transporters

Science.gov (United States)

2011-07-01

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

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

Science.gov (United States)

2013-01-01

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

In-silico screening and validation of high-affinity tetra-peptide inhibitor of Leishmania donovani O-acetyl serine sulfhydrylase (OASS).

Science.gov (United States)

Kant, Vishnu; Vijayakumar, Saravanan; Sahoo, Ganesh Chandra; Chaudhery, Shailendra S; Das, Pradeep

2018-02-07

OASS is a specific enzyme that helps Leishmania parasite to survive the oxidative stress condition in human macrophages. SAT C-terminal peptides in several organisms, including Leishmania, were reported to inhibit or reduce the activity of OASS. Small peptide and small molecules mimicking the SAT C-terminal residues are designed and tested for the inhibition of OASS in different organisms. Hence, in this study, all the possible tetra-peptide combinations were designed and screened based on the docking ability with Leishmania donovani OASS (Ld-OASS). The top ranked peptides were further validated for the stability using 50 ns molecular dynamic simulation. In order to identify the better binding capability of the peptides, the top peptides complexed with Ld-OASS were also subjected to molecular dynamic simulation. The docking and simulation results favored the peptide EWSI to possess greater advantage than previously reported peptide (DWSI) in binding with Ld-OASS active site. Also, screening of non-peptide inhibitor of Asinex Biodesign library based on the shape similarity of EWSI and DWSI was performed. The top similar molecules of each peptides were docked on to Ld-OASS active site and subsequently simulated for 20 ns. The results suggested that the ligand that shares high shape similarity with EWSI possess better binding capability than the ligand that shares high shape similarity with DWSI. This study revealed that the tetra-peptide EWSI had marginal advantage over DWSI in binding with Ld-OASS, thereby providing basis for defining a pharmacophoric scaffold for the design of peptidomimetic inhibitors as well as non-peptide inhibitors of Ld-OASS.

Critical roles for LIGHT and its receptors in generating T cell-mediated immunity during Leishmania donovani infection.

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Amanda C Stanley

2011-10-01

Full Text Available LIGHT (TNFSF14 is a member of the TNF superfamily involved in inflammation and defence against infection. LIGHT signals via two cell-bound receptors; herpes virus entry mediator (HVEM and lymphotoxin-beta receptor (LTβR. We found that LIGHT is critical for control of hepatic parasite growth in mice with visceral leishmaniasis (VL caused by infection with the protozoan parasite Leishmania donovani. LIGHT-HVEM signalling is essential for early dendritic cell IL-12/IL-23p40 production, and the generation of IFNγ- and TNF-producing T cells that control hepatic infection. However, we also discovered that LIGHT-LTβR interactions suppress anti-parasitic immunity in the liver in the first 7 days of infection by mechanisms that restrict both CD4(+ T cell function and TNF-dependent microbicidal mechanisms. Thus, we have identified distinct roles for LIGHT in infection, and show that manipulation of interactions between LIGHT and its receptors may be used for therapeutic advantage.

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

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Daniel P Depledge

2010-09-01

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

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)

Characterization of dipeptidylcarboxypeptidase of Leishmania donovani: a molecular model for structure based design of antileishmanials

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Baig, Mirza Saqib; Kumar, Ashutosh; Siddiqi, Mohammad Imran; Goyal, Neena

2010-01-01

Leishmania donovani dipeptidylcarboxypeptidsae (LdDCP), an angiotensin converting enzyme (ACE) related metallopeptidase has been identified and characterized as a putative drug target for antileishmanial chemotherapy. The kinetic parameters for LdDCP with substrate, Hip-His-Leu were determined as, Km, 4 mM and Vmax, 1.173 μmole/ml/min. Inhibition studies revealed that known ACE inhibitors (captopril and bradykinin potentiating peptide; BPP1) were weak inhibitors for LdDCP as compared to human testicular ACE (htACE) with Ki values of 35.8 nM and 3.9 μM, respectively. Three dimensional model of LdDCP was generated based on crystal structure of Escherichia coli DCP (EcDCP) by means of comparative modeling and assessed using PROSAII, PROCHECK and WHATIF. Captopril docking with htACE, LdDCP and EcDCP and analysis of molecular electrostatic potentials (MEP) suggested that the active site domain of three enzymes has several minor but potentially important structural differences. These differences could be exploited for designing selective inhibitor of LdDCP thereby antileishmanial compounds either by denovo drug design or virtual screening of small molecule databases.

Development of a Species-Specific PCR Assay for Detection of Leishmania donovani in Clinical Samples from Patients with Kala-Azar and Post-Kala-Azar Dermal Leishmaniasis

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Salotra, Poonam; Sreenivas, G.; Pogue, Gregory P.; Lee, Nancy; Nakhasi, Hira L.; Ramesh, V.; Negi, N. S.

2001-01-01

We have developed a PCR assay that is capable of amplifying kinetoplast DNA (kDNA) of Leishmania donovani in a species-specific manner among Old World leishmanias. With Indian strains and isolates of L. donovani the assay was sensitive enough to detect kDNA in an amount equivalent to a single parasite or less. The extreme sensitivity of the assay was reflected in its ability to detect parasite DNA from small volumes of peripheral blood of patients with kala-azar (KA) and from skin lesions from patients with post-KA dermal leishmaniasis (PKDL). A total of 107 clinical leishmaniasis samples were analyzed. Of these 102 (95.3%) were positive by PCR. The test provided a diagnosis of KA with 96% sensitivity using patient whole-blood samples instead of bone marrow or spleen aspirates that are obtained by invasive procedures. The assay was also successful in the diagnosis of 45 of 48 PKDL cases (93.8%). Cross-reactions with pathogens prevalent in the area of endemicity, viz., Mycobacterium tuberculosis, Mycobacterium leprae, and Plasmodium spp., could be ruled out. Eighty-one control samples, including dermal scrapings from healthy portions of skin from patients with PKDL were all negative. Two of twenty controls from the area of endemicity were found positive by PCR assay; however, there was a good possibility that these two were asymptomatic carriers since they were serologically positive for KA. Thus, this PCR assay represents a tool for the diagnosis of KA and PKDL in Indian patients in a noninvasive manner, with simultaneous species identification of parasites in clinical samples. PMID:11230394

A high affinity Ca2(+)-ATPase on the surface membrane of Leishmania donovani promastigote

International Nuclear Information System (INIS)

Ghosh, J.; Ray, M.; Sarkar, S.; Bhaduri, A.

1990-01-01

A Ca2(+)-dependent ATP-hydrolytic activity was detected in the crude membrane ghost of the promastigote or vector form of the protozoal parasite Leishmania donovani, the pathogen responsible for kala azar. The Ca2(+)-ATPase was purified to apparent homogeneity after solubilization with deoxycholate. The enzyme consists of two subunits of Mr = 51,000 and 57,000 and has an apparent molecular weight of 215,000 +/- 12,000. The enzyme activity is exclusively dependent on Ca2+, and the pure enzyme can hydrolyze 1.6 mumol of ATP/min/mg of protein. The apparent Km for Ca2+ is 35 nM, which is further reduced to 12 nM in the presence of heterologous calmodulin. The enzyme is sensitive to vanadate, but is insensitive to oligomycin and ouabain. The enzyme is strongly associated with the plasma membrane and has its catalytic site oriented toward the cytoplasmic face. The enzyme spans across the plasma membrane as surface labeling with radioiodine shows considerable radioactivity in the completely purified enzyme. The localization and orientation of this high affinity, calmodulin-sensitive Ca2(+)-ATPase suggest some role of this enzyme in Ca2+ movement in the life cycle of this protozoal parasite

A high affinity Ca2(+)-ATPase on the surface membrane of Leishmania donovani promastigote

Energy Technology Data Exchange (ETDEWEB)

Ghosh, J.; Ray, M.; Sarkar, S.; Bhaduri, A. (Indian Institute of Chemical Biology, Calcutta (India))

1990-07-05

A Ca2(+)-dependent ATP-hydrolytic activity was detected in the crude membrane ghost of the promastigote or vector form of the protozoal parasite Leishmania donovani, the pathogen responsible for kala azar. The Ca2(+)-ATPase was purified to apparent homogeneity after solubilization with deoxycholate. The enzyme consists of two subunits of Mr = 51,000 and 57,000 and has an apparent molecular weight of 215,000 +/- 12,000. The enzyme activity is exclusively dependent on Ca2+, and the pure enzyme can hydrolyze 1.6 mumol of ATP/min/mg of protein. The apparent Km for Ca2+ is 35 nM, which is further reduced to 12 nM in the presence of heterologous calmodulin. The enzyme is sensitive to vanadate, but is insensitive to oligomycin and ouabain. The enzyme is strongly associated with the plasma membrane and has its catalytic site oriented toward the cytoplasmic face. The enzyme spans across the plasma membrane as surface labeling with radioiodine shows considerable radioactivity in the completely purified enzyme. The localization and orientation of this high affinity, calmodulin-sensitive Ca2(+)-ATPase suggest some role of this enzyme in Ca2+ movement in the life cycle of this protozoal parasite.

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

Science.gov (United States)

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

2013-03-01

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

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

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

2016-06-01

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

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

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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.

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

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B Bastrenta

2003-03-01

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

Presence of amastigotes in the central nervous system of hamsters infected with Leishmania sp. Presença de amastigotas em sistema nervoso central de hamster infectado com Leishmania sp.

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Elisangela de Oliveira

2011-06-01

Full Text Available Visceral leishmaniasis (VL is a severe chronic disease caused by Leishmania (Leishmania infantum chagasi. Better knowledge on the effects caused by this disease can help develop adequate clinical management and treatment. Parasitological and immunohistochemical studies were performed golden hamsters Mesocricetus auratus infected with bone marrow from individuals with VL in the State of Mato Grosso do Sul, central-west Brazil. The effects of parasitism in the spleen, liver, kidneys, lungs, heart and brain of the animals were examined. Eighteen hamsters were inoculated intraperitoneally, and six healthy animals were used as negative controls. The animals were kept in the animal house and checked for clinical signs. Specimens of each organ were examined for the presence of amastigotes. Immunohistochemical technique was performed in all brain specimens and organs negative on the direct examination of parasites. Direct examination of amastigotes was positive in the spleen and liver of all infected animals; 33.3% showed the parasite in the kidneys and lungs, and 16.7% in the heart. Parasitic forms were seen in 83.3% (15/18 of the brain examined. Immunohistochemistry confirmed the results of the direct examination, except in two specimens of lung tissue and in the brain specimens. Other studies are needed to further clarify the effect of the parasite in the central nervous system.A leishmaniose visceral (LV é uma doença crônica grave, causada pelo parasito Leishmania (Leishmania infantum chagasi. Esclarecer as alterações provocadas pela doença é fundamental para que se adotem condutas clínicas e de tratamento adequadas. Com o objetivo de analisar a infecção experimental em hamsters da linhagem golden, Mesocricetus auratus, infectados com tecido de medula óssea de pacientes com LV no Estado de Mato Grosso do Sul, foram realizados estudos parasitológicos e de imunomarcação. Foi verificada a distribuição do parasitismo no baço, f

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

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

2012-06-07

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

Novel protein–protein interaction between spermidine synthase and S-adenosylmethionine decarboxylase from Leishmania donovani

Energy Technology Data Exchange (ETDEWEB)

Mishra, Arjun K.; Agnihotri, Pragati; Srivastava, Vijay Kumar; Pratap, J. Venkatesh, E-mail: jvpratap@cdri.res.in

2015-01-09

Highlights: • L. donovani spermidine synthase and S-adenosylmethionine decarboxylase have been cloned and purified. • S-adenosylmethionine decarboxylase has autocatalytic property. • GST pull down assay shows the two proteins to form a metabolon. • Isothermal titration calorimetry shows that binding was exothermic having K{sub d} value of 0.4 μM. • Interaction confirmed by fluorescence spectroscopy and size exclusion chromatography. - Abstract: Polyamine biosynthesis pathway has long been considered an essential drug target for trypanosomatids including Leishmania. S-adenosylmethionine decarboxylase (AdoMetDc) and spermidine synthase (SpdSyn) are enzymes of this pathway that catalyze successive steps, with the product of the former, decarboxylated S-adenosylmethionine (dcSAM), acting as an aminopropyl donor for the latter enzyme. Here we have explored the possibility of and identified the protein–protein interaction between SpdSyn and AdoMetDc. The protein–protein interaction has been identified using GST pull down assay. Isothermal titration calorimetry reveals that the interaction is thermodynamically favorable. Fluorescence spectroscopy studies also confirms the interaction, with SpdSyn exhibiting a change in tertiary structure with increasing concentrations of AdoMetDc. Size exclusion chromatography suggests the presence of the complex as a hetero-oligomer. Taken together, these results suggest that the enzymes indeed form a heteromer. Computational analyses suggest that this complex differs significantly from the corresponding human complex, implying that this complex could be a better therapeutic target than the individual enzymes.

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

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Willian Marinho Dourado Coelho

2016-06-01

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

In vitro activity of the beta-carboline alkaloids harmane, harmine, and harmaline toward parasites of the species Leishmania infantum.

Science.gov (United States)

Di Giorgio, C; Delmas, F; Ollivier, E; Elias, R; Balansard, G; Timon-David, P

2004-01-01

Harmane, harmine, and harmaline were investigated for their in vitro antileishmanial activity toward parasites of the species Leishmania infantum. Harmane and Harmine displayed a moderate antiproliferative activity toward human monocytes and exerted a weak antileishmanial activity toward both the promastigote and the amastigote forms of the parasite. Their mechanism of action on the promastigote form of the parasite involved interactions with DNA metabolism leading to an accumulation of parasites in the S-G(2)M phases of the cell-cycle. Harmaline, at the contrary, was deprived from toxicity toward human cells and Leishmania promastigotes, however it exerted a strong antileishmanial activity toward the intracellular amastigote form of the parasite. This property was shown to partly result from the capacity of the molecule to prevent parasite internalization within macrophages by inhibiting Leishmania PKC activity.

Leishmania resistant to sodium stibogluconate: drug-associated macrophage-dependent killing

DEFF Research Database (Denmark)

Ibrahim, M E; Hag-Ali, M; el-Hassan, A M

1994-01-01

A total of 17 Leishmania isolates, 6 of them isolated from antimony-resistant patients, were collected in the Sudan and tested for their sensitivity to sodium stibogluconate (Pentostam) as promastigotes. Six of those isolates were tested as amastigotes infecting a murine macrophage cell line...

[Infecting glial cells with antimony resistant Leishmania tropica: A new ex-vivo model].

Science.gov (United States)

Zorbozan, Orçun; Harman, Mehmet; Evren, Vedat; Erdoğan, Mümin Alper; Kılavuz, Aslı; Tunalı, Varol; Çavuş, İbrahim; Yılmaz, Özlem; Özbilgin, Ahmet; Turgay, Nevin

2018-01-01

Leishmaniasis is a vector-borne zoonotic disease that shows different clinical features like cutaneous, mucocutaneous, visceral and viscerotropic forms. The protocols used in the treatment of leishmaniasis are toxic and have many limitations during administration. One of the limitations of treatment is the resistance against the protocols in practice. There is also a need to define new treatment options especially for resistant patients. Ex-vivo models using primary cell cultures may be a good source for evaluating new drug options in patients with antimony resistance, in addition to in-vitro and in-vivo studies. In this study, it was aimed to define a new ex-vivo culture model to evaluate treatment options in patients with cutaneous leishmaniasis who did not respond to treatment. In our experimental model of ex-vivo infection, Leishmania tropica promastigotes isolated from a case previously diagnosed with cutaneous leishmaniasis were used. The primary astroglial cell culture used for the ex-vivo model was prepared from 2-3 days old neonatal Sprague Dawley rat brains under sterile conditions by the modification McCarthy's method. The astroglia cells, which reached sufficient density, were infected with antimony resistant L.tropica promastigotes. After 24 hours of incubation, the supernatant on the cells were collected, the cell culture plate was dried at room temperature, then fixed with methyl alcohol and stained with Giemsa to search for L.tropica amastigotes. Amastigotes were intensely observed in glia cells in primary cell cultures infected with L.tropica promastigotes. No promastigotes were seen on Giemsa stained preparations of the precipitates prepared from the bottom sediment after the centrifugation of the liquid medium removed from the infected plates. In this study, promastigotes from a cutaneous leishmaniasis patient unable to respond to pentavalent antimony therapy were shown to infect rat glia cells and converted to amastigote form. This amastigote

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

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

2016-01-01

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

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

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

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Klinger Antonio da Franca Rodrigues

2013-01-01

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

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

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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.

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

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Carreira João Carlos

2012-06-01

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

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

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

2009-01-01

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

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

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Mário A. P. Moraes

1994-10-01

to Leishmania (Leishmania amazonensis are reported. In this form, less known than the diffuse one caused by the same species, the clinical manifestations are identical to those produced by other Leishmania species of the subgenus Viannia. There is, however, in the localized infection by L (L. amazonensis, a peculiar feature, only recently discovered: about 50% of the affected individuals are Montenegro-negatives. The main histologic change observed in the skin sections was the presence of groups of macrophages with a large vacuole in the cytoplasm, containing many amastigotes. The microscopic picture is similar to that found in the diffuse form of the disease, the difference being only quantitative. When in large numbers, the macrophages suffers necrosis, which generally starts at the center of the groups. First, in this process, the membrane of the parasitized cells ruptures, and the amastigotes become free; later, both cells and parasites are destroyed. The picture can be seen either in Montenegro-negative or in Montenegro-positive patients. The macrophages with amastigotes may persist in tissues for as long as 6-7 months, while in the infections due to L (V. braziliensis the parasites usually disappear in a few weeks.

IFN-γ, IL-2, IP-10, and MIG as Biomarkers of Exposure to Leishmania spp., and of Cure in Human Visceral Leishmaniasis

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Ana V. Ibarra-Meneses

2017-05-01

Full Text Available New biomarkers are needed for monitoring the effectiveness of treatment for visceral leishmaniasis (VL. They might also improve the detection of the asymptomatic population in Leishmania-endemic areas. This paper examines the IL-2, IFN-γ, IFN-γ-induced protein 10 (IP-10, and monokine-induced-by-IFN-γ (MIG levels in whole blood—stimulated in vitro with soluble Leishmania antigen (SLA—taken from asymptomatic individuals and patients treated for VL living in a post-outbreak (Leishmania infantum area in Spain, and in an endemic (Leishmania donovani area of Bangladesh. IP-10 was found to be an accurate global marker of asymptomatic subjects with positive cellular/humoral tests, while MIG was found to be a better marker of contact with L. donovani than IL-2 but no for those with L. infantum. Determining IP-10, MIG, and IFN-γ levels proved useful in monitoring the cellular immune response following treatment for active disease caused by L. infantum.

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

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

2017-04-01

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

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

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Ming Jang Chua

2017-04-01

Full Text Available Malaria, schistosomiasis and leishmaniases are among the most prevalent tropical parasitic diseases and each requires new innovative treatments. Targeting essential parasite pathways, such as those that regulate gene expression and cell cycle progression, is a key strategy for discovering new drug leads. In this study, four clinically approved anti-cancer drugs (Vorinostat, Belinostat, Panobinostat and Romidepsin that target histone/lysine deacetylase enzymes were examined for in vitro activity against Plasmodium knowlesi, Schistosoma mansoni, Leishmania amazonensis and L. donovani parasites and two for in vivo activity in a mouse malaria model. All four compounds were potent inhibitors of P. knowlesi malaria parasites (IC50 9–370 nM, with belinostat, panobinostat and vorinostat having 8–45 fold selectivity for the parasite over human neonatal foreskin fibroblast (NFF or human embryonic kidney (HEK 293 cells, while romidepsin was not selective. Each of the HDAC inhibitor drugs caused hyperacetylation of P. knowlesi histone H4. None of the drugs was active against Leishmania amastigote or promastigote parasites (IC50 > 20 μM or S. mansoni schistosomula (IC50 > 10 μM, however romidepsin inhibited S. mansoni adult worm parings and egg production (IC50 ∼10 μM. Modest in vivo activity was observed in P. berghei infected mice dosed orally with vorinostat or panobinostat (25 mg/kg twice daily for four days, with a significant reduction in parasitemia observed on days 4–7 and 4–10 after infection (P < 0.05, respectively.

Experimental induction of paromomycin resistance in antimony-resistant strains of L. donovani: outcome dependent on in vitro selection protocol.

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Sarah Hendrickx

Full Text Available Paromomycin (PMM has recently been introduced for treatment of visceral leishmaniasis in India. Although no clinical resistance has yet been reported, proactive vigilance should be warranted. The present in vitro study compared the outcome and stability of experimental PMM-resistance induction on promastigotes and intracellular amastigotes. Cloned antimony-resistant L. donovani field isolates from India and Nepal were exposed to stepwise increasing concentrations of PMM (up to 500 µM, either as promastigotes or intracellular amastigotes. One resulting resistant strain was cloned and checked for stability of resistance by drug-free in vitro passage as promastigotes for 20 weeks or a single in vivo passage in the golden hamster. Resistance selection in promastigotes took about 25 weeks to reach the maximal 97 µM inclusion level that did not affect normal growth. Comparison of the IC(50 values between the parent and the selected strains revealed a 9 to 11-fold resistance for the Indian and 3 to 5-fold for the Nepalese strains whereby the resistant phenotype was also maintained at the level of the amastigote. Applying PMM pressure to intracellular amastigotes produced resistance after just two selection cycles (IC(50 = 199 µM compared to the parent strain (IC(50 = 45 µM. In the amastigote-induced strains/clones, lower PMM susceptibilities were seen only in amastigotes and not at all in promastigotes. This resistance phenotype remained stable after serial in vitro passage as promastigote for 20 weeks and after a single in vivo passage in the hamster. This study clearly demonstrates that a different PMM-resistance phenotype is obtained whether drug selection is applied to promastigotes or intracellular amastigotes. These findings may have important relevance to resistance mechanism investigations and the likelihood of resistance development and detection in the field.

Induction of a stringent metabolic response in intracellular stages of Leishmania mexicana leads to increased dependence on mitochondrial metabolism.

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Eleanor C Saunders

2014-01-01

Full Text Available Leishmania parasites alternate between extracellular promastigote stages in the insect vector and an obligate intracellular amastigote stage that proliferates within the phagolysosomal compartment of macrophages in the mammalian host. Most enzymes involved in Leishmania central carbon metabolism are constitutively expressed and stage-specific changes in energy metabolism remain poorly defined. Using (13C-stable isotope resolved metabolomics and (2H2O labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state. This state is characterized by a global decrease in the uptake and utilization of glucose and amino acids, a reduced secretion of organic acids and increased fatty acid β-oxidation. Isotopomer analysis showed that catabolism of hexose and fatty acids provide C4 dicarboxylic acids (succinate/malate and acetyl-CoA for the synthesis of glutamate via a compartmentalized mitochondrial tricarboxylic acid (TCA cycle. In vitro cultivated and intracellular amastigotes are acutely sensitive to inhibitors of mitochondrial aconitase and glutamine synthetase, indicating that these anabolic pathways are essential for intracellular growth and virulence. Lesion-derived amastigotes exhibit a similar metabolism to in vitro differentiated amastigotes, indicating that this stringent response is coupled to differentiation signals rather than exogenous nutrient levels. Induction of a stringent metabolic response may facilitate amastigote survival in a nutrient-poor intracellular niche and underlie the increased dependence of this stage on hexose and mitochondrial metabolism.

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

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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

Benzaldehyde thiosemicarbazone derived from limonene complexed with copper induced mitochondrial dysfunction in Leishmania amazonensis.

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Elizandra Aparecida Britta

Full Text Available BACKGROUND: Leishmaniasis is a major health problem that affects more than 12 million people. Treatment presents several problems, including high toxicity and many adverse effects, leading to the discontinuation of treatment and emergence of resistant strains. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the in vitro antileishmanial activity of benzaldehyde thiosemicarbazone derived from limonene complexed with copper, termed BenzCo, against Leishmania amazonensis. BenzCo inhibited the growth of the promastigote and axenic amastigote forms, with IC(50 concentrations of 3.8 and 9.5 µM, respectively, with 72 h of incubation. Intracellular amastigotes were inhibited by the compound, with an IC(50 of 10.7 µM. BenzCo altered the shape, size, and ultrastructure of the parasites. Mitochondrial membrane depolarization was observed in protozoa treated with BenzCo but caused no alterations in the plasma membrane. Additionally, BenzCo induced lipoperoxidation and the production of mitochondrial superoxide anion radicals in promastigotes and axenic amastigotes of Leishmania amazonensis. CONCLUSION/SIGNIFICANCE: Our studies indicated that the antileishmania activity of BenzCo might be associated with mitochondrial dysfunction and oxidative damage, leading to parasite death.

Genetic Diversity and Phylogenetic Analysis of the Iranian Leishmania Parasites Based on HSP70 Gene PCR-RFLP and Sequence Analysis.

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Nemati, Sara; Fazaeli, Asghar; Hajjaran, Homa; Khamesipour, Ali; Anbaran, Mohsen Falahati; Bozorgomid, Arezoo; Zarei, Fatah

2017-08-01

Despite the broad distribution of leishmaniasis among Iranians and animals across the country, little is known about the genetic characteristics of the causative agents. Applying both HSP70 PCR-RFLP and sequence analyses, this study aimed to evaluate the genetic diversity and phylogenetic relationships among Leishmania spp. isolated from Iranian endemic foci and available reference strains. A total of 36 Leishmania isolates from almost all districts across the country were genetically analyzed for the HSP70 gene using both PCR-RFLP and sequence analysis. The original HSP70 gene sequences were aligned along with homologous Leishmania sequences retrieved from NCBI, and subjected to the phylogenetic analysis. Basic parameters of genetic diversity were also estimated. The HSP70 PCR-RFLP presented 3 different electrophoretic patterns, with no further intraspecific variation, corresponding to 3 Leishmania species available in the country, L. tropica, L. major, and L. infantum. Phylogenetic analyses presented 5 major clades, corresponding to 5 species complexes. Iranian lineages, including L. major, L. tropica, and L. infantum, were distributed among 3 complexes L. major, L. tropica, and L. donovani. However, within the L. major and L. donovani species complexes, the HSP70 phylogeny was not able to distinguish clearly between the L. major and L. turanica isolates, and between the L. infantum, L. donovani, and L. chagasi isolates, respectively. Our results indicated that both HSP70 PCR-RFLP and sequence analyses are medically applicable tools for identification of Leishmania species in Iranian patients. However, the reduced genetic diversity of the target gene makes it inevitable that its phylogeny only resolves the major groups, namely, the species complexes.

Evidence that leishmania donovani utilizes a mannose receptor on human mononuclear phagocytes to establish intracellular parasitism

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Wilson, M.E.; Pearson, R.D.

1986-01-01

The pathogenic protozoan Leishmania donovani must gain entrance into mononuclear phagocytes to successfully parasitize man. The parasite's extracellular promastigote stage is ingested by human peripheral blood monocytes or monocyte-derived macrophages in the absence of serum, in a manner characteristic of receptor-mediated endocytosis. Remarkable similarities have been found between the macrophage receptor(s) for promastigotes and a previously characterized eucaryotic receptor system, the mannose/fucose receptor (MFR), that mediates the binding of zymosan particles and mannose- or fucose-terminal glycoconjugates to macrophages. Ingestion of promastigotes by monocyte-derived macrophages was inhibited by several MFR ligands; that is mannan, mannose-BSA and fucose-BSA. In contrast, promastigote ingestion by monocytes was unaffected by MFR ligands. Furthermore, attachment of promastigotes to macrophages, assessed by using cytochalasin D to prevent phagocytosis, was reduced 49.8% by mannan. Reorientation of the MFR to the ventral surface of the cell was achieved by plating macrophages onto mannan-coated coverslips, reducing MFR activity on the exposed cell surface by 94% as assessed by binding of 125 I-mannose-BSA. Under these conditions, ingestion of promastigotes was inhibited by 71.4%. Internalization of the MFR by exposure of macrophages to zymosan before infection with promastigotes resulted in a 62.3% decrease in parasite ingestion. Additionally, NH 4 Cl decreased macrophage ingestion of promastigotes by 38.2%. Subinhibitory concentration of NH 4 Cl (10 mM) and of mannan (0.25 mg/ml) together inhibited parsite ingestion by 76.4%

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

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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

Impact of phlebotomine sand flies on U.S. military operations at Tallil Air Base, Iraq: 4. Detection and identification of leishmania parasites in sand flies.

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Coleman, Russell E; Hochberg, Lisa P; Swanson, Katherine I; Lee, John S; McAvin, James C; Moulton, John K; Eddington, David O; Groebner, Jennifer L; O'Guinn, Monica L; Putnam, John L

2009-05-01

Sand flies collected between April 2003 and November 2004 at Tallil Air Base, Iraq, were evaluated for the presence of Leishmania parasites using a combination of a real-time Leishmania-generic polymerase chain reaction (PCR) assay and sequencing of a 360-bp fragment of the glucose-6-phosphate-isomerase (GPI) gene. A total of 2,505 pools containing 26,574 sand flies were tested using the real-time PCR assay. Leishmania DNA was initially detected in 536 pools; however, after extensive retesting with the real-time PCR assay, a total of 456 pools were considered positive and 80 were considered indeterminate. A total of 532 samples were evaluated for Leishmania GPI by sequencing, to include 439 PCR-positive samples, 80 PCR-indeterminate samples, and 13 PCR-negative samples. Leishmania GPI was detected in 284 samples that were sequenced, to include 281 (64%) of the PCR-positive samples and 3 (4%) of the PCR-indeterminate samples. Of the 284 sequences identified as Leishmania, 261 (91.9%) were L. tarentolae, 18 (6.3%) were L. donovani-complex parasites, 3 (1.1%) were L. tropica, and 2 were similar to both L. major and L. tropica. Minimum field infection rates were 0.09% for L. donovani-complex parasites, 0.02% for L. tropica, and 0.01% for the L. major/tropica-like parasite. Subsequent sequencing of a 600-bp region of the "Hyper" gene of 12 of the L. donovani-complex parasites showed that all 12 parasites were L. infantum. These data suggest that L. infantum was the primary leishmanial threat to U.S. military personnel deployed to Tallil Air Base. The implications of these findings are discussed.

Intradermal Immunization of Leishmania donovani Centrin Knock-Out Parasites in Combination with Salivary Protein LJM19 from Sand Fly Vector Induces a Durable Protective Immune Response in Hamsters.

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Jacqueline Araújo Fiuza

2016-01-01

Full Text Available Visceral leishmaniasis (VL is a neglected tropical disease and is fatal if untreated. There is no vaccine available against leishmaniasis. The majority of patients with cutaneous leishmaniasis (CL or VL develop a long-term protective immunity after cure from infection, which indicates that development of an effective vaccine against leishmaniasis is possible. Such protection may also be achieved by immunization with live attenuated parasites that do not cause disease. We have previously reported a protective response in mice, hamsters and dogs with Leishmania donovani centrin gene knock-out parasites (LdCen-/-, a live attenuated parasite with a cell division specific centrin1 gene deletion. In this study we have explored the effects of salivary protein LJM19 as an adjuvant and intradermal (ID route of immunization on the efficacy of LdCen-/- parasites as a vaccine against virulent L. donovani.To explore the potential of a combination of LdCen-/- parasites and salivary protein LJM19 as vaccine antigens, LdCen-/- ID immunization followed by ID challenge with virulent L. donovani were performed in hamsters in a 9-month follow up study. We determined parasite burden (serial dilution, antibody production (ELISA and cytokine expression (qPCR in these animals. Compared to controls, animals immunized with LdCen-/- + LJM19 induced a strong antibody response, a reduction in spleen and liver parasite burden and a higher expression of pro-inflammatory cytokines after immunization and one month post-challenge. Additionally, a low parasite load in lymph nodes, spleen and liver, and a non-inflamed spleen was observed in immunized animals 9 months after the challenge infection.Our results demonstrate that an ID vaccination using LdCen-/-parasites in combination with sand fly salivary protein LJM19 has the capability to confer long lasting protection against visceral leishmaniasis that is comparable to intravenous or intracardial immunization.

Anti Leishmania activity of Lucilia sericata and Calliphora vicina maggots in laboratory models.

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Sanei-Dehkordi, Alireza; Khamesipour, Ali; Akbarzadeh, Kamran; Akhavan, Amir Ahmad; Mir Amin Mohammadi, Akram; Mohammadi, Younes; Rassi, Yavar; Oshaghi, Mohammad Ali; Alebrahim, Zahra; Eskandari, Seyed Ebrahim; Rafinejad, Javad

2016-11-01

Use of sterile fly larvae (maggots) of blow flies for the treatment of many different types of skin and soft tissue wounds is called Maggot debridement therapy (MDT). The larvae of blow flies secrete a broad spectrum of compounds with diverse mechanisms of action in the gut and salivary glands called excretion/secretion (ES) products which showed to have antimicrobial activities against Gram negative and positive bacteria. Cutaneous leishmaniasis (CL) which is the common form of leishmaniasis is difficult to treat. In this study, the effect of ES from 2 nd and 3 rd stages of L. sericata and C. vicina larvae on in vitro Leishmania major amastigote growth in macrophage was evaluated. The effect of ES on Leishmania growth was estimated by assessing the rate of macrophage infection and the number of amastigotes per infected macrophages. In addition, the anti Leishmania activities of larval and ES of L. sericata and C. vicina on the skin lesion induced by L. major infection was evaluated in susceptible BALB/c mice. The results showed that ES of both flies reduced the number of infected macrophages; 2.6 and 1.5-fold using L. sericata ES and C. vicina ES, respectively, and inhibited amastigotes growth in macrophages; 2.03 and 1.36-fold by L. sericata ES and C. vicina ES, respectively as compared to the control group. The results showed that L. sericata ES was significantly more effective than C. vicina ES to inhibit in vitro L. major amastigotes growth, The size of lesion was significantly smaller in BALB/c mice treated with L. sericata ES than treated with C. vicina ES. The results of in vivo experiments suggested that pre-treatment with ES derived from L. sericata may have some protective effects on the development of L. major lesion. Therefore, it seems that maggot ES might be considered as a possible candidate for the treatment of cutaneous leishmaniasis. Copyright © 2016 Elsevier Inc. All rights reserved.

Interaccion de promastigotes de Leishmania donovani con celulas de exudado peritonial de ratones mediante la influencia de la quimotripsina

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Lucila Arcay

1986-06-01

Full Text Available Se hace un estudio de la interacción de promastigotes de Leishmania donovani con células de exudado peritoneal de ratón (c e p mediante la influencia de la quimotripsina. La adhesión de los promastigotes a las c e p fue terminal y marginal, y en observaciones hechas a partir de los 10 minutos de enfrentamiento, esta adhesión fue nula hasta los 30 minutos en el grupo tratado, y sólo a las das horas hubo un pequeño incremento (2.4% con respecto al control. Se observa marcada disminución en todos los parámetro medidos, tales como enlace, penetración, multiplicación intracelular, división de formas flageladas, en el grupo tratado. La quimotripsina favorece la formación de formas intermedias flageladas, heciendose el parásito piriforme y esférico, apareciendo una forma aberrante de extremo anterior cilindrico que semeja a una forma coanoflagelada. Se sospecha que la enzima reduce efectivamente fragmentos proteicos o péptidos, los cuales pueden haber sido tan pequeños como para esconder a otros ligandos relacionados con la adhesión macrófago-parásito.

SOLiD™ sequencing of genomes of clinical isolates of Leishmania donovani from India confirm leptomonas co-infection and raise some key questions.

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Neeloo Singh

Full Text Available BACKGROUND: Known as 'neglected disease' because relatively little effort has been applied to finding cures, leishmaniasis kills more than 150,000 people every year and debilitates millions more. Visceral leishmaniasis (VL, also called Kala Azar (KA or black fever in India, claims around 20,000 lives every year. Whole genome analysis presents an excellent means to identify new targets for drugs, vaccine and diagnostics development, and also provide an avenue into the biological basis of parasite virulence in the L. donovani complex prevalent in India. METHODOLOGY/PRINCIPAL FINDINGS: In our presently described study, the next generation SOLiD™ platform was successfully utilized for the first time to carry out whole genome sequencing of L. donovani clinical isolates from India. We report the exceptional occurrence of insect trypanosomatids in clinical cases of visceral leishmaniasis (Kala Azar patients in India. We confirm with whole genome sequencing analysis data that isolates which were sequenced from Kala Azar (visceral leishmaniasis cases were genetically related to Leptomonas. The co-infection in splenic aspirate of these patients with a species of Leptomonas and how likely is it that the infection might be pathogenic, are key questions which need to be investigated. We discuss our results in the context of some important probable hypothesis in this article. CONCLUSIONS/SIGNIFICANCE: Our intriguing results of unusual cases of Kala Azar found to be most similar to Leptomonas species put forth important clinical implications for the treatment of Kala Azar in India. Leptomonas have been shown to be highly susceptible to several standard leishmaniacides in vitro. There is very little divergence among these two species viz. Leishmania sp. and L. seymouri, in terms of genomic sequence and organization. A more extensive perception of the phenomenon of co-infection needs to be addressed from molecular pathogenesis and eco

Cryopreservation of Leishmania Species in Manisa Province.

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

2017-09-01

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

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

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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

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

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Mukhopadhyay, Aakash Gautam; Dey, Chinmoy Sankar

2017-11-01

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

Pharmacological activities of cilantro’s aliphatic Aldehydes against leishmania donovani

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Leishmaniasis is a chronic infectious disease caused by different Leishmania species. Global occurrences of this disease are primarily limited to tropical and subtropical regions. Treatments are available; however, patients complain of side effects. Different species of plants have been screened as ...

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

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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

Development of a rapid loop-mediated isothermal amplification assay for diagnosis and assessment of cure of Leishmania infection.

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Verma, Sandeep; Singh, Ruchi; Sharma, Vanila; Bumb, Ram Avtar; Negi, Narendra Singh; Ramesh, V; Salotra, Poonam

2017-03-23

Leishmaniasis is a spectrum of diseases with great relevance to public health. Conventional diagnostic methods are time consuming, needing trained personnel. A robust, rapid and cost effective diagnostic test is warranted for on-time diagnosis and field application. We have developed a loop mediated isothermal amplification (LAMP) assay with primers (n = 6) based on Leishmania donovani kDNA for detection of Leishmania infection, using a closed tube to prevent cross-contamination. The assay was used to detect Leishmania infection in biological samples obtained from patients of visceral leishmaniasis (VL), post kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL). The assay was positive for L. donovani, L. tropica and L. major parasites, with the highest sensitivity towards L. donovani (1 fg DNA). The high sensitivity of the assay for detection of L. donovani was reflected in its ability to detect parasite DNA within 30 min of amplification time with a threshold detection limit of ≥25 copies per reaction. The assay detected parasite in 64 of 66 VL blood samples (sensitivity, 96.9%; 95% CI: 89.6-99.2%), 15 of 15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI:79.6-100%), 65 of 67 PKDL tissue biopsy samples (sensitivity, 97%; 95% CI:89.7-99.2%). The assay was evaluated in a few cases of CL wherein it was found positive in 8 of 10 tissue biopsies (sensitivity, 80%; 95% CI: 49-94.3%). The assay was negative in all control blood (n = 76) and tissue biopsy (n = 24) samples (specificity, 100%; 95% CI: 96.3-100%). Further, the assay was evaluated for its utility in assessment of cure in treated VL and PKDL patients. The assay detected parasite DNA in 2 of 20VL blood samples and 2 of 21 PKDL tissue samples. Out of 4 cases that were positive for parasite DNA at post treatment stage, 2 patients (1VL and 1 PKDL) returned with relapse. The study demonstrated a Leishmania genus specific closed tube LAMP assay for reliable and rapid

Estudio ultraestructural de la fagocitosis de promastigotes y amastigotes de Leishmania mexicana por la línea de células dendríticas FSDC

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Ladys Sarmiento

2006-10-01

Full Text Available Introducción. Las células dendríticas están presentes en la mayoría de los tejidos, ellas capturany presentan antígenos para activar a los linfocitos T. Objetivo. Se describe ultraestructuralmente la fagocitosis de Leishmania mexicana por lalínea de células dendríticas FSDC, una línea de células de Langerhans obtenida de la epidermisfetal de ratón, e inmortalizada por la transducción retroviral del oncogen v-myc. Materiales y métodos. Se obtuvieron amastigotes de la lesión de ratones Balb/c y promastigotesa partir del cultivo (24°C de la lesión. Las FSDC se cultivaron con los parásitos en unaproporción de 5 parásitos por célula, en medio IMDM, durante 24 horas. Los cultivos infectadosy los controles se procesaron para microscopía electrónica de transmisión. Se hicieron cortessemifinos contrastados con azul de toluidina para evaluar porcentaje de fagocitosis y finos,contrastados con acetato de uranilo y citrato de plomo. Resultados. El 13,42% de las FSDC fagocitaron promastigotes; de ellas el 8% contenían unparásito y el restante 5,2% fagocitó dos o más. El 20% de las FSDC fagocitaron amastigotes;10% contenían un parásito y 10% dos o más. Ultraestructuralmente se observaron promastigotesen contacto con las células por el flagelo o por el polo posterior. Los fagosomas que conteníanpromastigotes eran organelos estrechos con uno ó dos parásitos. Los que conteníanamastigotes eran de gran tamaño (8 μm con uno o varios parásitos, libres o adosados a lamembrana del fagosoma por su polo posterior. Conclusión. La infección de las FSDC se caracterizó por una baja tasa de células infectadasal ser expuestas a promastigotes o amastigotes. La vacuola parasitofora presentó característicassimilares a las de los macrófagos. En su mayoría las FSDC presentaban 1 a 3 parásitos porcélula. Las observaciones plantean la necesidad de estudiar la relación entre capacidad defagocitosis y función.

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

In Vitro Study on effects of Amiodarone and Ketoconazole on Leishmania infantum

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Mohammad Hosein Razi Jalali

2014-09-01

Full Text Available Background & objectives: The leishmaniases are considered among the major infectious diseases affecting public health in several regions. There are many chemical agents which are effective in treatment of visceral leishmaniasis. But, overall treatment of visceral leishmaniasis is often difficult. Thus, identification of new chemotherapeutic agents is important for treatment of disease. Since targeting of the ergosterol synthesis pathway of Leishmania may be useful therapeutically, the aim of this study was to investigate the effect of alone or in combination of amiodarone and ketoconazole on Leishmania infantum.   Methods : To obtain logarithmic promastigotes of L. infantum, the parasites were cultured in BHI medium with FCS 10% together with antibiotics of penicillin and streptomycin and incubated at 24° C. Amastigote forms were obtained in BHI medium supplemented with 20% FCS at pH of 5.5 which incubated in 37° C. L.infantum susceptibility to amiodarone and ketoconazole was evaluated by proliferation of parasites in the absence or presence of these drugs with MTT assay. For evaluation of antiproliferative synergism against promastigotes and axenic amastigotes, fractional inhibitory concentrations (FIC were calculated. An isobologram curve was constructed too.   Results: Amiodarone produced a marked reduction in the viability of L.infantum promastigotes and axenic amastigotes. On the other hand ketoconazole induced a dose dependent effect on the parasites proliferation for promastigotes and axenic amastigotes. When the drugs were used in combination, the results indicated clear synergistic as shown by a concave isobologram and FIC value.   Conclusion: The present study represents the evidence that the combination of amiodarone plus ketoconazole acts synergistically in controlling L. infantume in vitro. It is possible that amiodarone could be used in combination with ketoconazole to combat infection at low doses, thus reducing its side

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

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

2017-10-01

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

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

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Willian Marinho Dourado Coelho

2010-12-01

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

Synthesis, X-ray crystal structure and theoretical calculations of antileishmanial neolignan analogues

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Nascimento, Josenaide P. do; Santos, Lourivaldo S.; Carmo, Maria Carolina L. do; Brasil, Davi S.B.; Alves, Claudio N., E-mail: nahum@ufpa.b [Universidade Federal do Para (UFPA), Belem, PA (Brazil). Inst. de Ciencias Exatas e Naturais; Santos, Regina Helena A.; Tozzo, Erica; Ferreira, Janaina G. [Universidade de Sao Paulo (IQSC/USP), Sao Carlos, SP (Brazil). Inst. de Quimica

2010-07-01

The synthesis and X-ray crystal diffraction structure of two analogues of neolignans, 2-(4-chlorophenyl)-1-phenylethanone (20) and 2-[(4-chlorophenyl)thio]-1-(3,4-dimethoxyphenyl) propan-1-one (12) is described. The compound 12 presents activity against intracellular Leishmania donovani and Leishmania amazonensis amastigotes that cause cutaneous and visceral leishmaniasis. In addition, the density functional theory (DFT) with the B3LYP hybrid functional was employed to calculate a set of molecular descriptors for nineteen synthetic analogues of neolignans with antileishmanial activities. Afterwards, the stepwise discriminant analysis was performed to investigate possible relationship between the molecular descriptors and biological activities. Through this analysis the compounds were classified into two groups active and inactive according to their degree of biological activities, and the more important properties were charges on some key atoms, electronic affinity and ClogP. (author)

Synthesis, X-ray crystal structure and theoretical calculations of antileishmanial neolignan analogues

International Nuclear Information System (INIS)

Nascimento, Josenaide P. do; Santos, Lourivaldo S.; Carmo, Maria Carolina L. do; Brasil, Davi S.B.; Alves, Claudio N.; Santos, Regina Helena A.; Tozzo, Erica; Ferreira, Janaina G.

2010-01-01

The synthesis and X-ray crystal diffraction structure of two analogues of neolignans, 2-(4-chlorophenyl)-1-phenylethanone (20) and 2-[(4-chlorophenyl)thio]-1-(3,4-dimethoxyphenyl) propan-1-one (12) is described. The compound 12 presents activity against intracellular Leishmania donovani and Leishmania amazonensis amastigotes that cause cutaneous and visceral leishmaniasis. In addition, the density functional theory (DFT) with the B3LYP hybrid functional was employed to calculate a set of molecular descriptors for nineteen synthetic analogues of neolignans with antileishmanial activities. Afterwards, the stepwise discriminant analysis was performed to investigate possible relationship between the molecular descriptors and biological activities. Through this analysis the compounds were classified into two groups active and inactive according to their degree of biological activities, and the more important properties were charges on some key atoms, electronic affinity and ClogP. (author)

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.

The Leishmania donovani UMP Synthase Is Essential for Promastigote Viability and Has an Unusual Tetrameric Structure That Exhibits Substrate-controlled Oligomerization

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French, Jarrod B.; Yates, Phillip A.; Soysa, D.Radika; Boitz, Jan M.; Carter, Nicola S.; Chang, Bailey; Ullman, Buddy; Ealick, Steven E. (Oregon HSU); (Cornell)

2011-08-09

The final two steps of de novo uridine 5'-monophosphate (UMP) biosynthesis are catalyzed by orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC). In most prokaryotes and simple eukaryotes these two enzymes are encoded by separate genes, whereas in mammals they are expressed as a bifunctional gene product called UMP synthase (UMPS), with OPRT at the N terminus and OMPDC at the C terminus. Leishmania and some closely related organisms also express a bifunctional enzyme for these two steps, but the domain order is reversed relative to mammalian UMPS. In this work we demonstrate that L. donovani UMPS (LdUMPS) is an essential enzyme in promastigotes and that it is sequestered in the parasite glycosome. We also present the crystal structure of the LdUMPS in complex with its product, UMP. This structure reveals an unusual tetramer with two head to head and two tail to tail interactions, resulting in two dimeric OMPDC and two dimeric OPRT functional domains. In addition, we provide structural and biochemical evidence that oligomerization of LdUMPS is controlled by product binding at the OPRT active site. We propose a model for the assembly of the catalytically relevant LdUMPS tetramer and discuss the implications for the structure of mammalian UMPS.

Detection and characterization of Leishmania in tissues of patients with post kala-azar dermal leishmaniasis using a specific monoclonal antibody

DEFF Research Database (Denmark)

Ismail, A; Kharazmi, A; Permin, H

1997-01-01

Sections from skin lesions and draining lymph nodes of patients with post kala-azar dermal leishmaniasis were examined using an immunoperoxidase method and a monoclonal antibody directed against Leishmania donovani. Parasites were detected in 22 of 25 biopsies (88%). In parallel sections stained...

Identification and biochemical characterization of Leishmania strains isolated in Peru, Mexico, and Spain.

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Rodríguez-González, Isabel; Marín, Clotilde; Vargas, Franklin; Córdova, Ofelia; Barrera, Mario; Gutiérrez-Sánchez, Ramón; Alunda, Jose María; Sánchez-Moreno, Manuel

2006-01-01

Eight Leishmania promastigotes were isolated from different geographical areas: three (LP1, LP2, and LP3) from the provincial department La Libertad and the fourth (LP4) from the department of Cajamarca (northern Peru); another three (LM1, LM2, and LM3) in the province of Campeche (Mexico); and the last (LS1) from a clinical case of a dog in Madrid (Spain). The isolates were characterized by carbohydrate cell-surface residues using agglutinations with four purified lectins, by isoenzyme analysis using different isoenzymes, by analysis of kinetoplast DNA (kDNA) restriction fragment length polymorphism using four different restriction endonucleases and by the final metabolite patterns after in vitro culture. These isolates were compared with four reference strains and typified as: Leishmania (Leishmania) donovani, two strains of L. (L.) infantum, and one species of L. (Viania) peruviana. According to our results and the statistical study, the Peruvian isolates represent three different strains: one would be L. (V.) peruviana, another the strain isolated in Cajamarca (LP4) and the third would include the three strains from the department of La Libertad (LP1, LP2, and LP3), these latter three isolates being phylogenetically closer to the reference strain L. (L.) donovani. Meanwhile, the three isolates from Mexico form a group with close phylogenetic relationships to each other. The isolate from Spain belongs to the species L. (L.) infantum. Thus, a close correlation was drawn between the identity of each strain and its geographical origin.

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

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Adrien Weingärtner

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

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

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

2017-11-01

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

Successful Therapy of Murine Visceral Leishmaniasis with Astrakurkurone, a Triterpene Isolated from the Mushroom Astraeus hygrometricus, Involves the Induction of Protective Cell-Mediated Immunity and TLR9

Science.gov (United States)

Mallick, Suvadip; Dutta, Aritri; Chaudhuri, Ankur; Mukherjee, Debasri; Dey, Somaditya; Halder, Subhadra; Ghosh, Joydip; Mukherjee, Debarati; Sultana, Sirin Salma; Biswas, Gunjan; Lai, Tapan Kumar; Patra, Pradyumna; Sarkar, Indranil; Chakraborty, Sibani; Saha, Bhaskar; Acharya, Krishnendu

2016-01-01

In our previous report, we showed that astrakurkurone, a triterpene isolated from the Indian mushroom Astraeus hygrometricus (Pers.) Morgan, induced reactive oxygen species, leading to apoptosis in Leishmania donovani promastigotes, and also was effective in inhibiting intracellular amastigotes at the 50% inhibitory concentration of 2.5 μg/ml. The aim of the present study is to characterize the associated immunomodulatory potentials and cellular activation provided by astrakurkurone, leading to effective antileishmanial activity in vitro and in vivo. Astrakurkurone-mediated antileishmanial activity was evaluated by real-time PCR and flow cytometry. The involvement of Toll-like receptor 9 (TLR9) was studied by in vitro assay in the presence of a TLR9 agonist and antagonist and by in silico modeling of a three-dimensional structure of the ectodomain of TLR9 and its interaction with astrakurkurone. Astrakurkurone caused a significant increase in TLR9 expression of L. donovani-infected macrophages along with the activation of proinflammatory responses. The involvement of TLR9 in astrakurkurone-mediated amastigote killing has been evidenced from the fact that a TLR9 agonist (CpG, ODN 1826) in combination with astrakurkurone enhanced the amastigote killing, while a TLR9 antagonist (bafilomycin A1) alone or in combination with astrakurkurone curbed the amastigote killing, which could be further justified by in silico evidence of docking between mouse TLR9 and astrakurkurone. Astrakurkurone was found to reduce the parasite burden in vivo by inducing protective cytokines, gamma interferon and interleukin 17. Moreover, astrakurkurone was nontoxic toward peripheral blood mononuclear cells of immunocompromised patients with visceral leishmaniasis. Astrakurkurone, a nontoxic antileishmanial, enhances the immune efficiency of host cells, leading to parasite clearance in vitro and in vivo. PMID:26883702

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

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

2012-08-10

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

In vitro and in vivo activity of an organic tellurium compound on Leishmania (Leishmania chagasi.

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Isabella Aparecida Salerno Pimentel

Full Text Available Tellurium compounds have shown several biological properties and recently the leishmanicidal effect of one organotellurane was demonstrated. These findings led us to test the effect of the organotellurium compound RF07 on Leishmania (Leishmania chagasi, the agent of visceral leishmaniasis in Latin America. In vitro assays were performed in L. (L. chagasi-infected bone marrow derived macrophages treated with different concentrations of RF07. In in vivo experiments Golden hamsters were infected with L. (L. chagasi and injected intraperitoneally with RF07 whereas control animals received either Glucantime or PBS. The effect of RF07 on cathepsin B activity of L. (L. chagasi amastigotes was assayed spectrofluorometrically using fluorogenic substrates. The main findings were: 1 RF07 showed significant leishmanicidal activity against intracellular parasites at submicromolar concentrations (IC50 of 529.7±26.5 nM, and the drug displayed 10-fold less toxicity to macrophages (CC50 of 5,426±272.8 nM; 2 kinetics assays showed an increasing leishmanicidal action of RF07 at longer periods of treatment; 3 one month after intraperitoneal injection of RF07 L. (L. chagasi-infected hamsters showed a reduction of 99.6% of parasite burden when compared to controls that received PBS; 4 RF07 inhibited the cathepsin B activity of L. (L. chagasi amastigotes. The present results demonstrated that the tellurium compound RF07 is able to destroy L. (L. chagasi in vitro and in vivo at concentrations that are non toxic to the host. We believe these findings support further study of the potential of RF07 as a possible alternative for the chemotherapy of visceral leishmaniasis.

Interferon-¿ and interleukin-4 production by human T cells recognizing Leishmania donovani antigens separated by SDS-PAGE

DEFF Research Database (Denmark)

Bahrenscheer, J; Kemp, M; Kurtzhals, J A

1995-01-01

of proliferation and interferon-gamma (IFN-gamma) production in cultures of peripheral blood mononuclear cells (PBMC) from individuals who had recovered from visceral leishmaniasis caused by L. donovani. The release of interleukin-4 (IL-4) by PBMC stimulated with the isolated L. donovani antigen fractions...... was measured after treatment with phorbol-myristate-acetate and ionomycin. The cells proliferated in response to all protein fractions with molecular weights in the range production...... was infrequently observed. The results show that T cells from individuals who have been cured of visceral leishmaniasis recognize and respond to a wide range of leishmanial antigens. There was no evidence of particular fractions constantly giving either IFN-gamma or IL-4-producing responses....

Evaluación del efecto del ácido nalidíxico, ampicilina, kanamicina, penicilina G y polimixina B en los cultivos de promastigotes de leishmania

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Sofía Duque Beltrán

1992-06-01

Full Text Available Se evaluó el efecto de diferentes concentraciones de ácido nalidíxico, ampicilina, kanarnicina, penicilina G y polimixina B, sobre la población de promastigotes de Leishmania braziliensis braziliensis , Leishmania donovani chagasi y Leishmania mexicana amazonensis in vitro. La penicilina G y la ampicilina se pueden utilizar hasta concentraciones de 1000 ug/ml y 500 ug/ml respectivamente en cultivo de promastigotes de cualquier cepa de Leishmania sin que éstos se afecten. La polimixina B disminuye la población de prosmastigotes por lo cual es preferible no usarse en cultivos de Leishmania. El ácido nalidíxico y kanamicina pueden ser utilizados in vitro pero teniéndose en cuenta la especie de Leishmania y la concentración de anfimicrobiano recomendado para la misma.

Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi

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Víctor T Contreras

2002-12-01

Full Text Available Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU medium at 37°C for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90% of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts.

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

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Veras, Patrícia Sampaio Tavares; Bezerra de Menezes, Juliana Perrone

2016-08-19

Leishmania is a protozoan parasite that causes a wide range of different clinical manifestations in mammalian hosts. It is a major public health risk on different continents and represents one of the most important neglected diseases. Due to the high toxicity of the drugs currently used, and in the light of increasing drug resistance, there is a critical need to develop new drugs and vaccines to control Leishmania infection. Over the past few years, proteomics has become an important tool to understand the underlying biology of Leishmania parasites and host interaction. The large-scale study of proteins, both in parasites and within the host in response to infection, can accelerate the discovery of new therapeutic targets. By studying the proteomes of host cells and tissues infected with Leishmania, as well as changes in protein profiles among promastigotes and amastigotes, scientists hope to better understand the biology involved in the parasite survival and the host-parasite interaction. This review demonstrates the feasibility of proteomics as an approach to identify new proteins involved in Leishmania differentiation and intracellular survival.

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

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Márcia Mariza Gomes Jusi

2011-09-01

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

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

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

2017-02-01

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

Molecular Characterization of Leishmania Species Isolated from Cutaneous Leishmaniasis in Yemen

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

2010-01-01

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

Production of interferon-¿ and interleukin-4 by human T cells recognizing Leishmania lipophosphoglycan-associated protein

DEFF Research Database (Denmark)

Kemp, M; Kurtzhals, J A; Christensen, C B

1993-01-01

The Leishmania protein LPGAP which is co-isolated with lipophosphoglycan is a specific activator of T cells from individuals who have recovered from American leishmaniasis. We have tested the effect of LPGAP on peripheral blood mononuclear cells (PBMC) from Kenyan donors cured from L. donovani in....... The results show that both IFN-gamma producing (Th1-like) and IL-4 producing (Th2-like) T cells recognizing LPGAP are expanded after infection with L. donovani in humans....... infections. LPGAP induced vigorous proliferation and production of interferon-gamma (IFN-gamma) by the cells. In addition PBMC incubated with LPGAP released interleukin-4 (IL-4) after pulsing with ionomycin and phorbol myristate acetate. Single cells were isolated from LPGAP-stimulated cell lines...

Leishmania infantum Asparagine Synthetase A Is Dispensable for Parasites Survival and Infectivity.

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Faria, Joana; Loureiro, Inês; Santarém, Nuno; Macedo-Ribeiro, Sandra; Tavares, Joana; Cordeiro-da-Silva, Anabela

2016-01-01

A growing interest in asparagine (Asn) metabolism has currently been observed in cancer and infection fields. Asparagine synthetase (AS) is responsible for the conversion of aspartate into Asn in an ATP-dependent manner, using ammonia or glutamine as a nitrogen source. There are two structurally distinct AS: the strictly ammonia dependent, type A, and the type B, which preferably uses glutamine. Absent in humans and present in trypanosomatids, AS-A was worthy of exploring as a potential drug target candidate. Appealingly, it was reported that AS-A was essential in Leishmania donovani, making it a promising drug target. In the work herein we demonstrate that Leishmania infantum AS-A, similarly to Trypanosoma spp. and L. donovani, is able to use both ammonia and glutamine as nitrogen donors. Moreover, we have successfully generated LiASA null mutants by targeted gene replacement in L. infantum, and these parasites do not display any significant growth or infectivity defect. Indeed, a severe impairment of in vitro growth was only observed when null mutants were cultured in asparagine limiting conditions. Altogether our results demonstrate that despite being important under asparagine limitation, LiAS-A is not essential for parasite survival, growth or infectivity in normal in vitro and in vivo conditions. Therefore we exclude AS-A as a suitable drug target against L. infantum parasites.

Leishmania infantum Asparagine Synthetase A Is Dispensable for Parasites Survival and Infectivity.

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Joana Faria

2016-01-01

Full Text Available A growing interest in asparagine (Asn metabolism has currently been observed in cancer and infection fields. Asparagine synthetase (AS is responsible for the conversion of aspartate into Asn in an ATP-dependent manner, using ammonia or glutamine as a nitrogen source. There are two structurally distinct AS: the strictly ammonia dependent, type A, and the type B, which preferably uses glutamine. Absent in humans and present in trypanosomatids, AS-A was worthy of exploring as a potential drug target candidate. Appealingly, it was reported that AS-A was essential in Leishmania donovani, making it a promising drug target. In the work herein we demonstrate that Leishmania infantum AS-A, similarly to Trypanosoma spp. and L. donovani, is able to use both ammonia and glutamine as nitrogen donors. Moreover, we have successfully generated LiASA null mutants by targeted gene replacement in L. infantum, and these parasites do not display any significant growth or infectivity defect. Indeed, a severe impairment of in vitro growth was only observed when null mutants were cultured in asparagine limiting conditions. Altogether our results demonstrate that despite being important under asparagine limitation, LiAS-A is not essential for parasite survival, growth or infectivity in normal in vitro and in vivo conditions. Therefore we exclude AS-A as a suitable drug target against L. infantum parasites.

Multilocus microsatellite typing (MLMT of strains from Turkey and Cyprus reveals a novel monophyletic L. donovani sensu lato group.

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Evi Gouzelou

Full Text Available BACKGROUND: New foci of human CL caused by strains of the Leishmania donovani (L. donovani complex have been recently described in Cyprus and the Çukurova region in Turkey (L. infantum situated 150 km north of Cyprus. Cypriot strains were typed by Multilocus Enzyme Electrophoresis (MLEE using the Montpellier (MON system as L. donovani zymodeme MON-37. However, multilocus microsatellite typing (MLMT has shown that this zymodeme is paraphyletic; composed of distantly related genetic subgroups of different geographical origin. Consequently the origin of the Cypriot strains remained enigmatic. METHODOLOGY/PRINCIPAL FINDINGS: The Cypriot strains were compared with a set of Turkish isolates obtained from a CL patient and sand fly vectors in south-east Turkey (Çukurova region; CUK strains and from a VL patient in the south-west (Kuşadasi; EP59 strain. These Turkish strains were initially analyzed using the K26-PCR assay that discriminates MON-1 strains by their amplicon size. In line with previous DNA-based data, the strains were inferred to the L. donovani complex and characterized as non MON-1. For these strains MLEE typing revealed two novel zymodemes; L. donovani MON-309 (CUK strains and MON-308 (EP59. A population genetic analysis of the Turkish isolates was performed using 14 hyper-variable microsatellite loci. The genotypic profiles of 68 previously analyzed L. donovani complex strains from major endemic regions were included for comparison. Population structures were inferred by combination of bayesian model-based and distance-based approaches. MLMT placed the Turkish and Cypriot strains in a subclade of a newly discovered, genetically distinct L. infantum monophyletic group, suggesting that the Cypriot strains may originate from Turkey. CONCLUSION: The discovery of a genetically distinct L. infantum monophyletic group in the south-eastern Mediterranean stresses the importance of species genetic characterization towards better understanding

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

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

2016-02-15

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

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

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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.

Cytotoxicity and anti-Leishmania amazonensis activity of Citrus sinensis leaf extracts.

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Garcia, Andreza R; Amaral, Ana Claudia F; Azevedo, Mariana M B; Corte-Real, Suzana; Lopes, Rosana C; Alviano, Celuta S; Pinheiro, Anderson S; Vermelho, Alane B; Rodrigues, Igor A

2017-12-01

Leishmania amazonensis is the main agent of diffuse cutaneous leishmaniasis, a disease characterized by lesional polymorphism and the commitment of skin surface. Previous reports demonstrated that the Citrus genus possess antimicrobial activity. This study evaluated the anti-L. amazonensis activity of Citrus sinensis (L.) Osbeck (Rutaceae) extracts. Citrus sinensis dried leaves were subjected to maceration with hexane (CH), ethyl acetate (CEA), dichloromethane/ethanol (CD/Et - 1:1) or ethanol/water (CEt/W - 7:3). Leishmania amazonensis promastigotes were treated with C. sinensis extracts (1-525 μg/mL) for 120 h at 27 °C. Ultrastructure alterations of treated parasites were evaluated by transmission electron microscopy. Cytotoxicity of the extracts was assessed on RAW 264.7 and J774.G8 macrophages after 48-h treatment at 37 °C using the tetrazolium assay. In addition, Leishmania-infected macrophages were treated with CH and CD/Et (10-80 μg/mL). CH, CD/Et and CEA displayed antileishmanial activity with 50% inhibitory activity (IC 50 ) of 25.91 ± 4.87, 54.23 ± 3.78 and 62.74 ± 5.04 μg/mL, respectively. Parasites treated with CD/Et (131.2 μg/mL) presented severe alterations including mitochondrial swelling, lipid body formation and intense cytoplasmic vacuolization. CH and CD/Et demonstrated cytotoxic effects similar to that of amphotericin B in the anti-amastigote assays (SI of 2.16, 1.98 and 1.35, respectively). Triterpene amyrins were the main substances in CH and CD/Et extracts. In addition, 80 μg/mL of CD/Et reduced the number of intracellular amastigotes and the percentage of infected macrophages in 63% and 36%, respectively. The results presented here highlight C. sinensis as a promising source of antileishmanial agents.

Leishmania donovani-reactive Th1- and Th2-like T-cell clones from individuals who have recovered from visceral leishmaniasis

DEFF Research Database (Denmark)

Kemp, M; Kurtzhals, J A; Bendtzen, K

1993-01-01

analyzed in a panel of L. donovani-reactive CD4+ human T-cell clones generated from individuals who had recovered from VL after antimonial treatment. Two of the T-cell clones produced large amounts of IL-4 without production of IFN-gamma, seven clones produced both IFN-gamma and IL-4, and eight produced...... by interleukin-4 (IL-4)-producing Th2 cells, or cure may result by Th1 cells secreting gamma interferon (IFN-gamma). The present study examined the potential of human T cells to generate Th1 or Th2 responses to L. donovani. The profiles of IFN-gamma, IL-4, and lymphotoxin secretion after antigen stimulation were...... only IFN-gamma. This is the first report of a Th1- and Th2-type response in human leishmaniasis. These results suggest that in analogy with murine models, there is a dichotomy in the human T-cell response to L. donovani infections. Preferential activation of IL-4-producing Th2-like cells may...

T7 polymerase-driven transcription is downregulated in metacyclic promastigotes and amastigotes of Leishmania mexicana

Czech Academy of Sciences Publication Activity Database

Ishemgulova, A.; Kraeva, N.; Faktorová, Drahomíra; Podešvová, L.; Lukeš, Julius; Yurchenko, V.

2016-01-01

Roč. 63, MAY 18 (2016), č. článku 016. ISSN 1803-6465 Institutional support: RVO:60077344 Keywords : gene expression * untranslated regions * Tet-inducible system * Leishmania mexicana Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.082, year: 2016

Immunization with Live Attenuated Leishmania donovani Centrin−/− Parasites Is Efficacious in Asymptomatic Infection

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Nevien Ismail

2017-12-01

Full Text Available Currently, there is no vaccine against visceral leishmaniasis (VL. Toward developing an effective vaccine, we have reported extensively on the immunogenicity of live attenuated LdCentrin−/− mutants in naive animal models. In VL endemic areas, asymptomatic carriers outnumber symptomatic cases of VL and are considered to be a reservoir of infection. Vaccination of asymptomatic cases represents a viable strategy to eliminate VL. Immunological correlates of protection thus derived might have limited applicability in conditions where the immunized host has prior exposure to virulent infection. To examine whether LdCen−/− parasites can induce protective immunity in experimental hosts that have low-level parasitemia from a previous exposure mimicking an asymptomatic condition, we infected C57Bl/6 mice with wild-type Leishmania donovani parasites expressing LLO epitope (LdWTLLO 103, i.v.. After 3 weeks, the mice with low levels of parasitemia were immunized with LdCen−/− parasites expressing 2W epitope (LdCen−/−2W 3 × 106 i.v. to characterize the immune responses in the same host. Antigen experienced CD4+ T cells from the asymptomatic (LdWTLLO infected LdCen−/−2W immunized, and other control groups were enriched using LLO- and 2W-specific tetramers, followed by Flow cytometric analysis. Our analysis showed that comparable CD4+ T cell proliferation and CD4+ memory T cell responses (TCM represented by CD62Lhi, CCR7+, and IL-7R+ T cell populations were induced with LdCen−/−2W in both asymptomatic and naive animals that received LdCen−/− immunization. Upon restimulation with peptide, TCM cells differentiated into effector T cells and there was no significant difference in the recall response in animals with asymptomatic infection. Following virulent challenge, comparable reduction in splenic parasite burden was observed in both asymptomatic and naive LdCen−/− immunized animals concomitant with the development of

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

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

2011-02-01

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

Kharon1 null mutants of Leishmania mexicana are avirulent in mice and exhibit a cytokinesis defect within macrophages.

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Khoa D Tran

Full Text Available In a variety of eukaryotes, flagella play important roles both in motility and as sensory organelles that monitor the extracellular environment. In the parasitic protozoan Leishmania mexicana, one glucose transporter isoform, LmxGT1, is targeted selectively to the flagellar membrane where it appears to play a role in glucose sensing. Trafficking of LmxGT1 to the flagellar membrane is dependent upon interaction with the KHARON1 protein that is located at the base of the flagellar axoneme. Remarkably, while Δkharon1 null mutants are viable as insect stage promastigotes, they are unable to survive as amastigotes inside host macrophages. Although Δkharon1 promastigotes enter macrophages and transform into amastigotes, these intracellular parasites are unable to execute cytokinesis and form multinucleate cells before dying. Notably, extracellular axenic amastigotes of Δkharon1 mutants replicate and divide normally, indicating a defect in the mutants that is only exhibited in the intra-macrophage environment. Although the flagella of Δkharon1 amastigotes adhere to the phagolysomal membrane of host macrophages, the morphology of the mutant flagella is often distorted. Additionally, these null mutants are completely avirulent following injection into BALB/c mice, underscoring the critical role of the KHARON1 protein for viability of intracellular amastigotes and disease in the animal model of leishmaniasis.

Characterization of Leishmania Parasites Isolated From Kala- azar Patients in Kohgiloyeh and Boyerahmad, Using Semi-Nested PCR

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B Sarkari

2006-04-01

Full Text Available ABSTRACT: Introduction & Objective: Visceral leishmaniasis (VL is a disease commonly known as Kala-azar caused by protozoan parasites of the genus Leishmania including L. donovani, L. infantum and L. chagasi. VL is sporadic in many areas of Iran and is endemic in a few provinces such as Fars, Azarbayjan, Bushehr, Ardabil and Qom. VL has been reported from some areas of Kohgiloyeh and Boyerahmad and this study aimed to characterize the causative agent of VL in this region. Materials & Methods: Bone marrow sample was obtained from 6 VL patients from children department in Imam Sajad hospital in Yasuj. DNA was extracted from the obtained samples and was checked by semi-nested PCR to determine the species of the parasite. To do that, a segment of minicircle kinetoplast DNA was amplified, using LINR4 and LIN17 primers. Products of PCR were evaluated by electrophoresis, using 1.5% agarose and stained with ethidium bromide. Results: Parasitologically examination of bone marrow smears demonstrated amastigotes form of the parasite in the samples. For mass cultivation, isolated parasites were cultured in diphasic NNN followed by RPMI 1640 media. All the samples produced a 720 bp band in PCR assay. The isolates were compared with referent strains and it was revealed that all the isolates were L. infantum. Conclusion: Findings of this study demonstrated that the causative agent of VL in Kohgiloyeh and Boyerahmad was L. infantum. Further study is needed to explore other aspects of VL in this region.

Functional and genetic evidence that nucleoside transport is highly conserved in Leishmania species: Implications for pyrimidine-based chemotherapy

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Khalid J.H. Alzahrani

2017-08-01

Full Text Available Leishmania pyrimidine salvage is replete with opportunities for therapeutic intervention with enzyme inhibitors or antimetabolites. Their uptake into cells depends upon specific transporters; therefore it is essential to establish whether various Leishmania species possess similar pyrimidine transporters capable of drug uptake. Here, we report a comprehensive characterization of pyrimidine transport in L. major and L. mexicana. In both species, two transporters for uridine/adenosine were detected, one of which also transported uracil and the antimetabolites 5-fluoruracil (5-FU and 5F,2′deoxyuridine (5F,2′dUrd, and was designated uridine-uracil transporter 1 (UUT1; the other transporter mediated uptake of adenosine, uridine, 5F,2′dUrd and thymidine and was designated Nucleoside Transporter 1 (NT1. To verify the reported L. donovani model of two NT1-like genes encoding uridine/adenosine transporters, and an NT2 gene encoding an inosine transporter, we cloned the corresponding L. major and L. mexicana genes, expressing each in T. brucei. Consistent with the L. donovani reports, the NT1-like genes of either species mediated the adenosine-sensitive uptake of [3H]-uridine but not of [3H]-inosine. Conversely, the NT2-like genes mediated uptake of [3H]-inosine but not [3H]-uridine. Among pyrimidine antimetabolites tested, 5-FU and 5F,2′dUrd were the most effective antileishmanials; resistance to both analogs was induced in L. major and L. mexicana. In each case it was found that the resistant cells had lost the transport capacity for the inducing drug. Metabolomics analysis found that the mechanism of action of 5-FU and 5F-2′dUrd was similar in both Leishmania species, with major changes in deoxynucleotide metabolism. We conclude that the pyrimidine salvage system is highly conserved in Leishmania species - essential information for the development of pyrimidine-based chemotherapy. Keywords: Leishmania, Pyrimidine metabolism, Uracil

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

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Ameer Khusro

2017-07-01

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

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

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

2015-01-01

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

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

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Erico Vinícius de Souza Carmo

2010-11-01

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

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

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Smandi Sondos

2012-01-01

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

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

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Messlinger, Helena; Sebald, Heidi; Heger, Lukas; Dudziak, Diana; Bogdan, Christian; Schleicher, Ulrike

2018-01-01

Activated natural killer (NK) cells release interferon (IFN)-γ, which is crucial for the control of intracellular pathogens such as Leishmania. In contrast to experimental murine leishmaniasis, the human NK cell response to Leishmania is still poorly characterized. Here, we investigated the interaction of human blood NK cells with promastigotes of different Leishmania species (Leishmania major, Leishmania mexicana, Leishmania infantum, and Leishmania donovani). When peripheral blood mononuclear cells or purified NK cells and monocytes (all derived from healthy blood donors from Germany without a history of leishmaniasis) were exposed to promastigotes, NK cells showed increased surface expression of the activation marker CD69. The extent of this effect varied depending on the Leishmania species; differences between dermotropic and viscerotropic L. infantum strains were not observed. Upregulation of CD69 required direct contact between monocytes and Leishmania and was partly inhibitable by anti-interleukin (IL)-18. Unexpectedly, IL-18 was undetectable in most of the supernatants (SNs) of monocyte/parasite cocultures. Confocal fluorescence microscopy of non-permeabilized cells revealed that Leishmania-infected monocytes trans-presented IL-18 to NK cells. Native, but not heat-treated SNs of monocyte/Leishmania cocultures also induced CD69 on NK cells, indicating the involvement of a soluble heat-labile factor other than IL-18. A role for the NK cell-activating cytokines IL-1β, IL-2, IL-12, IL-15, IL-21, and IFN-α/β was excluded. The increase of CD69 was not paralleled by NK cell IFN-γ production or enhanced cytotoxicity. However, prior exposure of NK cells to Leishmania parasites synergistically increased their IFN-γ release in response to IL-12, which was dependent on endogenous IL-18. CD1c+ dendritic cells were identified as possible source of Leishmania-induced IL-12. Finally, we observed that direct contact between Leishmania and NK cells reduced the

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

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Helena Messlinger

2018-01-01

Full Text Available Activated natural killer (NK cells release interferon (IFN-γ, which is crucial for the control of intracellular pathogens such as Leishmania. In contrast to experimental murine leishmaniasis, the human NK cell response to Leishmania is still poorly characterized. Here, we investigated the interaction of human blood NK cells with promastigotes of different Leishmania species (Leishmania major, Leishmania mexicana, Leishmania infantum, and Leishmania donovani. When peripheral blood mononuclear cells or purified NK cells and monocytes (all derived from healthy blood donors from Germany without a history of leishmaniasis were exposed to promastigotes, NK cells showed increased surface expression of the activation marker CD69. The extent of this effect varied depending on the Leishmania species; differences between dermotropic and viscerotropic L. infantum strains were not observed. Upregulation of CD69 required direct contact between monocytes and Leishmania and was partly inhibitable by anti-interleukin (IL-18. Unexpectedly, IL-18 was undetectable in most of the supernatants (SNs of monocyte/parasite cocultures. Confocal fluorescence microscopy of non-permeabilized cells revealed that Leishmania-infected monocytes trans-presented IL-18 to NK cells. Native, but not heat-treated SNs of monocyte/Leishmania cocultures also induced CD69 on NK cells, indicating the involvement of a soluble heat-labile factor other than IL-18. A role for the NK cell-activating cytokines IL-1β, IL-2, IL-12, IL-15, IL-21, and IFN-α/β was excluded. The increase of CD69 was not paralleled by NK cell IFN-γ production or enhanced cytotoxicity. However, prior exposure of NK cells to Leishmania parasites synergistically increased their IFN-γ release in response to IL-12, which was dependent on endogenous IL-18. CD1c+ dendritic cells were identified as possible source of Leishmania-induced IL-12. Finally, we observed that direct contact between Leishmania and NK cells

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

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

2016-10-01

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

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

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

1999-05-01

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

Microculture for the isolation of Leishmania parasites from cutaneous lesions -- Sri Lankan experience.

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Ihalamulla, R L; Rajapaksa, U S; Karunaweera, N D

2005-09-01

Novy, McNeal and Nicolle (NNN) medium and Evans' modified Tobie's medium are two conventional media for the isolation of Leishmania parasites in in-vitro cultures. Both are biphasic, with a solid layer of blood agar, and are normally prepared in glass test-tubes. In Sri Lanka at least, a monophasic microcapillary culture, based solely on RPMI 1640 medium supplemented with foetal calf serum, has been found simpler, more economical and more sensitive, for the isolation of L. donovani from skin lesions, than the use of Evans' modified Tobie's medium.

Avaliação clínica e parasitológica de cães naturalmente infectados por Leishmania (Leishmania chagasi submetidos a tratamento com antimoniato de meglumina e alopurinol

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Fabiana Augusta Ikeda-Garcia

2010-06-01

Full Text Available Aiming to assess the efficacy of the treatment, to verify the occurrence of possible disease relapses and to search for the presence of parasites after the treatment, seven dogs naturally infected by Leishmania sp., were submitted to a treatment with meglumine antimoniate and allopurinol. For this, lymph node and bone marrow aspiration biopsies were carried out at seven moments. After the end of the six-month observation period all dogs were submitted to euthanasia. Then, spleen and liver "imprints" and in vitro cultures were carried out to search for amastigote forms of the parasite. All animals presented remission of the symptoms and during all the observation period no dog presented relapse of the disease, although amastigote forms of the parasite were observed in two of the animals at the end of the experiment. Thus, it was possible to conclude that the treatment promotes clinical healing but it does not eliminate the parasites completely.

Dynamics of sterol synthesis during development of Leishmania spp. parasites to their virulent form.

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Yao, Chaoqun; Wilson, Mary E

2016-04-12

The Leishmania spp. protozoa, the causative agents of the "neglected" tropical disease leishmaniasis, are transmitted to mammals by sand fly vectors. Within the sand fly, parasites transform from amastigotes to procyclic promastigotes, followed by development of virulent (metacyclic) promastigote forms. The latter are infectious to mammalian hosts. Biochemical components localized in the parasite plasma membrane such as proteins and sterols play a pivotal role in Leishmania pathogenesis. Leishmania spp. lack the enzymes for cholesterol synthesis, and the dynamics of sterol acquisition and biosynthesis in parasite developmental stages are not understood. We hypothesized that dynamic changes in sterol composition during metacyclogenesis contribute to the virulence of metacyclic promastigotes. Sterols were extracted from logarithmic phase or metacyclic promastigotes grown in liquid culture with or without cholesterol, and analyzed qualitatively and quantitatively by gas chromatograph-mass spectrometry (GC-MS). TriTrypDB was searched for identification of genes involved in Leishmania sterol biosynthetic pathways. In total nine sterols were identified. There were dynamic changes in sterols during promastigote metacyclogenesis. Cholesterol in the culture medium affected sterol composition in different parasite stages. There were qualitative and relative quantitative differences between the sterol content of virulent versus avirulent parasite strains. A tentative sterol biosynthetic pathway in Leishmania spp. promastigotes was identified. Significant differences in sterol composition were observed between promastigote stages, and between parasites exposed to different extracellular cholesterol in the environment. These data lay the foundation for further investigating the role of sterols in the pathogenesis of Leishmania spp. infections.

Immunopathological Features of Canine Myocarditis Associated with Leishmania infantum Infection

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Alessandro Costagliola

2016-01-01

Full Text Available Myocarditis associated with infectious diseases may occur in dogs, including those caused by the protozoa Neospora caninum, Trypanosoma cruzi, Babesia canis, and Hepatozoon canis. However, although cardiac disease due to Leishmania infection has also been documented, the immunopathological features of myocarditis have not been reported so far. The aim of this study was to examine the types of cellular infiltrates and expression of MHC classes I and II in myocardial samples obtained at necropsy from 15 dogs with an established intravitam diagnosis of visceral leishmaniasis. Pathological features of myocardium were characterized by hyaline degeneration of cardiomyocytes, necrosis, and infiltration of mononuclear inflammatory cells consisting of lymphocytes and macrophages, sometimes with perivascular pattern; fibrosis was also present in various degrees. Immunophenotyping of inflammatory cells was performed by immunohistochemistry on cryostat sections obtained from the heart of the infected dogs. The predominant leukocyte population was CD8+ with a fewer number of CD4+ cells. Many cardiomyocytes expressed MHC classes I and II on the sarcolemma. Leishmania amastigote forms were not detected within macrophages or any other cell of the examined samples. Our study provided evidence that myocarditis in canine visceral leishmaniasis might be related to immunological alterations associated with Leishmania infection.

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

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

2015-10-01

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

Susceptibilidad in vitro a infección por Leishmania y sensibilidad a medicamentos difiere según tipo de macrófagos The susceptibility to infection by Leishmania panamensis and the sensitivity to drugs differ according to the macrophage type

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Carol V. Mesa

2010-12-01

Full Text Available Introducción: Los sistemas in vitro son útiles en la evaluación de compuestos con actividad biológica, permitiendo por ejemplo, determinar el potencial citotóxico y leishmanicida de un compuesto. Objetivo: Identificar el tipo de célula mamífera que permita una óptima infección in vitro por Leishmania y que constituya el sistema adecuado para el tamizaje in vitro de compuestos con actividad anti-Leishmania. Métodos: La susceptibilidad de las células a infección por L. panamensis se evalúo según la Concentración Infectiva 50 determinada por microscopía de luz y citometría de flujo; la supervivencia intracelular de los amastigotes se evaluó por microscopía de fluorescencia y la sensibilidad de las células a anfotericina B y antimoniato de meglumina se evalúo por espectrofotometría. Resultados: Los cultivos primarios son más susceptibles a la infección por L. panamensis in vitro. Sin embargo, la supervivencia intracelular del parásito fue mejor en U-937. Por su parte, la sensibilidad de las células a anfotericina B y antimoniato de meglumina vario según el tipo de célula. Conclusión: Las células U-937 son las adecuadas para la infección por Leishmania porque: a presentan crecimiento ilimitado y se les puede inducir transformación a macrófagos. b la susceptibilidad a la infección por Leishmania es similar a la observada en cultivos primarios de macrófagos y c permiten mayor supervivencia de los amastigotes luego de la infección. Adicionalmente, las células U-937 son menos sensibles a la acción de los fármacos comúnmente utilizados como control en la detección de compuestos con actividad leishmanicida. Salud UIS 2010; 42: 200-211Introduction: In vitro systems are useful in the evaluation of compounds with biological activity determining the cytotoxic and leishmanicidal activity of the candidates. Objective: To identify the mammalian cell that allows the optimal in vitro infection by Leishmania and therefore

Understanding the transmission dynamics of Leishmania donovani to provide robust evidence for interventions to eliminate visceral leishmaniasis in Bihar, India

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Molecular tools enable the collection of accurate estimates of human blood index (HBI) in P. argentipes. The refinement of a metacyclic-specific qPCR assay to identify L. donovani in P. argentipes would enable quantification of the entomological inoculation rate (EIR) for the first time. Likewise, a...

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

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

2014-01-01

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

Functional and genetic evidence that nucleoside transport is highly conserved in Leishmania species: Implications for pyrimidine-based chemotherapy.

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Alzahrani, Khalid J H; Ali, Juma A M; Eze, Anthonius A; Looi, Wan Limm; Tagoe, Daniel N A; Creek, Darren J; Barrett, Michael P; de Koning, Harry P

2017-08-01

Leishmania pyrimidine salvage is replete with opportunities for therapeutic intervention with enzyme inhibitors or antimetabolites. Their uptake into cells depends upon specific transporters; therefore it is essential to establish whether various Leishmania species possess similar pyrimidine transporters capable of drug uptake. Here, we report a comprehensive characterization of pyrimidine transport in L. major and L. mexicana. In both species, two transporters for uridine/adenosine were detected, one of which also transported uracil and the antimetabolites 5-fluoruracil (5-FU) and 5F,2'deoxyuridine (5F,2'dUrd), and was designated uridine-uracil transporter 1 (UUT1); the other transporter mediated uptake of adenosine, uridine, 5F,2'dUrd and thymidine and was designated Nucleoside Transporter 1 (NT1). To verify the reported L. donovani model of two NT1-like genes encoding uridine/adenosine transporters, and an NT2 gene encoding an inosine transporter, we cloned the corresponding L. major and L. mexicana genes, expressing each in T. brucei. Consistent with the L. donovani reports, the NT1-like genes of either species mediated the adenosine-sensitive uptake of [ 3 H]-uridine but not of [ 3 H]-inosine. Conversely, the NT2-like genes mediated uptake of [ 3 H]-inosine but not [ 3 H]-uridine. Among pyrimidine antimetabolites tested, 5-FU and 5F,2'dUrd were the most effective antileishmanials; resistance to both analogs was induced in L. major and L. mexicana. In each case it was found that the resistant cells had lost the transport capacity for the inducing drug. Metabolomics analysis found that the mechanism of action of 5-FU and 5F-2'dUrd was similar in both Leishmania species, with major changes in deoxynucleotide metabolism. We conclude that the pyrimidine salvage system is highly conserved in Leishmania species - essential information for the development of pyrimidine-based chemotherapy. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights

The development of post-kala-azar dermal leishmaniasis (PKDL) is associated with acquisition of Leishmania reactivity by peripheral blood mononuclear cells (PBMC)

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Gasim, S; Elhassan, A M; Kharazmi, A

2000-01-01

PKDL develops in about 50% of Sudanese patients treated for visceral leishmaniasis (kala-azar). Patients with kala-azar were entered into this study and followed for a period of up to 2 years. During follow up 12 patients developed PKDL and eight did not. Proliferative responses and cytokine...... production to Leishmania donovani and control antigens were measured in vitro using PBMC isolated at the time of diagnosis of kala-azar, after treatment of visceral leishmaniasis, during follow up, and at the time of diagnosis of PKDL. Proliferative responses and interferon-gamma (IFN-gamma) production were...... assays. There were no differences in Leishmania antigen-induced production of IL-4, IL-5 and IL-10 between or within the two groups. We have previously shown that Leishmania parasites spread to the skin during visceral leishmaniasis and proposed that PKDL was the result of an immunological attack...

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

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

1999-01-01

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

Changes to cholesterol trafficking in macrophages by Leishmania parasites infection.

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

2017-08-01

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

Molecular detection of Leishmania infection in sand flies in border line of Iran-Turkmenistan: restricted and permissive vectors.

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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.

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

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

2018-02-27

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

Leishmania donovani isolates with antimony-resistant but not -sensitive phenotype inhibit sodium antimony gluconate-induced dendritic cell activation.

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Arun Kumar Haldar

2010-05-01

Full Text Available The inability of sodium antimony gluconate (SAG-unresponsive kala-azar patients to clear Leishmania donovani (LD infection despite SAG therapy is partly due to an ill-defined immune-dysfunction. Since dendritic cells (DCs typically initiate anti-leishmanial immunity, a role for DCs in aberrant LD clearance was investigated. Accordingly, regulation of SAG-induced activation of murine DCs following infection with LD isolates exhibiting two distinct phenotypes such as antimony-resistant (Sb(RLD and antimony-sensitive (Sb(SLD was compared in vitro. Unlike Sb(SLD, infection of DCs with Sb(RLD induced more IL-10 production and inhibited SAG-induced secretion of proinflammatory cytokines, up-regulation of co-stimulatory molecules and leishmanicidal effects. Sb(RLD inhibited these effects of SAG by blocking activation of PI3K/AKT and NF-kappaB pathways. In contrast, Sb(SLD failed to block activation of SAG (20 microg/ml-induced PI3K/AKT pathway; which continued to stimulate NF-kappaB signaling, induce leishmanicidal effects and promote DC activation. Notably, prolonged incubation of DCs with Sb(SLD also inhibited SAG (20 microg/ml-induced activation of PI3K/AKT and NF-kappaB pathways and leishmanicidal effects, which was restored by increasing the dose of SAG to 40 microg/ml. In contrast, Sb(RLD inhibited these SAG-induced events regardless of duration of DC exposure to Sb(RLD or dose of SAG. Interestingly, the inhibitory effects of isogenic Sb(SLD expressing ATP-binding cassette (ABC transporter MRPA on SAG-induced leishmanicidal effects mimicked that of Sb(RLD to some extent, although antimony resistance in clinical LD isolates is known to be multifactorial. Furthermore, NF-kappaB was found to transcriptionally regulate expression of murine gammaglutamylcysteine synthetase heavy-chain (mgammaGCS(hc gene, presumably an important regulator of antimony resistance. Importantly, Sb(RLD but not Sb(SLD blocked SAG-induced mgammaGCS expression in DCs by

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

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

2018-06-12

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

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

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

2015-11-21

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

Expression of Leishmania major LmSTI1 in Yeast Pichia Pastoris

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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.

Amiloidosis en Didelphis marsupialis infectado experimentalmente con Leishmania chagasi.

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Diana Milena Roa

2002-09-01

Full Text Available Un Didelphis marsupialis macho capturado en Teruel, Huila, se inoculó intraperitonealmente con 1x106 promastigotes de Leishmania chagasi (MHOM/CO/84/CL044B. El animal desarrolló leishmaniosis visceral con abundantes amastigotes fagocitados en las células de Kupffer y en los macrófagos esplénicos; murió 5 semanas después de la inoculación. La autopsia reveló amplios depósitos de amiloide en el hígado y en el bazo, demostrados por sus características tintoriales y ultraestructurales. La consideramos como una amiloidosis secundaria a la infección experimental, que ocurrió en el transcurso de 5 semanas luego de inoculado el animal.

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

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Yeda L. Nogueira

2006-12-01

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

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

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Marie Samanovic

2009-01-01

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

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

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

2017-09-01

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

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

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Miguel Angel Minero

2004-03-01

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

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

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

2014-03-01

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

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

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Hossein Mahmoudvand

2014-03-01

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

Action of Bothrops moojeni venom and its L-amino acid oxidase fraction, treated with {sup 60}Co gamma rays, in Leishmania spp; Acao do veneno de Bothrops moojeni e sua fracao L-aminoacido oxidase, submetida ao tratamento com raios gama de {sup 60}Co, em Leishmania spp

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Cardoso, Andre Gustavo Tempone

1999-07-01

Bothrops moojeni venom showed an anti leishmania activity in vitro, as determined by a cell viability assay using the reduction of MTT. After venom purification, by chromatography techniques, the fractions with anti leishmania and L-amino acid oxidase activities, eluted in the same positions. The molecular weight of the enzyme was estimated to be 140 kDa by molecular exclusion chromatography, and 69 kDa, by SDS-PAGE, migrating as a single band, with an isoelectric point of 4.8 as determined by isoelectric focusing. The purified LAO from B. moojeni venom, 135-fold more active than crude venom, showed homo dimeric constitution, and was active against Leishmania spp from the New World, with an effective concentration against L(L). amazonensis of 1.80 {mu}g/ml (EC{sub 50}), L.(V.) panamensis (0.78 |{mu}g/ml) and L.(L.) chagasi (0.63 ({mu}g/ml). Ultrastructural studies of promastigotes affected by LAO demonstrated cell death, with edema in several organelles such as mitochondria and nuclear membrane, before cell disruption and necrosis. The action of LAO was demonstrated to be hydrogen peroxide-dependent. Studies with LLCMK-2 cells, treated with LAO, showed a toxic effect, with an EC{sub 50} of 11|{mu}g/ml. Irradiation of LAO with 6{sup 0C}o gamma rays, did not affect its whole oxidative activity, neither detoxified the enzyme. Amastigotes treated with LAO were not affected by its hydrogen peroxide, otherwise, the exogenous product, killed amastigotes with an EC{sub 50} of 0.67mM. These data could be of help in the development of alternative therapeutic approaches to the treatment of leishmaniasis. (author)

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

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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.

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

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Zampieri, Ricardo Andrade; Laranjeira-Silva, Maria Fernanda; Muxel, Sandra Marcia; Stocco de Lima, Ana Carolina; Shaw, Jeffrey Jon; Floeter-Winter, Lucile Maria

2016-02-01

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.

Imidazole-containing phthalazine derivatives inhibit Fe-SOD performance in Leishmania species and are active in vitro against visceral and mucosal leishmaniasis.

Science.gov (United States)

Sánchez-Moreno, M; Gómez-Contreras, F; Navarro, P; Marín, C; Ramírez-Macías, I; Rosales, M J; Campayo, L; Cano, C; Sanz, A M; Yunta, M J R

2015-07-01

The in vitro leishmanicidal activity of a series of imidazole-containing phthalazine derivatives 1-4 was tested on Leishmania infantum, Leishmania braziliensis and Leishmania donovani parasites, and their cytotoxicity on J774·2 macrophage cells was also measured. All compounds tested showed selectivity indexes higher than that of the reference drug glucantime for the three Leishmania species, and the less bulky monoalkylamino substituted derivatives 2 and 4 were clearly more effective than their bisalkylamino substituted counterparts 1 and 3. Both infection rate measures and ultrastructural alterations studies confirmed that 2 and 4 were highly leishmanicidal and induced extensive parasite cell damage. Modifications to the excretion products of parasites treated with 2 and 4 were also consistent with substantial cytoplasmic alterations. On the other hand, the most active compounds 2 and 4 were potent inhibitors of iron superoxide dismutase enzyme (Fe-SOD) in the three species considered, whereas their impact on human CuZn-SOD was low. Molecular modelling suggests that 2 and 4 could deactivate Fe-SOD due to a sterically favoured enhanced ability to interact with the H-bonding net that supports the antioxidant features of the enzyme.

Action of Bothrops moojeni venom and its L-amino acid oxidase fraction, treated with 60Co gamma rays, in Leishmania spp

International Nuclear Information System (INIS)

Cardoso, Andre Gustavo Tempone

1999-01-01

Bothrops moojeni venom showed an anti leishmania activity in vitro, as determined by a cell viability assay using the reduction of MTT. After venom purification, by chromatography techniques, the fractions with anti leishmania and L-amino acid oxidase activities, eluted in the same positions. The molecular weight of the enzyme was estimated to be 140 kDa by molecular exclusion chromatography, and 69 kDa, by SDS-PAGE, migrating as a single band, with an isoelectric point of 4.8 as determined by isoelectric focusing. The purified LAO from B. moojeni venom, 135-fold more active than crude venom, showed homo dimeric constitution, and was active against Leishmania spp from the New World, with an effective concentration against L(L). amazonensis of 1.80 μg/ml (EC 50 ), L.(V.) panamensis (0.78 |μg/ml) and L.(L.) chagasi (0.63 (μg/ml). Ultrastructural studies of promastigotes affected by LAO demonstrated cell death, with edema in several organelles such as mitochondria and nuclear membrane, before cell disruption and necrosis. The action of LAO was demonstrated to be hydrogen peroxide-dependent. Studies with LLCMK-2 cells, treated with LAO, showed a toxic effect, with an EC 50 of 11|μg/ml. Irradiation of LAO with 6 0C o gamma rays, did not affect its whole oxidative activity, neither detoxified the enzyme. Amastigotes treated with LAO were not affected by its hydrogen peroxide, otherwise, the exogenous product, killed amastigotes with an EC 50 of 0.67mM. These data could be of help in the development of alternative therapeutic approaches to the treatment of leishmaniasis. (author)

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

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Sara María Robledo Restrepo

2000-03-01

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

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

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Badirzadeh, Alireza; Taheri, Tahereh; Taslimi, Yasaman; Abdossamadi, Zahra; Heidari-Kharaji, Maryam; Gholami, Elham; Sedaghat, Baharehsadat; Niyyati, Maryam; Rafati, Sima

2017-07-01

Proliferation of Leishmania (L.) parasites depends on polyamine availability, which can be generated by the L-arginine catabolism and the enzymatic activity of arginase (ARG) of the parasites and of the mammalian hosts. In the present study, we characterized and compared the arginase (arg) genes from pathogenic L. major and L. tropica and from non-pathogenic L. tarentolae. We quantified the level of the ARG activity in promastigotes and macrophages infected with pathogenic L. major and L. tropica and non-pathogenic L. tarentolae amastigotes. The ARG's amino acid sequences of the pathogenic and non-pathogenic Leishmania demonstrated virtually 98.6% and 88% identities with the reference L. major Friedlin ARG. Higher ARG activity was observed in all pathogenic promastigotes as compared to non-pathogenic L. tarentolae. In vitro infection of human macrophage cell line (THP1) with pathogenic and non-pathogenic Leishmania spp. resulted in increased ARG activities in the infected macrophages. The ARG activities present in vivo were assessed in susceptible BALB/c and resistant C57BL/6 mice infected with L. major, L. tropica and L. tarentolae. We demonstrated that during the development of the infection, ARG is induced in both strains of mice infected with pathogenic Leishmania. However, in L. major infected BALB/c mice, the induction of ARG and parasite load increased simultaneously according to the time course of infection, whereas in C57BL/6 mice, the enzyme is upregulated solely during the period of footpad swelling. In L. tropica infected mice, the footpads' swellings were slow to develop and demonstrated minimal cutaneous pathology and ARG activity. In contrast, ARG activity was undetectable in mice inoculated with the non-pathogenic L. tarentolae. Our data suggest that infection by Leishmania parasites can increase ARG activity of the host and provides essential polyamines for parasite salvage and its replication. Moreover, the ARG of Leishmania is vital for parasite

Cell migration induced by Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) major and Leishmania (Viannia) braziliensis into the peritoneal cavity of BALB/c mice

OpenAIRE

DT Wakimoto; KV Gaspareto; TGV Silveira; MVC Lonardoni; SMA Aristides

2010-01-01

In American cutaneous leishmaniasis, the initial infection phase is characterized by recruitment of neutrophils and monocytes. The migration of these cells in response to the presence of Leishmania in the peritoneum of affected animals remains unclear. The objective of this study was to investigate cell migration to the peritoneum of BALB/c mice after infection with Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis and Leishmania (Leishmania) major. Initially, Leishmania ...

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

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Rubén E Varela-M

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

Effects of trans-stilbene and terphenyl compounds on different strains of Leishmania and on cytokines production from infected macrophages.

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Bruno, Federica; Castelli, Germano; Vitale, Fabrizio; Giacomini, Elisa; Roberti, Marinella; Colomba, Claudia; Cascio, Antonio; Tolomeo, Manlio

2018-01-01

Most of the antileishmanial modern therapies are not satisfactory due to high toxicity or emergence of resistance and high cost of treatment. Previously, we observed that two compounds of a small library of trans-stilbene and terphenyl derivatives, ST18 and TR4, presented the best activity and safety profiles against Leishmania infantum promastigotes and amastigotes. In the present study we evaluated the effects of ST18 and the TR4 in 6 different species of Leishmania and the modifications induced by these two compounds in the production of 8 different cytokines from infected macrophages. We observed that TR4 was potently active in all Leishmania species tested in the study showing a leishmanicidal activity higher than that of ST18 and meglumine antimoniate in the most of the species. Moreover, TR4 was able to decrease the levels of IL-10, a cytokine able to render the host macrophage inactive allowing the persistence of parasites inside its phagolysosome, and increase the levels of IL-1β, a cytokine important for host resistance to Leishmania infection by inducible iNOS-mediated production of NO, and IL-18, a cytokine implicated in the development of Th1-type immune response. Copyright © 2017 Elsevier Inc. All rights reserved.

Factores dermicos que condicionan la infeccion de Lutzomyia townsendi (Ortiz, 1959 por Leishmania spp. de Venezuela Dermal factors which condition infection of Lutzomyia townsendi (Ortiz, 1959 by Leishmania spp. of Venezuela

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Maritza Carnevali

1982-12-01

Full Text Available Se estudia la susceptibilidad de Lutzomyia townsendi a la infección con Leishmania spp. sobre lesiones experimentales de hamsteres. Se estudia la frecuencia y distribución de los amastigotos en la dermis, relacionándola con la profundidad que alcanza el estilete bucal del insecto. Una correlación positiva, con significante coeficiente de correlación, se establece entre la frecuencia de los parásitos a una profundidad de 100-150 nm en la dermis y el éxito de la infección de los flebótomos.The susceptibility of Lutzomyia townsendi to infection with amastigotes of Leishmania spp. from experimental lesions in hamsters is studied. A good correlation is found when the number of infected sandflies are compared with the distribution and density of parasites in the dermis, taking into account the size of the fascicle, particularly the length of labrun and dental depth in maxillae. Considerations are made on the susceptibilities of some anthropophilic sandflies from America that are considered as good vectors.

Concurrent cutaneous, visceral and ocular leishmaniasis caused by Leishmania (Viannia braziliensis in a kidney transplant patient

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Gontijo Célia MF

2002-01-01

Full Text Available Although cases of leishmaniasis co-infection have been described in acquired immunodeficiency syndrome patients as well as those who have undergone organ transplants, to our knowledge, the present report is the first documented case of simultaneous cutaneous, visceral and ocular leishmaniasis due to Leishmania (Viannia braziliensis in a transplant patient. The patient had been using immunosuppressive drugs since receiving a transplanted kidney. The first clinical signs of leishmaniasis included fever, thoracic pain, hepatosplenomegaly, leucopenia and anemia. The cutaneous disease was revealed by the presence of amastigotes in the skin biopsy. After three months, the patient presented fever with conjunctive hyperemia, intense ocular pain and low visual acuity. Parasites isolated from iliac crest, aqueous humor and vitreous body were examined using a range of molecular techniques. The same strain of L. (V. braziliensis was responsible for the different clinical manifestations. The immunosuppressive drugs probably contributed to the dissemination of Leishmania.

The paratransgenic sand fly: a platform for control of Leishmania transmission.

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Hurwitz, Ivy; Hillesland, Heidi; Fieck, Annabeth; Das, Pradeep; Durvasula, Ravi

2011-05-19

Leishmania donovani is transmitted by the bite of the sand fly, Phlebotomus argentipes. This parasite is the agent of visceral leishmaniasis (VL), an endemic disease in Bihar, India, where prevention has relied mainly on DDT spraying. Pesticide resistance in sand fly populations, environmental toxicity, and limited resources confound this approach. A novel paratransgenic strategy aimed at control of vectorial transmission of L. donovani is presented using Bacillus subtilis, a commensal bacterium isolated from the sand fly gut. In this work, B. subtilis expressing Green Fluorescent Protein (GFP) was added to sterilized larval chow. Control pots contained larval chow spiked either with untransformed B. subtilis or phosphate-buffered saline. Fourth-instar P. argentipes larvae were transferred into the media and allowed to mature. The number of bacterial colony forming units, relative abundance and the mean microbial load were determined per developmental stage. Addition of B. subtilis to larval chow did not affect sand fly emergence rates. B. cereus and Lys fusiformis were identified at each developmental stage, revealing transstadial passage of endogenous microbes. Larvae exposed to an exogenous bolus of B. subtilis harbored significantly larger numbers of bacteria. Bacterial load decreased to a range comparable to sand flies from control pots, suggesting an upper limit to the number of bacteria harbored. Emerging flies reared in larval chow containing transformed B. subtilis carried large numbers of these bacteria in their gut lumens. Strong GFP expression was detected in these paratransgenic flies with no spread of transformed bacteria to other compartments of the insects. This is the first demonstration of paratransgenic manipulation of P. argentipes. Paratransgenic manipulation of P. argentipes appears feasible. Expression of leishmanicidal molecules via commensal bacteria commonly found at breeding sites of P. argentipes could render adult sand flies refractory

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...

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

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Lívia O Santos

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

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

Science.gov (United States)

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

2017-01-01

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

Molecular and serological detection of tick-borne pathogens in dogs from an area endemic for Leishmania infantumin Mato Grosso do Sul, Brazil

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Keyla Carstens Marques de Sousa

Full Text Available Tick-borne pathogens affect a wide range of vertebrate hosts. To identify tick-borne pathogens among dogs from Campo Grande, MS, Brazil testing seropositive for Leishmania infantum (syn. L. chagasi, a serological and molecular study was conducted to detectEhrlichia canis, Anaplasma platys and Babesia vogeli in 60 serum and spleen samples. A confirmatory diagnosis ofL. infantum based on serological and molecular assays was also performed, as was sequence alignment and phylogenetic analysis to assess the identity of the parasite species infecting these animals. IgG antibodies toEhrlichia spp., B. vogeli and L. infantum were found, respectively, in 39 (65%, 49 (81.6% and 60 (100% of the sampled dogs. Twenty-seven (45%, fifty-four (90%, fifty-three (88.3%, two (3.3% and one (1.6% dog were positive, respectively, forE. canis, Leishmania spp., Leishmania donovani complex, Babesia sp. and Anaplasma sp. in PCR assays. After sequencing, the amplicons showed 99% of identity with E. canis, B. vogeli, A. platys andLeishmania chagasi isolates. The findings of this study indicate that L. infantum-seropositive dogs from Campo Grande are exposed to multiple tick-borne pathogens, which should therefore be included in the differential diagnosis of dogs with clinical suspicion of leishmaniasis.

Identification of Leishmania by Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry Using a Free Web-Based Application and a Dedicated Mass-Spectral Library.

Science.gov (United States)

Lachaud, Laurence; Fernández-Arévalo, Anna; Normand, Anne-Cécile; Lami, Patrick; Nabet, Cécile; Donnadieu, Jean Luc; Piarroux, Martine; Djenad, Farid; Cassagne, Carole; Ravel, Christophe; Tebar, Silvia; Llovet, Teresa; Blanchet, Denis; Demar, Magalie; Harrat, Zoubir; Aoun, Karim; Bastien, Patrick; Muñoz, Carmen; Gállego, Montserrat; Piarroux, Renaud

2017-10-01

Human leishmaniases are widespread diseases with different clinical forms caused by about 20 species within the Leishmania genus. Leishmania species identification is relevant for therapeutic management and prognosis, especially for cutaneous and mucocutaneous forms. Several methods are available to identify Leishmania species from culture, but they have not been standardized for the majority of the currently described species, with the exception of multilocus enzyme electrophoresis. Moreover, these techniques are expensive, time-consuming, and not available in all laboratories. Within the last decade, mass spectrometry (MS) has been adapted for the identification of microorganisms, including Leishmania However, no commercial reference mass-spectral database is available. In this study, a reference mass-spectral library (MSL) for Leishmania isolates, accessible through a free Web-based application (mass-spectral identification [MSI]), was constructed and tested. It includes mass-spectral data for 33 different Leishmania species, including species that infect humans, animals, and phlebotomine vectors. Four laboratories on two continents evaluated the performance of MSI using 268 samples, 231 of which were Leishmania strains. All Leishmania strains, but one, were correctly identified at least to the complex level. A risk of species misidentification within the Leishmania donovani , L. guyanensis , and L. braziliensis complexes was observed, as previously reported for other techniques. The tested application was reliable, with identification results being comparable to those obtained with reference methods but with a more favorable cost-efficiency ratio. This free online identification system relies on a scalable database and can be implemented directly in users' computers. Copyright © 2017 American Society for Microbiology.

Uveitis associated to the infection by Leishmania chagasi in dog from the Olinda city, Pernambuco, Brazil

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Brito Fábio Luiz da Cunha

2004-01-01

Full Text Available Among the parasitic diseases, Canine Visceral Leishmaniasis (CVL is included in the systemic illnesses of chronic evolution that attack men and dogs, presenting varied clinical manifestations as cachexia, dermatologic lesions, peripheral lymphadenopathies, besides the ocular lesions. This work report the case of a dog clinically suspected of having CVL, presenting skin lesions, cachexia, gryphosis, and ocular signs of uveitis. The parasitological diagnosis was accomplished for Canine Leishmaniasis through the visualization of amastigote forms of Leishmania chagasi in smears of bone marrow fluid aspirate, of non-lesioned, and lesioned skin. Alterations in the ocular structures are characterized mainly by mononuclear-plasmocitic infiltrate.

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

OpenAIRE

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

1999-01-01

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

Pathology of dogs in Campo Grande, MS, Brazil naturally co-infected with Leishmania infantum and Ehrlichia canis

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Gisele Braziliano Andrade

2014-12-01

Full Text Available Different parasites that commonly occur concomitantly can influence one another, sometimes with unpredictable effects. We evaluated pathological aspects of dogs naturally co-infected with Leishmania infantum and Ehrlichia canis. The health status of the dogs was investigated based on histopathological, hematological and biochemical analyses of 21 animals infected solely with L. infantum and 22 dogs co- infected with L. infantum and E. canis. The skin of both groups showed chronic, predominantly lymphohistioplasmacytic inflammatory reaction. The plasmacytosis in the lymphoid tissues was likely related with the hypergammaglobulinemia detected in all the dogs. The disorganization of extracellular matrix found in the reticular dermis of the inguinal region and ear, characterized by the substitution of thick collagen fibers for thin fibers, was attributed to the degree of inflammatory reaction, irrespective of the presence of parasites. In addition, the histopathological analysis revealed that twice as many dogs in the co-infected group presented Leishmania amastigotes in the ear skin than those infected solely with Leishmania, increasing the possibility of becoming infected through sand fly vectors. Our findings highlight the fact that the health of dogs infected concomitantly with L. infantum and E. canis is severely compromised due to their high levels of total plasma protein, globulins, alkaline phosphatase and creatine kinase, and severe anemia.

Histopathological and parasitological study of the gastrointestinal tract of dogs naturally infected with Leishmania infantum

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Pinto Aldair JW

2011-12-01

Full Text Available Abstract Background The aim of this study was to provide a systematic pathological and parasitological overview of the gastrointestinal tract (GIT, including the stomach, duodenum, jejunum, ileum, caecum and colon, of dogs naturally infected with Leishmania. Methods Twenty mongrel dogs naturally infected with Leishmania (Leishmania infantum and obtained from the Control Zoonosis Center of the Municipality of Ribeirão das Neves, Belo Horizonte Metropolitan area, Minas Gerais (MG state, Brazil, were analyzed. The dogs were divided into two groups: Group 1 comprised nine clinically normal dogs and group 2 comprised 11 clinically affected dogs. After necropsy, one sample was collected from each GIT segment, namely the stomach, duodenum, jejunum, ileum, caecum and colon. Furthermore, paraffin-embedded samples were used for histological and parasitological (immunohistochemistry evaluation and a morphometrical study were carried out to determine the parasite load (immunolabeled amastigote forms of Leishmania. The Friedman and the Mann Whitney tests were used for statistical analysis. The Friedman test was used to analyze each segment of the GIT within each group of dogs and the Mann Whitney test was used to compare the GIT segments between clinically unaffected and affected dogs. Results The infected dogs had an increased number of macrophages, plasma cells and lymphocytes, but lesions were generally mild. Parasite distribution in the GIT was evident in all intestinal segments and layers of the intestinal wall (mucosal, muscular and submucosal irrespective of the clinical status of the dogs. However, the parasite load was statistically higher in the caecum and colon than in other segments of the GIT. Conclusion The high parasite burden evident throughout the GIT mucosa with only mild pathological alterations led us to consider whether Leishmania gains an advantage from the intestinal immunoregulatory response (immunological tolerance.

Antileishmanial activity and tubulin polymerization inhibition of podophyllotoxin derivatives on Leishmania infantum

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José Miguel Escudero-Martínez

2017-12-01

Full Text Available Leishmania microtubules play an important role not only in cell division, but also in keeping the shape of the parasite and motility of its free-living stages. Microtubules result from the self-assembly of alpha and beta tubulins, two phylogenetically conserved and very abundant eukaryotic proteins in kinetoplastids. The colchicine binding domain has inspired the discovery and development of several drugs currently in clinical use against parasites. However, this domain is less conserved in kinetoplastids and may be selectively targeted by new compounds. This report shows the antileishmanial effect of several series of compounds (53, derived from podophyllotoxin (a natural cyclolignan isolated from rhizomes of Podophyllum spp. and podophyllic aldehyde, on a transgenic, fluorescence-emitting strain of Leishmania infantum. These compounds were tested on both promastigotes and amastigote-infected mouse splenocytes, and in mammalian – mouse non-infected splenocytes and liver HepG2 cells – in order to determine selective indexes of the drugs. Results obtained with podophyllotoxin derivatives showed that the hydroxyl group at position C-7α was a structural requisite to kill the parasites. On regards podophyllic aldehyde, derivatives with C9-aldehyde group integrated into a bicyclic heterostructure displayed more potent antileishmanial effects and were relatively safe for host cells. Docking studies of podophyllotoxin and podophyllic aldehyde derivatives showed that these compounds share a similar pattern of interaction at the colchicine site of Leishmania tubulin, thus pointing to a common mechanism of action. However, the results obtained suggested that despite tubulin is a remarkable target against leishmaniasis, there is a poor correlation between inhibition of tubulin polymerization and antileishmanial effect of many of the compounds tested, fact that points to alternative pathways to kill the parasites. Keywords: Leishmania, Tubulin, DNA

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

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Chen Hai-Tang

2011-05-01

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

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

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Maricel Labrada

1989-09-01

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

Molecular mechanisms of drug resistance in natural Leishmania populations vary with genetic background.

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Saskia Decuypere

Full Text Available The evolution of drug-resistance in pathogens is a major global health threat. Elucidating the molecular basis of pathogen drug-resistance has been the focus of many studies but rarely is it known whether a drug-resistance mechanism identified is universal for the studied pathogen; it has seldom been clarified whether drug-resistance mechanisms vary with the pathogen's genotype. Nevertheless this is of critical importance in gaining an understanding of the complexity of this global threat and in underpinning epidemiological surveillance of pathogen drug resistance in the field. This study aimed to assess the molecular and phenotypic heterogeneity that emerges in natural parasite populations under drug treatment pressure. We studied lines of the protozoan parasite Leishmania (L. donovani with differential susceptibility to antimonial drugs; the lines being derived from clinical isolates belonging to two distinct genetic populations that circulate in the leishmaniasis endemic region of Nepal. Parasite pathways known to be affected by antimonial drugs were characterised on five experimental levels in the lines of the two populations. Characterisation of DNA sequence, gene expression, protein expression and thiol levels revealed a number of molecular features that mark antimonial-resistant parasites in only one of the two populations studied. A final series of in vitro stress phenotyping experiments confirmed this heterogeneity amongst drug-resistant parasites from the two populations. These data provide evidence that the molecular changes associated with antimonial-resistance in natural Leishmania populations depend on the genetic background of the Leishmania population, which has resulted in a divergent set of resistance markers in the Leishmania populations. This heterogeneity of parasite adaptations provides severe challenges for the control of drug resistance in the field and the design of molecular surveillance tools for widespread

The Polymerase chain reaction as a tool of molecular diagnosis of Leishmania infection in the Sudan

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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

The Polymerase chain reaction as a tool of molecular diagnosis of Leishmania infection in the Sudan

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Hashim, Amna Osman Yousif [Department of Zoology, Faculty of Science, University of Khartoum, Khartoum (Sudan)

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

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

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

2017-06-01

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

In vitro anti-Leishmania infantum activity of essential oil from Piper angustifolium

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Lauriane S.S. Bosquiroli

Full Text Available Abstract Piper angustifolium Lam., Piperaceae, popularly known as "matito", "pimenta-de-macaco", "pimenta-longa" or "jagurandi" in Brazil, has been commonly used in the treatment of cutaneous leishmaniasis-associated lesions, but there are few studies on the activity against visceral leishmaniasis-associated species. This study demonstrates the first in vitro antileishmanial activity of the P. angustifolium essential oil, of which the phytochemical profile showed the presence of sesquiterpenes and monoterpenes. The main compounds were spathulenol (23.8% and caryophyllene oxide (13.1%. P. angustifolium essential oil was highly active [the half maximum inhibitory concentration = 1.43 μg/ml] against intracellular amastigotes of Leishmania infantum, the etiological agent of visceral leishmaniasis in the New and Old World. Activity was obtained 24 h after addition of the oil (6.25–50 μg/ml, with a reduction of 100% in the infection index at concentrations of 25 and 50 μg/ml. P. angustifolium essential oil showed low cytotoxicity for mammalian fibroblasts and macrophages (the half maximum inhibitory concentration values of 31.67 and 48.22 μg/ml, respectively, and it was 33 and 22 times more toxic to amastigotes than to mammalian cells, as indicated by selectivity indexes. The results demonstrated that P. angustifolium essential oil is a promising alternative for the study of potential drugs for visceral leishmaniasis.

Identification of a RAC/AKT-like gene in Leishmania parasites as a putative therapeutic target in leishmaniasis.

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Varela-M, Rubén E; Ochoa, Rodrigo; Muskus, Carlos E; Muro, Antonio; Mollinedo, Faustino

2017-10-10

Leishmaniasis is one of the world's most neglected diseases caused by at least 20 different species of the protozoan parasite Leishmania. Although new drugs have become recently available, current therapy for leishmaniasis is still unsatisfactory. A subgroup of serine/threonine protein kinases named as related to A and C protein kinases (RAC), or protein kinase B (PKB)/AKT, has been identified in several organisms including Trypanosoma cruzi parasites. PKB/AKT plays a critical role in mammalian cell signaling promoting cell survival and is a major drug target in cancer therapy. However, the role of protozoan parasitic PKB/AKT remains to be elucidated. We have found that anti-human AKT antibodies recognized a protein of about 57 kDa in Leishmania spp. parasites. Anti-human phospho-AKT(Thr308) antibodies identified a protein in extracts from Leishmania spp. that was upregulated following parasite exposure to stressful conditions, such as nutrient deprivation or heat shock. Incubation of AKT inhibitor X with Leishmania spp. promastigotes under stressful conditions or with Leishmania-infected macrophages led to parasite cell death. We have identified and cloned a novel gene from Leishmania donovani named Ld-RAC/AKT-like gene, encoding a 510-amino acid protein of approximately 57.6 kDa that shows a 26.5% identity with mammalian AKT1. Ld-RAC/AKT-like protein contains major mammalian PKB/AKT hallmarks, including the typical pleckstrin, protein kinase and AGC kinase domains. Unlike mammalian AKT that contains key phosphorylation sites at Thr308 and Ser473 in the activation loop and hydrophobic motif, respectively, Ld-RAC/AKT-like protein has a Thr residue in both motifs. By domain sequence comparison, we classified AKT proteins from different origins in four major subcategories that included different parasites. Our data suggest that Ld-RAC/AKT-like protein represents a Leishmania orthologue of mammalian AKT involved in parasite stress response and survival, and

Infective stages of Leishmania in the sandfly vector and some observations on the mechanism of transmission Formas infectante de Leishmania no vetor flebotomíneo e algumas observações sobre o mecanismo de transmissão

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Ralph Lainson

1987-09-01

Full Text Available Infective stages of Leishmania (Leishmania amazonensis, capable of producing amastigote infections in hamster skin, were shown to be present in the experimentally infected sandfly vector Lutzomyia flaviscutellata 15, 25, 40, 49, 70, 96 and 120 hours after the flies had received their infective blood-meal. Similarly, infective stages of Leishmania (L. chagasi were demonstrated in the experimentally infected vector Lu. longipalpis examined 38, 50, 63, 87, 110, 135, 171 and 221 hours following the infective blood-meal, by the intraperitoneal inoculation of the flagellates into hamsters. The question of whether or not transmission by the bite of the sandfly is dependent on the presence of [quot ]metacyclic[quot ] promastigotes in the mouthparts of the vector is discussed.Foi demonstrado através de infecção experimental, que estágios infectivos de Leishmania (L. amazonensis, capazes de produzir infecção na pele do hamster, encontram-se presentes no vetor flebotomíneo Lutzomyia flaviscutellata 15, 25, 40, 49, 70, 96 e 120 horas após o inseto ter recebido sua refeição sangüínea infectiva. Da mesma maneira, foi comprovada a presença de estágios infectivos de L. (L. chagasi em exemplares do vetor Lu. longipalpis, examinados 38, 50, 63, 87, 110, 135, 171 e 221 horas após o repasto sangüíneo infectivo - através da inoculação em hamster por via intraperitoneal dos flagelados obtidos desses fle botomíneos. A questão sobre a transmissão do gênero Leishmania pelo flebotomíneo ser ou não dependente da presença de promastigotos "metacíclios" na proboscis do vetor, é discutida.

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

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

2017-04-01

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

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

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

2002-01-01

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

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.

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

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

2007-01-01

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

In Vitro Antiparasitic and Apoptotic Effects of Antimony Sulfide Nanoparticles on Leishmania infantum

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Saied Soflaei

2012-01-01

Full Text Available Visceral leishmaniasis is one of the most important sever diseases in tropical and subtropical countries. In the present study the effects of antimony sulfide nanoparticles on Leishmania infantum in vitro were evaluated. Antimony sulfide NPs (Sb2S5 were synthesized by biological method from Serratia marcescens bacteria. Then the cytotoxicity effects of different concentrations (5, 10, 25, 50, and 100 μg/mL of this nanoparticle were assessed on promastigote and amastigote stages of L. infantum. MTT method was used for verification results of promastigote assay. Finally, the percentages of apoptotic, necrotic, and viable cells were determined by flow cytometry. The results indicated the positive effectiveness of antimony sulfide NPs on proliferation of promastigote form. The IC50 (50% inhibitory concentration of antimony sulfide NPs on promastigotes was calculated 50 μg/mL. The cytotoxicity effect was dose-dependent means by increasing the concentration of antimony sulfide NPs, the cytotoxicity curve was raised and the viability curve of the parasite dropped simultaneously. Moreover, the IC50 of antimony sulfide NPs on amastigote stage was calculated 25 μg/mL. On the other hand, however, antimony sulfide NPs have a low cytotoxicity effect on uninfected macrophages but it can induce apoptosis in promastigote stage at 3 of 4 concentrations.

Trypanosoma (Herpetosoma rangeli Tejera, 1920: intracellular amastigote stages of reproduction in white mice

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Servio Urdaneta-Morales

1986-06-01

Full Text Available The method, site, and stage of multiplication of Trypanosoma (Herpetosoma rangeli Tejera, 1920 has not hitherto been known. "We have now observed many intracellular nests or pseudocysts, containing amastigotes and trypomastigotes of this parasite in the heart, liver, and spleen of suckling (5.0 g male white mice (NMRI strain inoculated i.p. with 9 x 10(4 metatrypomastigotes/g body weight from a 12-day-old culture of the "Dog-82" strain of T. rangeli. At the peak of parasitemia (1.9 x 10(6 trypomastigotes/ml blood, 3 days post-inoculation various tissues were taken for sectioning and staining. The heart was most intensely parasitized. The amastigotes were rounded or ellipsoidal, with a rounded nucleus and the kinetoplast in the form of a straight or curved bar; the average maximum diameter of 50 measured amastigotes was 4.2 p. Binary fission was seen in the nucleus and kinetoplast of some amastigotes; no blood trypomastigotes were seen in division. The above characteristics, as well as the location of the pseudocysts in the tissues, are similar to T. cruzi. Comparison of these results with those reported for other Herpetosoma suggest study of the taxonomic position of T. rangeli.

Leishmania major infection in a dog with cutaneous manifestations.

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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

Leishmania eukaryotic initiation factor (LeIF inhibits parasite growth in murine macrophages.

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Olga Koutsoni

Full Text Available The leishmaniases constitute neglected global public health problems that require adequate control measures, prophylactic clinical vaccines and effective and non-toxic drug treatments. In this study, we explored the potential of Leishmania infantum eukaryotic initiation factor (LieIF, an exosomal protein, as a novel anti-infective therapeutic molecule. More specifically, we assessed the efficacy of recombinant LieIF, in combination with recombinant IFN-γ, in eliminating intracellular L. donovani parasites in an in vitro macrophage model. J774A.1 macrophages were initially treated with LieIF/IFN-γ prior to in vitro infection with L. donovani stationary phase promastigotes (pre-infection treatment, and resistance to infection was observed 72 h after infection. J774A.1 macrophages were also treated with LieIF/IFN-γ after L. donovani infection (post-infection treatment, and resistance to infection was also observed at both time points tested (19 h and 72 h after infection. To elucidate the LieIF/IFN-γ-induced mechanism(s that mediate the reduction of intracellular parasite growth, we examined the generation of potent microbicidal molecules, such as nitric oxide (NO and reactive oxygen species (ROS, within infected macrophages. Furthermore, macrophages pre-treated with LieIF/IFN-γ showed a clear up-regulation in macrophage inflammatory protein 1α (MIP-1α as well as tumor necrosis factor alpha (TNF-α expression. However, significant different protein levels were not detected. In addition, macrophages pre-treated with LieIF/IFN-γ combined with anti-TNF-α monoclonal antibody produced significantly lower amounts of ROS. These data suggest that during the pre-treatment state, LieIF induces intramacrophage parasite growth inhibition through the production of TNF-α, which induces microbicidal activity by stimulating NO and ROS production. The mechanisms of NO and ROS production when macrophages are treated with LieIF after infection are probably

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

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Camila M Adade

2014-10-01

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

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

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

2007-01-01

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

The lignan niranthin poisons Leishmania donovani topoisomerase IB and favours a Th1 immune response in mice

OpenAIRE

Chowdhury, Sayan; Mukherjee, Tulika; Mukhopadhyay, Rupkatha; Mukherjee, Budhaditya; Sengupta, Souvik; Chattopadhyay, Sharmila; Jaisankar, Parasuraman; Roy, Syamal; Majumder, Hemanta K

2012-01-01

Niranthin, a lignan isolated from the aerial parts of the plant Phyllanthus amarus, exhibits a wide spectrum of pharmacological activities. In the present study, we have shown for the first time that niranthin is a potent anti-leishmanial agent. The compound induces topoisomerase I-mediated DNA–protein adduct formation inside Leishmania cells and triggers apoptosis by activation of cellular nucleases. We also show that niranthin inhibits the relaxation activity of heterodimeric type IB topois...

The NAD+ metabolism of Leishmania, notably the enzyme nicotinamidase involved in NAD+ salvage, offers prospects for development of anti-parasite chemotherapy.

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Michels, Paul A M; Avilán, Luisana

2011-10-01

NAD+ plays multiple, essential roles in the cell. As a cofactor in many redox reactions it is key in the cellular energy metabolism and as a substrate it participates in many reactions leading to a variety of covalent modifications of enzymes with major roles in regulation of expression and metabolism. Cells may have the ability to produce this metabolite either via alternative de novo synthesis pathways and/or by different salvage pathways. In this issue of Molecular Microbiology, Gazanion et al. (2011) demonstrate that Leishmania species can only rely on the salvage of NAD+ building blocks. One of the enzymes involved, nicotinamidase, is absent from human cells. The enzyme is important for growth of Leishmania infantum and essential for establishing an infection. The crystal structure of the parasite protein has been solved and shows prospects for design of inhibitors to be used as leads for development of new drugs. Indeed, NAD+ metabolism is currently being considered as a promising drug target in various diseases and the vulnerability of Leishmania for interference of this metabolism has been proved in previous work by the same group, by showing that administration of NAD+ precursors has detrimental effect on the pathogenic, amastigote stage of this parasite. © 2011 Blackwell Publishing Ltd.

Journal of Biosciences | Indian Academy of Sciences

Indian Academy of Sciences (India)

Home; Journals; Journal of Biosciences. MONIDIPA GHOSH. Articles written in Journal of Biosciences. Volume 42 Issue 3 September 2017 pp 427-438 Article. Cholesterol-lowering drug, in combination with chromium chloride, induces early apoptotic signals in intracellular L. donovani amastigotes, leading to death.

Reactivity of various leishmanial antigens in a direct agglutination test and their value in differentiating post-kala azar dermal leishmaniasis from leprosy and other skin conditions

NARCIS (Netherlands)

El Harith, A.; Chowdhury, S.; Al-Masum, A.; Semião-Santos, S.; Das, P. K.; Akhter, S.; Vetter, J. C.; Haq, I.

1996-01-01

A direct agglutination test (DAT) for the detection of post-kala azar dermal leishmaniasis (PKDL) was evaluated in conditions that simulate the disease clinically or immunologically. A reference strain of Leishmania donovani (LEM 1399), and antigen preparations from Leishmania isolates from

Enhanced leishmanicidal activity of cryptopeptide chimeras from the active N1 domain of bovine lactoferrin

NARCIS (Netherlands)

Silva, T.; Abengózar, M.A.; Fernández-Reyes, M.; Andreu, D.; Nazmi, K.; Bolscher, J.G.M.; Bastos, M.; Rivas, L.

2012-01-01

wo antimicrobial cryptopeptides from the N1 domain of bovine lactoferrin, lactoferricin (LFcin17-30) and lactoferrampin (LFampin265-284), together with a hybrid version (LFchimera), were tested against the protozoan parasite Leishmania. All peptides were leishmanicidal against Leishmania donovani

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

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

2018-03-01

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

Interleukin-4 and interferon-¿ production by Leishmania stimulated peripheral blood mononuclear cells from nonexposed individuals

DEFF Research Database (Denmark)

Kurtzhals, J A; Kemp, M; Poulsen, L K

1995-01-01

of antigen stimulation suggesting a response due to antigen recognition. Both IL-4 and IFN-gamma production was abrogated by depletion of CD2+ or CD4+ but not CD8+ cells. CD2+ or CD4+ but not CD8+ enriched cultures produced cytokines as unseparated PBMC. Thus, in non-exposed individuals circulating...... call for studies of the importance of cytokine production by cross-reactive T cells for the outcome of L. donovani infections in humans and show that the method for IL-4 detection is useful for this purpose.......Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) production by Leishmania reactive peripheral blood mononuclear cells (PBMC) from non-exposed individuals was investigated. IFN-gamma was measured in culture supernatants after antigen stimulation. For the measurement of IL-4, antigen stimulated...

Coinfection of Leishmania guyanensis and Human Immunodeficiency Virus–Acquired Immune Deficiency Syndrome: Report of a Case of Disseminated Cutaneous Leishmaniasis in Ecuador

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Calvopina, Manuel; Aguirre, Cristina; Cevallos, William; Castillo, Alberto; Abbasi, Ibrahim; Warburg, Alon

2017-01-01

Reported herein is the first case of Leishmania–human immunodeficiency virus (HIV) coinfection in Ecuador. In Ecuador, HIV infections overlap endemic areas of leishmaniasis. Immunosuppression is a well-established risk factor for developing severe disease. This is a severe case of a 32-year-old man presenting with disseminated pleomorphic ulcers, papules, and cutaneous plaque-like lesions over his whole body. Numerous amastigotes were observed in both skin scrapings and biopsies. The sequence of the cytochrome b gene confirmed the presence of Leishmania guyanensis. The patient was treated but failed to respond to meglumine antimoniate and amphotericin B. Six months later, the patient died due to bacterial septic shock. PMID:28193741

A study of a self diagnostic platform for the detection of A2 biomarker for Leishmania donovani

Science.gov (United States)

Roche, Philip J. R.; Cheung, Maurice C.; Najih, Mohamed; McCall, Laura-Isobel; Fakih, Ibrahim; Chodavarapu, Vamsy P.; Ward, Brian; Ndao, Momar; Kirk, Andrew G.

2012-03-01

Visceral leishmaniasis (L.donovani) is a protozoan infection that attacks mononuclear phagocytes and causes the liver and spleen damage that can cause death. The investigation presented is a proof of concept development applying a plasmonic diagnostic platform with simple microfluidic sample delivery and optical readout. An immune-assay method is applied to the quantification of A2 protein, a highly immunogenic biomarker for the pathogen. Quantification of A2 was performed in the ng/ml range, analysis by ELISA suggested that a limit of 0.1ng/ml of A2 is approximate to 1 pathogen per ml and the sensing system shows the potential to deliver a similar level of quantification. Significant reduction in assay complexity as further enzyme linked enhancement is not required when applying a plasmonic methodology to an immunoassay. The basic instrumentation required for a portable device and potential dual optical readout where both plasmonic and photoluminescent response are assessed and investigated including consideration of the application of the device to testing where non-literate communication of results is considered and issues of performance are addressed.

Impact of leishmaniasis on public health

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L. B Camargo

2006-01-01

Full Text Available Leishmaniasis is a parasitic zoonosis caused by protozoans of the genus Leishmania transmitted by insects known as phlebotomines, which are found in wild or urban environments. It affects domestic and wild animals and transmission to man happens by accident. The disease occurs in tropical and sub-tropical areas, mainly in Asia, Europe, Africa, and the Americas. There are two forms that affect man: American cutaneous leishmaniasis (ACL and American visceral leishmaniasis (AVL. The latter is caused by three species of Leishmania: Leishmania (Leishmania donovani, Leishmania (Leishmania infantum, and Leishmania (Leishmania chagasi, which are grouped in the Leishmania (Leishmania donovani complex. Wild reservoir hosts of L. chagasi known so far are foxes and marsupials. In domestic environment, dogs are the most important reservoir hosts and sources of infection to the vectors Lutzomyia longipalpis. Leishmaniasis is difficult to control, causing epidemic outbreaks, thus being an important public health problem. Due to lesions caused by the mucocutaneous type and the severity of those caused by the visceral type in humans, visceral leishmaniasis is one of the main public health concerns. This paper is part of the monograph presented at the end of the residency program in the field of Zoonosis and Public Health at the School of Veterinary Sciences and Animal Husbandry, São Paulo State University, UNESP, Botucatu, São Paulo State, Brazil, in 2005.

An outbreak investigation of visceral leishmaniasis among residents of Dharan town, eastern Nepal, evidence for urban transmission of Leishmania donovani

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Uranw Surendra

2013-01-01

Full Text Available Abstract Background Visceral leishmaniasis (VL is a predominantly rural disease, common in the low lands of eastern Nepal. Since 1997 VL cases have also been reported among residents of the city of Dharan. Our main research objective was to find out whether there had been local transmission of VL inside the city. Methods We conducted an outbreak investigation including a case–control study; cases were all urban residents treated for VL between 2000 and 2008 at BP Koirala Institute of Health Sciences, a university hospital in the city. For each case, we selected four random controls, with no history of previous VL; frequency-matched for age. Cases and controls were subjected to a structured interview on the main exposures of interest and potential confounders; a binominal multilevel model was used to analyze the data. We also collected entomological data from all neighborhoods of the city. Results We enrolled 115 VL patients and 448 controls. Cases were strongly clustered, 70% residing in 3 out of 19 neighborhoods. We found a strong association with socio-economic status, the poorest being most at risk. Housing was a risk factor independent from socio-economic status, most at risk were those living in thatched houses without windows. ‘Sleeping upstairs’ and ‘sleeping on a bed’ were strongly protective, OR of 0.08 and 0.25 respectively; proximity to a case was a strong risk factor (OR 3.79. Sand flies were captured in all neighborhoods; in collections from several neighborhoods presence of L. donovani could be demonstrated by PCR. Conclusion The evidence found in this study is consistent with transmission of anthroponotic VL within the city. The vector P. argentipes and the parasite L. donovani have both been identified inside the town. These findings are highly relevant for policy makers; in VL endemic areas appropriate surveillance and disease control measures must be adopted not only in rural areas but in urban areas as well.

Mitochondria Superoxide Anion Production Contributes to Geranylgeraniol-Induced Death in Leishmania amazonensis

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Milene Valéria Lopes

2012-01-01

Full Text Available Here we demonstrate the activity of geranylgeraniol, the major bioactive constituent from seeds of Bixa orellana, against Leishmania amazonensis. Geranylgeraniol was identified through 1H and 13C nuclear magnetic resonance imaging and DEPT. The compound inhibited the promastigote and intracellular amastigote forms, with IC50 of 11±1.0 and 17.5±0.7 μg/mL, respectively. This compound was also more toxic to parasites than to macrophages and did not cause lysis in human blood cells. Morphological and ultrastructural changes induced by geranylgeraniol were observed in the protozoan by electronic microscopy and included mainly mitochondria alterations and an abnormal chromatin condensation in the nucleus. These alterations were confirmed by Rh 123 and TUNEL assays. Additionally, geranylgeraniol induces an increase in superoxide anion production. Collectively, our in vitro studies indicate geranylgeraniol as a selective antileishmanial that appears to be mediated by apoptosis-like cell death.

Profiling gene expression of antimony response genes in Leishmania (Viannia) panamensis and infected macrophages and its relationship with drug susceptibility.

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Barrera, Maria Claudia; Rojas, Laura Jimena; Weiss, Austin; Fernandez, Olga; McMahon-Pratt, Diane; Saravia, Nancy G; Gomez, Maria Adelaida

2017-12-01

The mechanisms of Leishmania resistance to antimonials have been primarily determined in experimentally derived Leishmania strains. However, their participation in the susceptibility phenotype in field isolates has not been conclusively established. Being an intracellular parasite, the activity of antileishmanials is dependent on internalization of drugs into host cells and effective delivery to the intracellular compartments inhabited by the parasite. In this study we quantified and comparatively analyzed the gene expression of nine molecules involved in mechanisms of xenobiotic detoxification and Leishmania resistance to antimonial drugs in resistant and susceptible laboratory derived and clinical L.(Viannia) panamensis strains(n=19). In addition, we explored the impact of Leishmania susceptibility to antimonials on the expression of macrophage gene products having putative functions in transport, accumulation and metabolism of antimonials. As previously shown for other Leishmania species, a trend of increased abcc3 and lower aqp-1 expression was observed in the laboratory derived Sb-resistant L.(V.) panamensis line. However, this was not found in clinical strains, in which the expression of abca2 was significantly higher in resistant strains as both, promastigotes and intracellular amastigotes. The effect of drug susceptibility on host cell gene expression was evaluated on primary human macrophages from patients with cutaneous leishmaniasis (n=17) infected ex-vivo with the matched L.(V.) panamensis strains isolated at diagnosis, and in THP-1 cells infected with clinical strains (n=6) and laboratory adapted L.(V.) panamensis lines. Four molecules, abcb1 (p-gp), abcb6, aqp-9 and mt2a were differentially modulated by drug resistant and susceptible parasites, and among these, a consistent and significantly increased expression of the xenobiotic scavenging molecule mt2a was observed in macrophages infected with Sb-susceptible L. (V.) panamensis. Our results

Decreased antimony uptake and overexpression of genes of thiol metabolism are associated with drug resistance in a canine isolate of Leishmania infantum

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Verónica Gómez Pérez

2016-08-01

Full Text Available Visceral leishmaniasis (VL caused by the protozoan parasite Leishmania infantum, is one of the most important zoonotic diseases affecting dogs and humans in the Mediterranean area. The presence of infected dogs as the main reservoir host of L. infantum is regarded as the most significant risk for potential human infection. We have studied the susceptibility profile to antimony and other anti-leishmania drugs (amphotericin B, miltefosine, paromomycin in Leishmania infantum isolates extracted from a dog before and after two therapeutic interventions with meglumine antimoniate (subcutaneous Glucantime®, 100 mg/kg/day for 28 days. After the therapeutic intervention, these parasites were significantly less susceptible to antimony than pretreatment isolate, presenting a resistance index of 6-fold to SbIII for promastigotes and >3-fold to SbIII and 3-fold to SbV for intracellular amastigotes. The susceptibility profile of this resistant L. infantum line is related to a decreased antimony uptake due to lower aquaglyceroporin-1 expression levels. Additionally, other mechanisms including an increase in thiols and overexpression of enzymes involved in thiol metabolism, such as ornithine decarboxylase, trypanothione reductase, mitochondrial tryparedoxin and mitochondrial tryparedoxin peroxidase, could contribute to the resistance as antimony detoxification mechanisms. A major contribution of this study in a canine L. infantum isolate is to find an antimony-resistant mechanism similar to that previously described in other human clinical isolates.

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

) and amastigotes (LDA), and the surface protease gp63. The proliferative responses to Leishmania antigens were lower in patients with severe disease than in patients with mild disease (P = 0.01-0.05), and such a difference was not observed in the response to purified protein derivative of tuberculin (PPD...... when the cells from the same donors were stimulated by TT and PPD. The percentages of patients with a Th1-like response pattern after stimulation by LMP in patients with severe and mild disease manifestations were 30% and 80%, respectively. This difference was statistically significant (P = 0.034)....

Actividad in vitro de la mezcla de alcaloides de Ervatamia coronaria (Jacq Staff. Apocynaceae sobre amastigotes de Leishmania braziliensis

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Amanda Moreno Rodríguez

Full Text Available A leishmaniose é considerada uma importante causa de morbidade e mortalidade a nível mundial, principalmente nos países tropicais. As formas cutânea e mucocutânea são causadas, entre outras espécies, por Leishmania braziliensis. Na procura de compostos leishmanicidas de origem natural, foi estudada a atividade da mistura de alcalóides de Ervatamia coronaria (Apocynaceae contra amastigotas de L. braziliensis em 6 concentrações diferentes (1, 10, 20, 25, 50 e 100 µg/mL. Foram tratados macrófagos de ratos da linha J774, infectados com promastigotas de L. braziliensis, com a mistura de alcalóides 1 hora após-infecção e diariamente por 3 dias sem mudança de meio. As experiências de citotoxicidade foram efetuadas sobre os macrófagos com azul tripam. Todos os cultivos foram feitos de forma triplicada e os grupos de controle não foram submetidos à mistura de alcalóides. Foi obtido que o composto adicionado exerce atividade doses/dependente sobre a parasita. No entanto, as concentrações mais altas (50 e 100 µg/mL, adicionado durante 3 dias, mostraram os maiores índices de infecção, provavelmente devido a diminuição no número de macrófagos, sobre os quais não foi observado efeito tóxico do tratamento durante 24 horas DL50/24h = 233,52 µg/mL. Os resultados dessa pesquisa revelaram uma nova atividade farmacológica de alcalóides da espécie Ervatamia coronaria sobre a forma amastigota de Leishmania braziliensis, com IC50 = 2,6 e 12,4 µg/mL sem mostrar toxicidade sobre a célula hospedeira.

Leishmania major Infection Activates NF-κB and Interferon Regulatory Factors 1 and 8 in Human Dendritic Cells▿

Science.gov (United States)

Jayakumar, Asha; Donovan, Michael J.; Tripathi, Vinita; Ramalho-Ortigao, Marcelo; McDowell, Mary Ann

2008-01-01

The salient feature of dendritic cells (DC) is the initiation of appropriate adaptive immune responses by discriminating between pathogens. Using a prototypic model of intracellular infection, we previously showed that Leishmania major parasites prime human DC for efficient interleukin-12 (IL-12) secretion. L. major infection is associated with self-limiting cutaneous disease and powerful immunity. In stark contrast, the causative agent of visceral leishmaniasis, Leishmania donovani, does not prime human DC for IL-12 production. Here, we report that DC priming by L. major infection results in the early activation of NF-κB transcription factors and the up-regulation and nuclear translocation of interferon regulatory factor 1 (IRF-1) and IRF-8. The inhibition of NF-κB activation by the pretreatment of DC with caffeic acid phenethyl ester blocks L. major-induced IRF-1 and IRF-8 activation and IL-12 expression. We further demonstrate that IRF-1 and IRF-8 obtained from L. major-infected human DC specifically bind to their consensus binding sites on the IL-12p35 promoter, indicating that L. major infection either directly stimulates a signaling cascade or induces an autocrine pathway that activates IRF-1 and IRF-8, ultimately resulting in IL-12 transcription. PMID:18316378

Clinical and laboratory alterations in dogs naturally infected by Leishmania chagasi

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José Cláudio Carneiro de Freitas

2012-02-01

Full Text Available INTRODUCTION: Canine visceral leishmaniasis (CVL is a zoonotic disease with different clinical manifestations. Parasitism often occurs in bone marrow, but changes have been observed in peripheral blood and serum biochemical parameters. The aim of this study was to evaluate the hematological and biochemical parameters in dogs naturally infected by Leishmania chagasi. METHODS: Eighty-five adult dogs of both sexes and various weights and ages from the Zoonosis Control Center of Fortaleza (CCZ were used, selected by immunofluorescence assay (IFA and considered positive with IFA titers greater than 1:40 and by visualizing amastigotes of Leishmania chagasi in smears obtained by bone marrow aspiration. The dogs (n = 85 were grouped according to clinical signs: negative (CN = 7, subclinical (CS = 10, and clinical (CC = 68. Blood samples were collected for determination of hematological and biochemical serum values. The experimental protocol was approved by the CEUA/UECE. RESULTS: The most frequent clinical signs were cachexia (77.9%, keratitis (61.8%, and lymphadenopathy (55.9%, and 86.8% of the animals showed more than one clinical sign characteristic of CVL. In CC were observed reductions in red blood cells (63%, hematocrit (72%, and hemoglobin (62%, as well as leukocytosis (33%, neutropenia (28%, thrombocytopenia (50%, uremia (45%, hyperproteinemia (53%, p<0.05, hypergammaglobulinemia (62%, p<0.01, and hypoalbuminemia (58%. CONCLUSIONS: Animals with the clinical form of the disease demonstrate hematological and biochemical changes consistent with anemia, uremia, hyperproteinemia, and hyperglobulinemia, which present themselves as strong clinical markers of visceral leishmaniasis associated with the signs previously reported.

Potentiating Effects of MPL on DSPC Bearing Cationic Liposomes Promote Recombinant GP63 Vaccine Efficacy: High Immunogenicity and Protection

Science.gov (United States)

Mazumder, Saumyabrata; Maji, Mithun; Ali, Nahid

2011-01-01

Background Vaccines that activate strong specific Th1-predominant immune responses are critically needed for many intracellular pathogens, including Leishmania. The requirement for sustained and efficient vaccination against leishmaniasis is to formulate the best combination of immunopotentiating adjuvant with the stable antigen (Ag) delivery system. The aim of the present study is to evaluate the effectiveness of an immunomodulator on liposomal Ag through subcutaneous (s.c.) route of immunization, and its usefulness during prime/boost against visceral leishmaniasis (VL) in BALB/c mice. Methodology/Principal Findings Towards this goal, we formulated recombinant GP63 (rGP63)-based vaccines either with monophosphoryl lipid A-trehalose dicorynomycolate (MPL-TDM) or entrapped within cationic liposomes or both. Combinatorial administration of liposomes with MPL-TDM during prime confers activation of dendritic cells, and induces an early robust T cell response. To investigate whether the combined formulation is required for optimum immune response during boost as well, we chose to evaluate the vaccine efficacy in mice primed with combined adjuvant system followed by boosting with either rGP63 alone, in association with MPL-TDM, liposomes or both. We provide evidences that the presence of either liposomal rGP63 or combined formulations during boost is necessary for effective Th1 immune responses (IFN-γ, IL-12, NO) before challenge infection. However, boosting with MPL-TDM in conjugation with liposomal rGP63 resulted in a greater number of IFN-γ producing effector T cells, significantly higher levels of splenocyte proliferation, and Th1 responses compared to mice boosted with liposomal rGP63, after virulent Leishmania donovani (L. donovani) challenge. Moreover, combined formulations offered superior protection against intracellular amastigote replication in macrophages in vitro, and hepatic and splenic parasite load in vivo. Conclusion Our results define the

Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection.

Science.gov (United States)

Tiwananthagorn, Saruda; Kato, Hirotomo; Yeewa, Ranchana; Muengpan, Amontip; Polseela, Raxsina; Leelayoova, Saovanee

2017-02-01

Leishmaniasis caused by Leishmania martiniquensis infection has been reported in human and domestic animals of Martinique Island, Germany, Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of disseminated cutaneous and visceral forms co-existence render the urgent need of specific diagnostic tool to identify the natural sand fly vectors for effective prevention and control strategies. Loop-mediated isothermal amplification (LAMP) of 18S rRNA gene as well as polymerase chain reaction (PCR) of minicircle kinetoplast DNA gene (PCR-mkDNA) have never been applied to detect L. martiniquensis and L. siamensis in sand fly vectors. The present study was aimed to validate malachite green-LAMP (MG-LAMP) and PCR-mkDNA techniques to detect L. martiniquensis in sand fly vectors, compared with the conventional PCR of internal transcribed spacer 1 (PCR-ITS1). We compared the validity of LAMP of 18S rRNA gene and PCR-mkDNA, to PCR-ITS1 in simulation model of L. martiniquensis infection in Sergentomyia gemmea sand flies. Attributable to the sensitivity and specificity, PCR-mkDNA was consecutively applied to detect L. martiniquensis in 380 female sand fly individuals captured in the newly identified affected region of Lamphun Province, Thailand. Results showed that PCR-mkDNA could detect at least one promastigote per sand fly, which was 10-time superior to LAMP and PCR-ITS1. In addition, PCR-mkDNA was more specific, able to differentiate L. martiniquensis from other viscerotropic Leishmania species, such as L. siamensis, L. (L.) donovani, and L. (L.) infantum. Consecutively, mass screening of L. martiniquensis in 380 female sand fly individuals by PCR-mkDNA was implemented in a new affected area of Thailand where a patient with leishmaniasis/HIV co-infection resides; however Leishmania DNA was undetected. In conclusion, PCR-mkDNA is a promising tool for molecular mass screening of L. martiniquensis infection in outbreak areas where several species of Leishmania

Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection

Science.gov (United States)

Tiwananthagorn, Saruda; Kato, Hirotomo; Yeewa, Ranchana; Muengpan, Amontip; Polseela, Raxsina; Leelayoova, Saovanee

2017-01-01

BACKGROUND Leishmaniasis caused by Leishmania martiniquensis infection has been reported in human and domestic animals of Martinique Island, Germany, Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of disseminated cutaneous and visceral forms co-existence render the urgent need of specific diagnostic tool to identify the natural sand fly vectors for effective prevention and control strategies. Loop-mediated isothermal amplification (LAMP) of 18S rRNA gene as well as polymerase chain reaction (PCR) of minicircle kinetoplast DNA gene (PCR-mkDNA) have never been applied to detect L. martiniquensis and L. siamensis in sand fly vectors. OBJECTIVE The present study was aimed to validate malachite green-LAMP (MG-LAMP) and PCR-mkDNA techniques to detect L. martiniquensis in sand fly vectors, compared with the conventional PCR of internal transcribed spacer 1 (PCR-ITS1). METHODS We compared the validity of LAMP of 18S rRNA gene and PCR-mkDNA, to PCR-ITS1 in simulation model of L. martiniquensis infection in Sergentomyia gemmea sand flies. Attributable to the sensitivity and specificity, PCR-mkDNA was consecutively applied to detect L. martiniquensis in 380 female sand fly individuals captured in the newly identified affected region of Lamphun Province, Thailand. FINDINGS AND MAIN CONCLUSIONS Results showed that PCR-mkDNA could detect at least one promastigote per sand fly, which was 10-time superior to LAMP and PCR-ITS1. In addition, PCR-mkDNA was more specific, able to differentiate L. martiniquensis from other viscerotropic Leishmania species, such as L. siamensis, L. (L.) donovani, and L. (L.) infantum. Consecutively, mass screening of L. martiniquensis in 380 female sand fly individuals by PCR-mkDNA was implemented in a new affected area of Thailand where a patient with leishmaniasis/HIV co-infection resides; however Leishmania DNA was undetected. In conclusion, PCR-mkDNA is a promising tool for molecular mass screening of L. martiniquensis

Leishmania major methionine sulfoxide reductase A is required for resistance to oxidative stress and efficient replication in macrophages.

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Fiona M Sansom

Full Text Available Leishmania are protozoan parasites that proliferate within the phagolysome of mammalian macrophages. While a number of anti-oxidant systems in these parasites have been shown to protect against endogenous as well as host-generated reactive oxygen species, the potential role of enzymes involved in the repair of oxidatively damaged proteins remains uncharacterized. The Leishmania spp genomes encode a single putative methionine sulfoxide reductase (MsrA that could have a role in reducing oxidized free and proteinogenic methionine residues. A GFP-fusion of L. major MsrA was shown to have a cytoplasmic localization by immunofluorescence microscopy and subcellular fractionation. An L. major msrA null mutant, generated by targeted replacement of both chromosomal allelles, was viable in rich medium but was unable to reduce exogenous methionine sulfoxide when cultivated in the presence of this amino acid, indicating that msrA encodes a functional MsrA. The ΔmsrA mutant exhibited increased sensitivity to H(2O(2 compared to wild type parasites and was unable to proliferate normally in macrophages. Wild type sensitivity to H(2O(2 and infectivity in macrophages was restored by complementation of the mutant with a plasmid encoding MsrA. Unexpectedly, the ΔmsrA mutant was able to induce normal lesions in susceptible BALB/c indicating that this protein is not essential for pathogenesis in vivo. Our results suggest that Leishmania MsrA contributes to the anti-oxidative defences of these parasites, but that complementary oxidative defence mechansims are up-regulated in lesion amastigotes.

The cytotoxic activity of miltefosine against Leishmania and macrophages is associated with dynamic changes in plasma membrane proteins.

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Fernandes, Kelly Souza; de Souza, Paulo Eduardo Narcizo; Dorta, Miriam Leandro; Alonso, Antonio

2017-01-01

In this study, we combined electron paramagnetic resonance (EPR) spectroscopy with an analysis of biophysical cellular parameters to study the mechanisms underlying the in vitro anti-leishmanial activity of miltefosine (MT). A thiol-specific spin label attached to membrane-bound proteins of Leishmania amazonensis and peritoneal macrophages indicated that MT may bind to plasma membrane proteins in large quantities via a detergent-like action and cause structural changes associated with a marked increase in dynamics and exposure to an aqueous environment. EPR spectra of a spin-labeled stearic acid indicated strong interactions between the probe and membrane proteins and a marked increase in the membrane fluidity of MT-treated cells. The cytotoxicity of MT was found to depend on the cell concentration used in the assay. This dependence was described by an equation involving the 50% inhibitory concentrations of MT in the aqueous medium (c w50 ) and the cell membrane (c m50 ) and the membrane-aqueous medium partition coefficient of MT (K). With a c w50 of 8.7μM, macrophages were less sensitive to MT than amastigotes and promastigotes of Leishmania, which had c w50 values of 2.4-3.1μM. The estimated c m50 of MT for Leishmania was 1.8M, which appears sufficient to cause ruptures or formation of pores in the plasma membrane. Additionally, we demonstrated that the changes in the plasma membrane detected by EPR spectroscopy occurred at cytotoxic concentrations of MT, as assessed through in vitro assays. Copyright © 2016 Elsevier B.V. All rights reserved.

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...

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

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

2017-11-01

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

Page Header USER Username Password Remember me Login FONT SIZE Make font size smallerMake font size defaultMake font size larger Journal Help NOTIFICATIONS View Subscribe JOURNAL CONTENT Search Search Scope Search Browse By Issue By Author By Title INFORMATION For Authors ARTICLE TOOLS Print this article Indexing metadata How to cite item Finding References Email this article (Login required) Email the author (Login required) KEYWORDS Angiogenesis Antibacterial activity Catalase Cytotoxicity. Endostatin Fritillaria imperialis, Traditional Persian Medicine, Hakim Momen Tonekaboni Herbs Hydatidosis Ischemia Liver Matrix solid-phase dispersion, HPLC, Ellagic acid, Pomegranate Nectaroscordum tripedale Nigella sativa L. Nigella sativa, Black cumin, Artemia salina, Cytotoxicity, Essential oil Olive leaf extract Renal Reperfusion Satureja khuzestanica essential oil, Leishmania major, Macrophage, Amastigote, Promastigote Scolicidal effect Superoxide dismutase Surgery HOME ABOUT LOGIN REGISTER SEARCH CURRENT ARCHIVES ANNOUNCEMENTS ONLINE SUBMISSION INSTRUCTIONS FOR AUTHORS INDEXING & ABSTRACTING ##LORESTAN UNIVERSITY OF MEDICAL SCIENCES## Home > Vol 1, No 1(December 2016) > Kheirandish THE CYTOTOXIC AND ANTILEISHMANIAL EFFECTS OF SATUREJA KHUZESTANICA ESSENTIAL OIL

OpenAIRE

Farnaz Kheirandish; Rouzbeh Chegeni; Bahram Delfan; Mahboubeh Jabari; Farzad Ebrahimzadeh; Marzieh Rashidipour

2017-01-01

Background and Aim: Leishmaniasis is endemic in 98 countries including Iran. Pentavalent antimony compounds resistance as first-line therapy is increasing in some local areas. Also side effects of these drugs are limited at the beginning of treatment, but the toxicity increases with time. The aim of this study was to assess the effect of Satureja khuzestanica essential oil (SKEO) on promastigote and amastigote Leishmania major forms. Materials and Methods: The components of S. khuzestanic...

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....

Clinical and parasitological protection in a Leishmania infantum-macaque model vaccinated with adenovirus and the recombinant A2 antigen.

Science.gov (United States)

Grimaldi, Gabriel; Teva, Antonio; Porrozzi, Renato; Pinto, Marcelo A; Marchevsky, Renato S; Rocha, Maria Gabrielle L; Dutra, Miriam S; Bruña-Romero, Oscar; Fernandes, Ana-Paula; Gazzinelli, Ricardo T

2014-06-01

Visceral leishmaniasis (VL) is a severe vector-born disease of humans and dogs caused by Leishmania donovani complex parasites. Approximately 0.2 to 0.4 million new human VL cases occur annually worldwide. In the new world, these alarming numbers are primarily due to the impracticality of current control methods based on vector reduction and dog euthanasia. Thus, a prophylactic vaccine appears to be essential for VL control. The current efforts to develop an efficacious vaccine include the use of animal models that are as close to human VL. We have previously reported a L. infantum-macaque infection model that is reliable to determine which vaccine candidates are most worthy for further development. Among the few amastigote antigens tested so far, one of specific interest is the recombinant A2 (rA2) protein that protects against experimental L. infantum infections in mice and dogs. Primates were vaccinated using three rA2-based prime-boost immunization regimes: three doses of rA2 plus recombinant human interleukin-12 (rhIL-12) adsorbed in alum (rA2/rhIL-12/alum); two doses of non-replicative adenovirus recombinant vector encoding A2 (Ad5-A2) followed by two boosts with rA2/rhIL-12/alum (Ad5-A2+rA2/rhIL12/alum); and plasmid DNA encoding A2 gene (DNA-A2) boosted with two doses of Ad5-A2 (DNA-A2+Ad5-A2). Primates received a subsequent infectious challenge with L. infantum. Vaccines, apart from being safe, were immunogenic as animals responded with increased pre-challenge production of anti-A2-specific IgG antibodies, though with some variability in the response, depending on the vaccine formulation/protocol. The relative parasite load in the liver was significantly lower in immunized macaques as compared to controls. Protection correlated with hepatic granuloma resolution, and reduction of clinical symptoms, particularly when primates were vaccinated with the Ad5-A2+rA2/rhIL12/alum protocol. The remarkable clinical protection induced by A2 in an animal model that is

Novel selective inhibitor of Leishmania (Leishmania) amazonensis arginase.

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

2015-11-01

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

In vitro evaluation of traditionally used Surinamese medicinal plants for their potential anti-leishmanial efficacy.

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Mans, D R A; Beerens, T; Magali, I; Soekhoe, R C; Schoone, G J; Oedairadjsingh, K; Hasrat, J A; van den Bogaart, E; Schallig, H D F H

2016-03-02

Plant-based preparations are extensively used in Surinamese folk medicine for treating leishmaniasis, but often without a scientific rationale. To evaluate 25 Surinamese medicinal plants for their potential efficacy against leishmaniasis. Concentrated plant extracts were evaluated for their effect on the viability of L. (V.) guyanensis AMC, L. (L.) major NADIM5, and L. (L.) donovani GEDII promastigotes, as well as intracellular amastigotes of L. (L.) donovani BHU814 in infected THP-1 cells. Selectivity was assessed by cytotoxicity against THP-1 cells. The only plant extract that showed potentially meaningful anti-leishmanial activity was that from Solanum lycocarpum that displayed mean IC50 values of about 51, 61, and 500 µg/mL against THP-1 cells. The Bryophyllum pinnatum, Inga alba, and Quassia amara extracts displayed moderate to high IC50 values against promastigotes (about 51 to >500 µg/mL) and/or amastigotes (about 224 to >500 µg/mL) but were relatively toxic to THP-1 cells (IC50 values plant extracts exhibited in many cases IC50 values close to, around, or above 500µg/mL against promastigotes, amastigotes, and THP-1 cells. The S. lycocarpum preparation may be useful against leishmaniasis and may have a good safety index, warranting further investigations into its active constituents and mechanism(s) of action. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

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

Science.gov (United States)

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

2016-01-01

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

In vitro trypanocidal activities of new S-adenosylmethionine decarboxylase inhibitors.

Science.gov (United States)

Brun, R; Bühler, Y; Sandmeier, U; Kaminsky, R; Bacchi, C J; Rattendi, D; Lane, S; Croft, S L; Snowdon, D; Yardley, V; Caravatti, G; Frei, J; Stanek, J; Mett, H

1996-01-01

A series of novel aromatic derivatives based on the structure of methylglyoxal bis(guanylhydrazone) (MGBG) was examined for in vitro antitrypanosomal activities and cytotoxicities for human cells. One-third of the compounds tested showed trypanocidal activity at concentrations below 0.5 microM after an incubation period of 72 h. Structure-activity analysis revealed that bicyclic compounds with homocyclic rings and unmodified termini were the most active compounds. Results obtained in three laboratories employing different methods and trypanosome populations consistently ranked compound CGP 40215A highest. This compound had a 50% inhibitory concentration of 0.0045 microM for Trypanosoma brucei rhodesiense, was also active against other trypanosome species, including a multidrug-resistant Trypanosoma brucei brucei, and was significantly less toxic than other compounds tested for a human adenocarcinoma cell line, with a 50% inhibitory concentration of 1.14 mM. The effect of CGP 40215A was time and dose dependent, and low concentrations of the compound required exposure times of > 2 days to exert trypanocidal activity. Compounds were inactive against Leishmania donovani and Trypanosoma cruzi amastigotes in murine macrophages in vitro. PMID:8726017

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

Science.gov (United States)

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

2016-12-01

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

Leishmania tarentolae molecular signatures in a 300 hundred-years-old human Brazilian mummy.

Science.gov (United States)

Novo, Shênia P C; Leles, Daniela; Bianucci, Raffaella; Araujo, Adauto

2015-02-04

L. tarentolae, the lizard-infecting species of Old World geckos, has been classified as non-pathogenic to man. While it has been demonstrated that L. tarentolae is capable of infecting human phagocytic cells and to differentiate into amastigote-like forms, there is no clear evidence for its efficient replication within macrophages. Here we provide first evidence for L. tarentolae ancient DNA sequences from bone marrow and intestines of a 300yo adult male. We identified molecular signatures of Leishmania tarentolae, the lizard-infecting species of Old World geckos, in hard and soft tissue biopsies from a Brazilian mummy (A74) uncovered in Itacambira (Brazil) and dating to the Colonial Period (end of 18th/beginning of the 19th century). Our results imply that efficient replication of the parasite occurred within human macrophage and to lead to a systemic spread and visceralization in this individual. The ancient sequences show a 100% similarity with those of isolated L. tarentolae parasites grown on artificial nutrient media and a 99% similarity with two modern sequences isolated from reptiles. De facto, our findings re-open the debate about the potential survival of ancient L. tarentolae strain within human macrophage and its ability to spread systemically. They also raise ecological issues since it is unknown whether this parasite circulates in the reptilian reservoir in modern day Brazil or not. Investigations on fossil fauna and arthropods are needed to shed light on the interactions between saurian Leishmania and lizards in Brazil's remote and recent past.

Macrophage and T-cell gene expression in a model of early infection with the protozoan Leishmania chagasi.

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Nicholas A Ettinger

2008-06-01

Full Text Available Visceral leishmaniasis is a potentially fatal infectious disease caused by the protozoan parasite Leishmania infantum/chagasi in the New World, or by L. donovani or L. infantum/chagasi in the Old World. Infection leads to a variety of outcomes ranging from asymptomatic infection to active disease, characterized by fevers, cachexia, hepatosplenomegaly and suppressed immune responses. We reasoned that events occurring during the initial few hours when the parasite encounters cells of the innate and adaptive immune systems are likely to influence the eventual immune response that develops. Therefore, we performed gene expression analysis using Affymetrix U133Plus2 microarray chips to investigate a model of early infection with human monocyte-derived macrophages (MDMs challenged with wild-type L. chagasi parasites, with or without subsequent co-culture with Leishmania-naïve, autologous T-cells. Microarray data generated from total RNA were analyzed with software from the Bioconductor Project and functional clustering and pathway analysis were performed with DAVID and Gene Set Enrichment Analysis (GSEA, respectively. Many transcripts were down-regulated by infection in cultures containing macrophages alone, and the pattern indicated a lack of a classically activated phenotype. By contrast, the addition of autologous Leishmania-naïve T cells to infected macrophages resulted in a pattern of gene expression including many markers of type 1 immune cytokine activation (IFN-gamma, IL-6, IL-1alpha, IL-1beta. There was simultaneous up-regulation of a few markers of immune modulation (IL-10 cytokine accumulation; TGF-beta Signaling Pathway. We suggest that the initial encounter between L. chagasi and cells of the innate and adaptive immune system stimulates primarily type 1 immune cytokine responses, despite a lack of classical macrophage activation. This local microenvironment at the site of parasite inoculation may determine the initial course of immune T

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

Science.gov (United States)

Bordbar, Ali; Parvizi, Parviz

2014-03-01

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

Biomarkers of safety and immune protection for genetically modified live attenuated leishmania vaccines against visceral leishmaniasis - discovery and implications.

Science.gov (United States)

Gannavaram, Sreenivas; Dey, Ranadhir; Avishek, Kumar; Selvapandiyan, Angamuthu; Salotra, Poonam; Nakhasi, Hira L

2014-01-01

Despite intense efforts there is no safe and efficacious vaccine against visceral leishmaniasis, which is fatal and endemic in many tropical countries. A major shortcoming in the vaccine development against blood-borne parasitic agents such as Leishmania is the inadequate predictive power of the early immune responses mounted in the host against the experimental vaccines. Often immune correlates derived from in-bred animal models do not yield immune markers of protection that can be readily extrapolated to humans. The limited efficacy of vaccines based on DNA, subunit, heat killed parasites has led to the realization that acquisition of durable immunity against the protozoan parasites requires a controlled infection with a live attenuated organism. Recent success of irradiated malaria parasites as a vaccine candidate further strengthens this approach to vaccination. We developed several gene deletion mutants in Leishmania donovani as potential live attenuated vaccines and reported extensively on the immunogenicity of LdCentrin1 deleted mutant in mice, hamsters, and dogs. Additional limited studies using genetically modified live attenuated Leishmania parasites as vaccine candidates have been reported. However, for the live attenuated parasite vaccines, the primary barrier against widespread use remains the absence of clear biomarkers associated with protection and safety. Recent studies in evaluation of vaccines, e.g., influenza and yellow fever vaccines, using systems biology tools demonstrated the power of such strategies in understanding the immunological mechanisms that underpin a protective phenotype. Applying similar tools in isolated human tissues such as PBMCs from healthy individuals infected with live attenuated parasites such as LdCen(-/-) in vitro followed by human microarray hybridization experiments will enable us to understand how early vaccine-induced gene expression profiles and the associated immune responses are coordinately regulated in normal

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

Science.gov (United States)

Mukhopadhyay, R; Madhubala, R

1995-01-01

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

Molecular characterization of a Leishmania donovani cDNA clone with similarity to human 20S proteasome a-type subunit

DEFF Research Database (Denmark)

Christensen, C B; Jørgensen, L; Jensen, A T

2000-01-01

Using plasma from patients infected or previously infected with Leishmania donovanii, we isolated a L. donovanii cDNA clone with similarity to the proteasome a-type subunit from humans and other eukaryotes. The cDNA clone, designated LePa, was DNA sequenced and Northern blot analysis of L....... donovanii poly(A(+))mRNA indicated the isolation of a full length cDNA clone with a transcript size of 1.9 kb. The expressed recombinant LePa fusion protein induced proliferation of peripheral blood mononuclear cells in one out of seven patients who had suffered from visceral leishmaniasis. Plasma from 16...

Metabolomics to unveil and understand phenotypic diversity between pathogen populations.

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Ruben t'Kindt

Full Text Available Leishmaniasis is a debilitating disease caused by the parasite Leishmania. There is extensive clinical polymorphism, including variable responsiveness to treatment. We study Leishmania donovani parasites isolated from visceral leishmaniasis patients in Nepal that responded differently to antimonial treatment due to differing intrinsic drug sensitivity of the parasites. Here, we present a proof-of-principle study in which we applied a metabolomics pipeline specifically developed for L. donovani to characterize the global metabolic differences between antimonial-sensitive and antimonial-resistant L. donovani isolates. Clones of drug-sensitive and drug-resistant parasite isolates from clinical samples were cultured in vitro and harvested for metabolomics analysis. The relative abundance of 340 metabolites was determined by ZIC-HILIC chromatography coupled to LTQ-Orbitrap mass spectrometry. Our measurements cover approximately 20% of the predicted core metabolome of Leishmania and additionally detected a large number of lipids. Drug-sensitive and drug-resistant parasites showed distinct metabolic profiles, and unsupervised clustering and principal component analysis clearly distinguished the two phenotypes. For 100 metabolites, the detected intensity differed more than three-fold between the 2 phenotypes. Many of these were in specific areas of lipid metabolism, suggesting that the membrane composition of the drug-resistant parasites is extensively modified. Untargeted metabolomics has been applied on clinical Leishmania isolates to uncover major metabolic differences between drug-sensitive and drug-resistant isolates. The identified major differences provide novel insights into the mechanisms involved in resistance to antimonial drugs, and facilitate investigations using targeted approaches to unravel the key changes mediating drug resistance.

Mitochondria and lipid raft-located FOF1-ATP synthase as major therapeutic targets in the antileishmanial and anticancer activities of ether lipid edelfosine.

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Janny A Villa-Pulgarín

2017-08-01

Full Text Available Leishmaniasis is the world's second deadliest parasitic disease after malaria, and current treatment of the different forms of this disease is far from satisfactory. Alkylphospholipid analogs (APLs are a family of anticancer drugs that show antileishmanial activity, including the first oral drug (miltefosine for leishmaniasis and drugs in preclinical/clinical oncology trials, but their precise mechanism of action remains to be elucidated.Here we show that the tumor cell apoptosis-inducer edelfosine was the most effective APL, as compared to miltefosine, perifosine and erucylphosphocholine, in killing Leishmania spp. promastigotes and amastigotes as well as tumor cells, as assessed by DNA breakdown determined by flow cytometry. In studies using animal models, we found that orally-administered edelfosine showed a potent in vivo antileishmanial activity and diminished macrophage pro-inflammatory responses. Edelfosine was also able to kill Leishmania axenic amastigotes. Edelfosine was taken up by host macrophages and killed intracellular Leishmania amastigotes in infected macrophages. Edelfosine accumulated in tumor cell mitochondria and Leishmania kinetoplast-mitochondrion, and led to mitochondrial transmembrane potential disruption, and to the successive breakdown of parasite mitochondrial and nuclear DNA. Ectopic expression of Bcl-XL inhibited edelfosine-induced cell death in both Leishmania parasites and tumor cells. We found that the cytotoxic activity of edelfosine against Leishmania parasites and tumor cells was associated with a dramatic recruitment of FOF1-ATP synthase into lipid rafts following edelfosine treatment in both parasites and cancer cells. Raft disruption and specific FOF1-ATP synthase inhibition hindered edelfosine-induced cell death in both Leishmania parasites and tumor cells. Genetic deletion of FOF1-ATP synthase led to edelfosine drug resistance in Saccharomyces cerevisiae yeast.The present study shows that the

Regulatory volume decrease in Leishmania mexicana: effect of anti-microtubule drugs

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Francehuli Dagger

2013-02-01

Full Text Available The trypanosomatid cytoskeleton is responsible for the parasite's shape and it is modulated throughout the different stages of the parasite's life cycle. When parasites are exposed to media with reduced osmolarity, they initially swell, but subsequently undergo compensatory shrinking referred to as regulatory volume decrease (RVD. We studied the effects of anti-microtubule (Mt drugs on the proliferation of Leishmania mexicana promastigotes and their capacity to undergo RVD. All of the drugs tested exerted antiproliferative effects of varying magnitudes [ansamitocin P3 (AP3> trifluoperazine > taxol > rhizoxin > chlorpromazine]. No direct relationship was found between antiproliferative drug treatment and RVD. Similarly, Mt stability was not affected by drug treatment. Ansamitocin P3, which is effective at nanomolar concentrations, blocked amastigote-promastigote differentiation and was the only drug that impeded RVD, as measured by light dispersion. AP3 induced 2 kinetoplasts (Kt 1 nucleus cells that had numerous flagella-associated Kts throughout the cell. These results suggest that the dramatic morphological changes induced by AP3 alter the spatial organisation and directionality of the Mts that are necessary for the parasite's hypotonic stress-induced shape change, as well as its recovery.

Effects of medicinal plant extracts on growth of Leishmania (L. amazonensis and Trypanosoma cruzi Efeito de extratos de plantas medicinais no crescimento de Leishmania (L. amazonensis e Trypanosoma cruzi

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Patrícia Shima Luize

2005-03-01

Full Text Available This study describes the screening of extracts obtained from 19 species of plants used in Brazilian traditional medicine for treatment of a variety of diseases. The extracts were tested against axenic amastigote and promastigote forms of Leishmania (L. amazonensis, and epimastigote forms of Trypanosoma cruzi in vitro at a concentration of 100 mg/ml. Baccharis trimera, Cymbopogon citratus, Matricaria chamomilla, Mikania glomerata, Ocimum gratissimum, Piper regnellii, Prunus domestica, Psidium guajava, Sambucus canadensis, Stryphnodendron adstringens, Tanacetum parthenium, and Tanacetum vulgare showed significant effects against one or both parasites, with a percentage of growth inhibition between 49.5 and 99%. The extracts showed no cytotoxic effect on sheep erythrocytes. These medicinal plants may be sources of new compounds that are clinically active against L. amazonensis and T. cruzi.Este estudo descreve a triagem de extratos obtidos de 19 espécies de plantas usadas na medicina tradicional brasileira para o tratamento de várias doenças. Os extratos foram testados contra formas amastigota axênica e promastigota de Leishmania (L. amazonensis, e formas epimastigota de Trypanosoma cruzi in vitro na concentração de 100 mg/ml. Baccharis trimera, Cymbopogon citratus, Matricaria chamomilla, Mikania glomerata, Ocimum gratissimum, Piper regnellii, Prunus domestica, Psidium guajava, Sambucus canadensis, Stryphnodendron adstringens, Tanacetum parthenium, e Tanacetum vulgare apresentaram efeito significante contra um ou ambos parasitas, com a porcentagem de inibição de crescimento entre 49,5 e 99%. Os extratos não mostraram efeito citotóxico em hemácias de carneiro. Essas plantas medicinais podem ser fontes alternativas de novos compostos clinicamente ativos contra L. amazonensis e T. cruzi.

Biomarkers of Safety and Immune Protection for Genetically Modified Live Attenuated Leishmania Vaccines Against Visceral Leishmaniasis – Discovery and Implications

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Gannavaram, Sreenivas; Dey, Ranadhir; Avishek, Kumar; Selvapandiyan, Angamuthu; Salotra, Poonam; Nakhasi, Hira L.

2014-01-01

Despite intense efforts there is no safe and efficacious vaccine against visceral leishmaniasis, which is fatal and endemic in many tropical countries. A major shortcoming in the vaccine development against blood-borne parasitic agents such as Leishmania is the inadequate predictive power of the early immune responses mounted in the host against the experimental vaccines. Often immune correlates derived from in-bred animal models do not yield immune markers of protection that can be readily extrapolated to humans. The limited efficacy of vaccines based on DNA, subunit, heat killed parasites has led to the realization that acquisition of durable immunity against the protozoan parasites requires a controlled infection with a live attenuated organism. Recent success of irradiated malaria parasites as a vaccine candidate further strengthens this approach to vaccination. We developed several gene deletion mutants in Leishmania donovani as potential live attenuated vaccines and reported extensively on the immunogenicity of LdCentrin1 deleted mutant in mice, hamsters, and dogs. Additional limited studies using genetically modified live attenuated Leishmania parasites as vaccine candidates have been reported. However, for the live attenuated parasite vaccines, the primary barrier against widespread use remains the absence of clear biomarkers associated with protection and safety. Recent studies in evaluation of vaccines, e.g., influenza and yellow fever vaccines, using systems biology tools demonstrated the power of such strategies in understanding the immunological mechanisms that underpin a protective phenotype. Applying similar tools in isolated human tissues such as PBMCs from healthy individuals infected with live attenuated parasites such as LdCen−/− in vitro followed by human microarray hybridization experiments will enable us to understand how early vaccine-induced gene expression profiles and the associated immune responses are coordinately regulated in normal

Development of PLGA–PEG encapsulated miltefosine based drug delivery system against visceral leishmaniasis

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Kumar, Rishikesh; Sahoo, Ganesh Chandra [Biomedical and Nanomedicine Department, Rajendra Memorial Research Institute Medical Science (ICMR) (India); Pandey, Krishna; Das, V.N.R.; Topno, Roshan K. [Clinical Medicine Department, Rajendra Memorial Research Institute Medical Science (ICMR) (India); Ansari, Md Yousuf [Pharmacoinformatics Department, National Institute Pharmaceutica Research and Education Industrial Area, Hajipur (India); Rana, Sindhuprava [Biomedical and Nanomedicine Department, Rajendra Memorial Research Institute Medical Science (ICMR) (India); Das, Pradeep [Molecular Biology Department, Rajendra Memorial Research Institute Medical Science (ICMR) (India)

2016-02-01

Targeted drug delivery systems are ideal technology to increase the maximum mechanism of action with smaller dose, we have developed miltefosine encapsulated PLGA–PEG nanoparticles (PPEM) to target macrophage of infected tissues against Leishmania donovani. The structural characterization of PLGA–PEG by transmission electron microscopy (TEM) has shown a size range of 10 to 15 nm. Synthesis and drug encapsulation confirmed by dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR) and confirmed NP encapsulation. The dose of nano encapsulated miltefosine decreased by fifty percent as compared to that of a conventional miltefosine and Amphoterecin B. The inhibition of amastigotes in the splenic tissue with nano encapsulated miltefosine (23.21 ± 23) was significantly more than the conventional miltefosine (89.22 ± 52.7) and Amphoterecin B (94.12 ± 55.1). This study signifies that there is an increased contact surface area of the nano encapsulated drug and significant reduction in size, improved the efficacy in both in vitro and in vivo study than that of the conventional miltefosine, Amphoterecin B. - Graphical abstract: The analyses of detailed structure characterized by TEM and DLS confirmed the nano-size of the particle 10–20 nm and FTIR confirmed for antileishmanial drug encapsulation in to PLGA–PEG. The dose of miltefosine is decreased by fifty percent as the IC50 value is decreased from 0.2 to 0.1 μg. Further inhibitions of amastigotes in the splenic tissue with these nanoparticles are significantly more than the conventional miltefosine and PLGA–PEG encapsulated Amphoterecin B (23.21 ± 23/89.09 ± 52.7/92.12 ± 55.1). - Highlights: • Synthesis of PLGA-PEG encapsulated miltefosine nanoparticles has been done. • An IC50 value of PPEM (0.1 ug/ml), miltefosine (0.2 μg/ml) and AmpB (1 μg/ml) were observed two fold better efficacies. • Inhibition of amastigotes of PPEM (23.21 ± 23) was significantly more than

Miltefosine and antimonial drug susceptibility of Leishmania Viannia species and populations in regions of high transmission in Colombia.

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Olga Lucía Fernández

2014-05-01

Full Text Available Pentavalent antimonials have been the first line treatment for dermal leishmaniasis in Colombia for over 30 years. Miltefosine is administered as second line treatment since 2005. The susceptibility of circulating populations of Leishmania to these drugs is unknown despite clinical evidence supporting the emergence of resistance.In vitro susceptibility was determined for intracellular amastigotes of 245 clinical strains of the most prevalent Leishmania Viannia species in Colombia to miltefosine (HePC and/or meglumine antimoniate (Sb(V; 163, (80% were evaluated for both drugs. Additionally, susceptibility to Sb(V was examined in two cohorts of 85 L. V. panamensis strains isolated between 1980-1989 and 2000-2009 in the municipality of Tumaco. Susceptibility to each drug differed among strains of the same species and between species. Whereas 68% of L. V. braziliensis strains presented in vitro resistance to HePC, 69% were sensitive to Sb(V. Resistance to HePC and Sb(V occurred respectively, in 20% y 21% of L. panamensis strains. Only 3% of L. V. guyanensis were resistant to HePC, and none to Sb(V. Drug susceptibility differed between geographic regions and time periods. Subpopulations having disparate susceptibility to Sb(V were discerned among L. V. panamensis strains isolated during 1980-1990 in Tumaco where resistant strains belonged to zymodeme 2.3, and sensitive strains to zymodeme 2.2.Large scale evaluation of clinical strains of Leishmania Viannia species demonstrated species, population, geographic, and epidemiologic differences in susceptibility to meglumine antimoniate and miltefosine, and provided baseline information for monitoring susceptibility to these drugs. Sensitive and resistant clinical strains within each species, and zymodeme as a proxy marker of antimony susceptibility for L. V. panamensis, will be useful in deciphering factors involved in susceptibility and the distribution of sensitive and resistant populations.

Immunoactivation and immunopathogeny during active visceral leishmaniasis Imunoativação e imunopatogenia durante leishmaniose visceral ativa

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Hiro Goto

2009-10-01

Full Text Available Visceral leishmaniasis is caused by protozoan parasites of the Leishmania donovani complex. During active disease in humans, high levels of IFN-γ and TNF-α detected in blood serum, and high expression of IFN-γ mRNA in samples of the lymphoid organs suggest that the immune system is highly activated. However, studies using peripheral blood mononuclear cells have found immunosuppression specific to Leishmania antigens; this poor immune response probably results from Leishmania antigen-engaged lymphocytes being trapped in the lymphoid organs. To allow the parasites to multiply, deactivating cytokines IL-10 and TGF-β may be acting on macrophages as well as anti-Leishmania antibodies that opsonize amastigotes and induce IL-10 production in macrophages. These high activation and deactivation processes are likely to occur mainly in the spleen and liver and can be confirmed through the examination of organ samples. However, an analysis of sequential data from studies of visceral leishmaniasis in hamsters suggests that factors outside of the immune system are responsible for the early inactivation of inducible nitric oxide synthase, which occurs before the expression of deactivating cytokines. In active visceral leishmaniasis, the immune system actively participates in non-lymphoid organ lesioning. While current views only consider immunocomplex deposition, macrophages, T cells, cytokines, and immunoglobulins by diverse mechanism also play important roles in the pathogenesis.A leishmaniose visceral é causada por protozoários do gênero do complexo Leishmania donovani. Durante a doença ativa no homem são detectados altos níveis de IFN-γ e de TNF-α no soro, e elevada expressão de mRNA de IFN-γ em amostras de órgãos linfóides sugerindo um estado intensamente ativado do sistema imunológico. A visão atual, no entanto, refere-se à imunossupressão específica aos antígenos de Leishmania com base em estudos utilizando células mononucleares

The Malnutrition-Related Increase in Early Visceralization of Leishmania donovani Is Associated with a Reduced Number of Lymph Node Phagocytes and Altered Conduit System Flow

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Ibrahim, Marwa K.; Barnes, Jeffrey L.; Anstead, Gregory M.; Jimenez, Fabio; Travi, Bruno L.; Peniche, Alex G.; Osorio, E. Yaneth; Ahuja, Seema S.; Melby, Peter C.

2013-01-01

In a murine model of moderate childhood malnutrition we found that polynutrient deficiency led to a 4–5-fold increase in early visceralization of L. donovani (3 days post-infection) following cutaneous infection and a 16-fold decrease in lymph node barrier function (pmalnutrition-related parasite dissemination we analyzed the cellularity, architecture, and function of the skin-draining lymph node. There was no difference in the localization of multiple cell populations in the lymph node of polynutrient deficient (PND) mice, but there was reduced cellularity with fewer CD11c+dendritic cells (DCs), fibroblastic reticular cells (FRCs), MOMA-2+ macrophages, and CD169+ subcapsular sinus macrophage (p<0.05 for all) compared to the well-nourished (WN) mice. The parasites were equally co-localized with DCs associated with the lymph node conduit network in the WN and PND mice, and were found in the high endothelial venule into which the conduits drain. When a fluorescent low molecular weight (10 kD) dextran was delivered in the skin, there was greater efflux of the marker from the lymph node conduit system to the spleens of PND mice (p<0.04), indicating that flow through the conduit system was altered. There was no evidence of disruption of the conduit or subcapsular sinus architecture, indicating that the movement of parasites into the subcortical conduit region was due to an active process and not from passive movement through a leaking barrier. These results indicate that the impaired capacity of the lymph node to act as a barrier to dissemination of L. donovani infection is associated with a reduced number of lymph node phagocytes, which most likely leads to reduced capture of parasites as they transit through the sinuses and conduit system. PMID:23967356

First Report on Isolation and Characterization of Leishmania major from Meriones hurrianae (Rodentia: Gerbillidae) of A Rural Cutaneous leishmaniasis Focus in South-Eastern Iran.

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Kassiri, Hamid; Naddaf, Saied Reza; Javadian, Ezat-Aldin; Mohebali, Mehdi

2013-09-01

Zoonotic Cutaneous Leishmaniasis (ZCL) is an endemic health problem in many rural areas of Iran, with doubled number of incidences over the last decade. Different species of rodents serve as natural reservoir host for ZCL. The disease is considered as a major health problem in rural areas of Mirjaveh, Chabahar, and Konarak Counties of Sistan va Baluchistan Province. This study describes the identity of Leishmania species, isolated from Meriones hurrianae from Chabahar County using RAPD-PCR methodology. Rodents were entrapped by live traps baited with roasted walnut, tomato, and cucumber during spring and summer. All rodents were identified based on external features including fur color, ears characteristics, tail length, hind feet, body measurements, and internal features of teeth and cranium. Giemsa-stained impressions from rodents' ears were examined for amastigotes microscopically. The samples from infected rodents were cultured in NNN+LIT medium and then the harvested parasites at the stationary phase were subjected to DNA extraction followed by amplification with RAPD-PCR. All the 28 entrapped animals were identified as M. hurrianae. Five animals showed to harbor Leishmania parasite by microscopy. Leishmania DNA isolated from five M. hurrianae produced distinctive bands of L. major with four primers. However, the products that were amplified with primers AB1-07, 327, and 329 were stable and reproducible. This is the first report on the isolation and identification of L. major from M. hurrianae from Iran. Regarding infection rate of 17.8%, M. hurrianae seems to play the major role in the maintenance and transmission of disease to humans in this area.

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

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Marcello Ceccarelli

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

Clinical and parasitological protection in a Leishmania infantum-macaque model vaccinated with adenovirus and the recombinant A2 antigen.

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Gabriel Grimaldi

2014-06-01

Full Text Available BACKGROUND: Visceral leishmaniasis (VL is a severe vector-born disease of humans and dogs caused by Leishmania donovani complex parasites. Approximately 0.2 to 0.4 million new human VL cases occur annually worldwide. In the new world, these alarming numbers are primarily due to the impracticality of current control methods based on vector reduction and dog euthanasia. Thus, a prophylactic vaccine appears to be essential for VL control. The current efforts to develop an efficacious vaccine include the use of animal models that are as close to human VL. We have previously reported a L. infantum-macaque infection model that is reliable to determine which vaccine candidates are most worthy for further development. Among the few amastigote antigens tested so far, one of specific interest is the recombinant A2 (rA2 protein that protects against experimental L. infantum infections in mice and dogs. METHODOLOGY/PRINCIPAL FINDINGS: Primates were vaccinated using three rA2-based prime-boost immunization regimes: three doses of rA2 plus recombinant human interleukin-12 (rhIL-12 adsorbed in alum (rA2/rhIL-12/alum; two doses of non-replicative adenovirus recombinant vector encoding A2 (Ad5-A2 followed by two boosts with rA2/rhIL-12/alum (Ad5-A2+rA2/rhIL12/alum; and plasmid DNA encoding A2 gene (DNA-A2 boosted with two doses of Ad5-A2 (DNA-A2+Ad5-A2. Primates received a subsequent infectious challenge with L. infantum. Vaccines, apart from being safe, were immunogenic as animals responded with increased pre-challenge production of anti-A2-specific IgG antibodies, though with some variability in the response, depending on the vaccine formulation/protocol. The relative parasite load in the liver was significantly lower in immunized macaques as compared to controls. Protection correlated with hepatic granuloma resolution, and reduction of clinical symptoms, particularly when primates were vaccinated with the Ad5-A2+rA2/rhIL12/alum protocol. CONCLUSIONS

Page Header USER Username Password Remember me Login FONT SIZE Make font size smallerMake font size defaultMake font size larger Journal Help NOTIFICATIONS View Subscribe JOURNAL CONTENT Search Search Scope Search Browse By Issue By Author By Title INFORMATION For Authors ARTICLE TOOLS Print this article Indexing metadata How to cite item Finding References Email this article (Login required Email the author (Login required KEYWORDS Angiogenesis Antibacterial activity Catalase Cytotoxicity. Endostatin Fritillaria imperialis, Traditional Persian Medicine, Hakim Momen Tonekaboni Herbs Hydatidosis Ischemia Liver Matrix solid-phase dispersion, HPLC, Ellagic acid, Pomegranate Nectaroscordum tripedale Nigella sativa L. Nigella sativa, Black cumin, Artemia salina, Cytotoxicity, Essential oil Olive leaf extract Renal Reperfusion Satureja khuzestanica essential oil, Leishmania major, Macrophage, Amastigote, Promastigote Scolicidal effect Superoxide dismutase Surgery HOME ABOUT LOGIN REGISTER SEARCH CURRENT ARCHIVES ANNOUNCEMENTS ONLINE SUBMISSION INSTRUCTIONS FOR AUTHORS INDEXING & ABSTRACTING ##LORESTAN UNIVERSITY OF MEDICAL SCIENCES## Home > Vol 1, No 1(December 2016 >\tKheirandish THE CYTOTOXIC AND ANTILEISHMANIAL EFFECTS OF SATUREJA KHUZESTANICA ESSENTIAL OIL

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Farnaz Kheirandish

2017-11-01

Full Text Available Background and Aim: Leishmaniasis is endemic in 98 countries including Iran. Pentavalent antimony compounds resistance as first-line therapy is increasing in some local areas. Also side effects of these drugs are limited at the beginning of treatment, but the toxicity increases with time. The aim of this study was to assess the effect of Satureja khuzestanica essential oil (SKEO on promastigote and amastigote Leishmania major forms. Materials and Methods: The components of S. khuzestanica oil were identified by gas chromatography/mass spectroscopy (GC/MS analysis. To evaluate antipromastigote activity the different concentrations of extract and glucantime were added to the wells that contained L. major. The plates were incubated at 26±1°C for a week. On days 1, 3 and 5, the number of live promastigotes in each well was counted. For assessment of SKEO effect on intracellular amastigotes, mouse peritoneal macrophages were isolated and infected with promastigotes. Different concentrations of the extract and glucantime were added to the cultures. The cultures were incubated at 37°C and CO2 5%. The number of infected macrophages and amastigotes within each macrophage were counted. Toxicity assessment of SKEO on macrophages was done by MTT method. Results and Conclusions: The mean number of promastigotes, infected macrophages and amastigotes in a macrophage in the control and treated groups had significantly difference. So that their number in the treated groups was less as a dose-dependent response. The present research showed potent antileishmanial activity of, SKEO; additionally this plant had no toxic effect on mammalian cells.

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

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Raquel S. Pacheco

1987-12-01

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

Improving serodiagnosis of human and canine leishmaniasis with recombinant Leishmania braziliensis cathepsin l-like protein and a synthetic peptide containing its linear B-cell epitope.

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Daniel Menezes-Souza

2015-01-01

Full Text Available The early and correct diagnosis of human leishmaniasis is essential for disease treatment. Another important step in the control of visceral leishmaniasis is the identification of infected dogs, which are the main domestic reservoir of L. infantum. Recombinant proteins and synthetic peptides based on Leishmania genes have emerged as valuable targets for serodiagnosis due to their increased sensitivity, specificity and potential for standardization. Cathepsin L-like genes are surface antigens that are secreted by amastigotes and have little similarity to host proteins, factors that enable this protein as a good target for serodiagnosis of the leishmaniasis.We mapped a linear B-cell epitope within the Cathepsin L-like protein from L. braziliensis. A synthetic peptide containing the epitope and the recombinant protein was evaluated for serodiagnosis of human tegumentary and visceral leishmaniasis, as well as canine visceral leishmaniasis.The recombinant protein performed best for human tegumentary and canine visceral leishmaniasis, with 96.30% and 89.33% accuracy, respectively. The synthetic peptide was the best to discriminate human visceral leishmaniasis, with 97.14% specificity, 94.55% sensitivity and 96.00% accuracy. Comparison with T. cruzi-infected humans and dogs suggests that the identified epitope is specific to Leishmania parasites, which minimizes the likelihood of cross-reactions.

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

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Nely Pinheiro Souza

2010-06-01

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

Genetic structure and evolution of the Leishmania genus in Africa and Eurasia: what does MLSA tell us.

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El Baidouri, Fouad; Diancourt, Laure; Berry, Vincent; Chevenet, François; Pratlong, Francine; Marty, Pierre; Ravel, Christophe

2013-01-01

Leishmaniasis is a complex parasitic disease from a taxonomic, clinical and epidemiological point of view. The role of genetic exchanges has been questioned for over twenty years and their recent experimental demonstration along with the identification of interspecific hybrids in natura has revived this debate. After arguing that genetic exchanges were exceptional and did not contribute to Leishmania evolution, it is currently proposed that interspecific exchanges could be a major driving force for rapid adaptation to new reservoirs and vectors, expansion into new parasitic cycles and adaptation to new life conditions. To assess the existence of gene flows between species during evolution we used MLSA-based (MultiLocus Sequence Analysis) approach to analyze 222 Leishmania strains from Africa and Eurasia to accurately represent the genetic diversity of this genus. We observed a remarkable congruence of the phylogenetic signal and identified seven genetic clusters that include mainly independent lineages which are accumulating divergences without any sign of recent interspecific recombination. From a taxonomic point of view, the strong genetic structuration of the different species does not question the current classification, except for species that cause visceral forms of leishmaniasis (L. donovani, L. infantum and L. archibaldi). Although these taxa cause specific clinical forms of the disease and are maintained through different parasitic cycles, they are not clearly distinct and form a continuum, in line with the concept of species complex already suggested for this group thirty years ago. These results should have practical consequences concerning the molecular identification of parasites and the subsequent therapeutic management of the disease.

Genetic Structure and Evolution of the Leishmania Genus in Africa and Eurasia: What Does MLSA Tell Us

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El Baidouri, Fouad; Diancourt, Laure; Berry, Vincent; Chevenet, François; Pratlong, Francine; Marty, Pierre; Ravel, Christophe

2013-01-01

Leishmaniasis is a complex parasitic disease from a taxonomic, clinical and epidemiological point of view. The role of genetic exchanges has been questioned for over twenty years and their recent experimental demonstration along with the identification of interspecific hybrids in natura has revived this debate. After arguing that genetic exchanges were exceptional and did not contribute to Leishmania evolution, it is currently proposed that interspecific exchanges could be a major driving force for rapid adaptation to new reservoirs and vectors, expansion into new parasitic cycles and adaptation to new life conditions. To assess the existence of gene flows between species during evolution we used MLSA-based (MultiLocus Sequence Analysis) approach to analyze 222 Leishmania strains from Africa and Eurasia to accurately represent the genetic diversity of this genus. We observed a remarkable congruence of the phylogenetic signal and identified seven genetic clusters that include mainly independent lineages which are accumulating divergences without any sign of recent interspecific recombination. From a taxonomic point of view, the strong genetic structuration of the different species does not question the current classification, except for species that cause visceral forms of leishmaniasis (L. donovani, L. infantum and L. archibaldi). Although these taxa cause specific clinical forms of the disease and are maintained through different parasitic cycles, they are not clearly distinct and form a continuum, in line with the concept of species complex already suggested for this group thirty years ago. These results should have practical consequences concerning the molecular identification of parasites and the subsequent therapeutic management of the disease. PMID:23785530

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

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

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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

Identification of immunodominant Leishmania major antigenic markers of the early C57BL/6 and BALB/c mice infection stages.

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Sassi, Atfa; Kaak, Olfa; Elgaaied, Amel Benammar

2015-08-24

The C57BL/6 mouse strain is resistant to Leishmania (L.) major infection and, unlike susceptible BALB/c, develops small self healing cutaneous lesions. The specific antibody responses of C57BL/6 and BALB/c mice were previously characterized by the predominance of IgG2a ("resistant" isotype associated with Th1) and IgG1 ("pathogenic" isotype associated with Th2) antibodies, respectively. In this study, we looked for the presence of antigens able to elicit an exclusive or predominant IgG1 production during the early stages of C57BL/6 lesion development and checked whether they are recognized or not by BALB/c mice. We demonstrate first that IgG2a predominance in C57BL/6 sera occurs only late after infection whereas in BALB/c, IgG1 antibodies dominate mostly in the early stages. Interestingly, soon after inoculation of live amastigotes, C57BL/6 displayed an exclusive IgG1 reactivity against particular L. major antigens but with MWs different from those identified in BALB/c. Furthermore, mice immunized with killed amastigotes displayed striking differences in their immunodetection profiles, particularly for the IgG1 isotype. Taken together, the observed differences in the specific antibody repertoires between infected mice resulted, at least in part, from immunological events independent from those triggered by the replicating parasite, and bring new insights into the selection of future vaccine candidates. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

The action of ionizing radiation on the morphology, physiology and growth of Leishmania Amazonensis, with evaluation of their immunogenic power in experimental models; Acao da radiacao ionizante sobre a morfologia, fisiologia e crescimento da Leishmania amazonensis, com avaliacao de seu poder imunogenico em modelos experimentais

Energy Technology Data Exchange (ETDEWEB)

Bonetti, Franco Claudio

2002-07-01

Leishmaniasis is a disease which affects thousands of people in the Tropical regions around the world, is caused by a protozoan of the genus Leishmania spp., with urban and wild mammals acting as reservoirs. In the mammal host, the amastigote form of the parasite infects and multiplies into macrophages. Treatments for leishmaniasis have a high cost and are long lasting, frequently resulting in therapy interruption. This procedure culminates with a selection of resistant parasite strains, inducing tolerance to the therapy. Either the control of vectors or the mammal host are difficult due the social and economic implications. Thus, the search for alternatives treatments against these protozoans have been stimulated. The gamma radiation ({sup 60}CO) shown to be an efficient toll to kill these parasites maintaining their immunogenicity. Cellular viability, Electronically microscopy and Multiplex-PCR techniques showed that, after irradiation, the parasites had their growth inhibited by cytoplasmatic and nucleic material disorganisation, appointing the gamma radiation as important in terms of immunogens improvement. (author)

Browse Title Index

African Journals Online (AJOL)

. K P Lander, R J E Stewart. Vol 31, No 1 ... M-C. Madekurozwa, M. Purton, J. S. Boyd. Vol 33, No 2 (2002), Refractoriness to Leishmania donovani and L. major in experimentally infected domestic and wild birds, Abstract.

Inoculação experimental de Equus asinus com Leishmania chagasi Cunha & Chagas, 1937 Experimental infection of Equus asinus with Leishmania chagasi Cunha & Chagas, 1937

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Elúzio José Lima Cerqueira

2003-12-01

Full Text Available Quatro Equus asinus foram inoculados com promastigotas de Leishmania chagasi Cunha & Chagas, 1937 e acompanhados durante 12 meses através de: pesquisa de amastigotas em esfregaços e culturas de sangue periférico em fragmentos de tecido do lábio inferior, medula óssea, baço e fígado e de testes de ELISA e TRALd. Estes foram positivos nos 8º, 10º e 12º meses após a inoculação. O exame histopatológico pós necropsia, demonstrou discreto número de amastigotas no fígado de dois dos eqüídeos inoculados. Apesar de desafiados com elevado número de promastigotas, os animais não desenvolveram infecções patentes e não infectaram experimentalmente a vetora Lutzomya longipalpis. Os resultados induzem a acreditar que os eqüídeos são desprovidos de importância como reservatórios na cadeia de transmissão da leishmaniose visceral, embora sirvam como boa fonte de alimentação sangüínea e proliferação da vetora Lutzomyia longipalpis.Four Equus asinus were challenged with promastigotes of Leishmania chagasi Cunha & Chagas, 1937, and followed up for 12 months. They were observed by means of direct testing for promastigotes in smears and culture of peripheral blood, fragments from inferior lip, bone marrow, spleen and liver and the immunological assays ELISA and TRALd. The post-necropsy histological examination demonstrated a small number of amastigotes in the liver of two animals. ELISA and TRALd tests were positive at the 8th, 10th and 12th month after inoculation. The results suggest that the donkeys were able to overcome the experimental leishmanial infection and did not infect the vector Lutzomyia longipalpis in the laboratory. Consequently they can not be considered an important reservoir in the epidemiological chain of transmission of visceral leishmaniasis, although they represent an important blood source for the vector and its proliferation.

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

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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.

The pathogenesis of post kala-azar dermal leishmaniasis from the field to the molecule: does ultraviolet light (UVB) radiation play a role?

DEFF Research Database (Denmark)

Ismail, A; Khalil, E A G; Musa, A M

2006-01-01

Post kala-azar dermal leishmaniasis (PKDL) is a dermatosis caused by persistence of Leishmania donovani parasites in the skin following apparently successful treatment of visceral leishmaniasis. The distribution of PKDL lesions in Sudanese patients often mirrors the clothing habits of those...

Observações sobre calazar em Jacobina, Bahia. VI - Investigações sobre reservatórios silvestres e comensais

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I. A. Sherlock

1988-03-01

Full Text Available Durante os anos de 1982 a 1986, a investigação sobre mamíferos comensais e silvestres, da periferia da cidade de Jacobina, Bahia, mostrou, ao lado do escasso número de exemplares, uma reduzida variedade específica dessa fauna. Capturou-se apenas 11 espécies, entre as quais, predominou o Didelphis albiventris, que abrangeu 44% dos 213 espécimens capturados. Entre os 193 com exames já concluídos, 84 eram exemplares de D. albiventris e 2 estavam infectados pela Leishmania donovani senso lato, 1 por L. mexicana amazonensis, 1 por L. braziliensis, subespécie e 3 por Trypanosoma cruzi Também foram observadas formas suspeitas de serem amastigotas de leishmanics, nos esfregaços de órgãos de 3 exemplares de Dasyprocta aguti, 1 Cercomys cunicularius - e 1 Oryzomys eliurus. 0 restante dos exemplares, inclusive 14 de Lycalopex vetulus, estava negativo para flagelados. Apesar de reforçado por outros indicadores epidemiológicos, como a predominância específica, a freqüência domiciliar, a atratividade para a vetora Lutzomyia longipalpis, e a concomitância com casos humanos nos mesmos locais, o índice de 2,3% de infecção natural do Didelphis albiventris, não autoriza a conclusão definitiva de ser o marsupial o mais importante reservatório natural da leishmaniose visceral em Jacobina.From 1982 to 1986, investigations on the natural infection with leishmanias of the sylvatic and commensal mammals were carried out around the city of Jacobina, Bahia, one of the oldest endemic foci of visceral leishmaniasis in Brazil. The species of Marsupialia Didelphis albiventris predominated, with the rate of 44%, over the total of 213 specimens belonging to only 11 different species of mammals collected there. Among the 84 specimens of D. albiventris examined, 2(2.3% were infected with Leishmania donovani sensu lato; 1 with L. mexicana amazonensis, 1 with L. braziliensis sub species, and 3 with Trypanosoma cruzi. Also, amastigote suspected bodies were

Direct detection of Leishmania from clinical samples.

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

2017-01-01

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

Epidemiology of visceral leishmaniasis

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Ready PD

2014-05-01

Full Text Available Paul D ReadyDisease Control Department, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UKAbstract: Leishmania species are the causative agents of leishmaniasis, a neglected tropical disease. These parasitic protozoans are usually transmitted between vertebrate hosts by the bite of blood sucking female phlebotomine sand flies. This review focuses on the two parasites causing most human visceral leishmaniasis (VL, which leads to substantial health problems or death for up to 400,000 people per year. Except for travel cases, Leishmania donovani infections are restricted to the (sub-tropics of Asia and Africa, where transmission is mostly anthroponotic, while Leishmania infantum occurs in the drier parts of Latin America as well as in the Mediterranean climate regions of the Old World, with the domestic dog serving as the main reservoir host. The prevalence of VL caused by L. infantum has been declining where living standards have improved. In contrast, infections of L. donovani continue to cause VL epidemics in rural areas on the Indian subcontinent and in East Africa. The current review compares and contrasts these continental differences and suggests priorities for basic and applied research that might improve VL control. Transmission cycles, pathogenesis, diagnosis, treatment and prognosis, prevention (including vector control, surveillance, transmission modeling, and international control efforts are all reviewed. Most case detection is passive, and so routine surveillance does not usually permit accurate assessments of any changes in the incidence of VL. Also, it is not usually possible to estimate the human inoculation rate of parasites by the sand fly vectors because of the limitations of survey methods. Consequently, transmission modeling rarely passes beyond the proof of principle stage, and yet it is required to help develop risk factor analysis for control programs. Anthroponotic VL

Inaccuracy of Enzyme-Linked Immunosorbent Assay Using Soluble and Recombinant Antigens to Detect Asymptomatic Infection by Leishmania infantum

Science.gov (United States)

Moreno, Elizabeth Castro; Gonçalves, Andréa Vieira; Chaves, Anderson Vieira; Melo, Maria Norma; Lambertucci, José Roberto; Andrade, Antero Silva Ribeiro; Negrão-Corrêa, Deborah; Antunes, Carlos Mauricio de Figueiredo; Carneiro, Mariângela

2009-01-01

Background One of the most important drawbacks in visceral leishmaniasis (VL) population studies is the difficulty of diagnosing asymptomatic carriers. The aim of this study, conducted in an urban area in the Southeast of Brazil, was to evaluate the performance of serology to identify asymptomatic VL infection in participants selected from a cohort with a two-year follow-up period. Methodology Blood samples were collected in 2001 from 136 cohort participants (97 positive and 39 negatives, PCR/hybridization carried out in 1999). They were clinically evaluated and none had progressed to disease from their asymptomatic state. As controls, blood samples from 22 control individuals and 8 patients with kala-azar were collected. Two molecular biology techniques (reference tests) were performed: PCR with Leishmania-generic primer followed by hybridization using L. infantum probe, and PCR with specific primer to L. donovani complex. Plasma samples were tested by ELISA using three different antigens: L. infantum and L. amazonensis crude antigens, and rK39 recombinant protein. Accuracy of the serological tests was evaluated using sensitivity, specificity, likelihood ratio and ROC curve. Findings The presence of Leishmania was confirmed, by molecular techniques, in all kala-azar patients and in 117 (86%) of the 136 cohort participants. Kala-azar patients showed high reactivity in ELISAs, whereas asymptomatic individuals presented low reactivity against the antigens tested. When compared to molecular techniques, the L. amazonensis and L. infantum antigens showed higher sensitivity (49.6% and 41.0%, respectively) than rK39 (26.5%); however, the specificity of rK39 was higher (73.7%) than L. amazonensis (52.6%) and L. infantum antigens (36.8%). Moreover, there was low agreement among the different antigens used (kappa<0.10). Conclusions Serological tests were inaccurate for diagnosing asymptomatic infections compared to molecular methods; this could lead to misclassification bias

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

Science.gov (United States)

Baneth, Gad; Yasur-Landau, Daniel; Gilad, Matan; Nachum-Biala, Yaarit

2017-03-13

Infection and clinical disease associated with Leishmania major and Leishmania tropica, two common agents of human cutaneous leishmaniosis, have rarely been reported in dogs. This study describes dogs infected with these Leishmania spp. prevalent in the Middle East and North Africa, and compares the serological response of dogs infected with Leishmania infantum, L. major or L. tropica to whole promastigote antigen enzyme-linked immunosorbent assay (ELISA) of each species and to rK39 dipstick. Leishmania major infection in a 5-month-old male dog was associated with alopecic and ulcerative periocular and limb skin lesions which responded to allopurinol treatment. Infection was detected by skin and blood polymerase chain reaction (PCR) and confirmed by DNA sequencing but the dog was seronegative. Leishmania tropica infection was detected in a 3-month-old female dog co-infected with Babesia vogeli and Anaplasma platys and with no skin lesions. PCR and DNA sequencing of the blood and parasite culture were positive for L. tropica. Sera from 11 dogs infected with L. infantum, L. major or L. tropica were reactive with all three Leishmania spp. antigens except for sera from a dog with L. major infection. No significant differences were found between reactivity of dog sera to the antigen of the infecting species, or to the other Leishmania spp. antigens. Sera from dogs infected with L. infantum and L. tropica were positive with the rK39 antigen kit, while dogs with L. major infection were seronegative. Skin lesions in L. major infected dogs from this study and previous reports (n = 2) were ulcerative and located on the muzzle, feet and foot pads and not associated with generalized lymphadenomegaly and splenomegaly. In previous L. tropica infections, skin lesions were proliferative mucocutaneous in young dogs (n = 2), or associated with widespread dermatitis, lymphadenomegaly and splenomegaly in older dogs with similarity to L. infantum infection (n = 2). This

Leishmanicidal activities of Artemisia annua leaf essential oil against Visceral Leishmaniasis

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Mohammad eIslamuddin

2014-11-01

Full Text Available Visceral leishmaniasis (VL, the second-most dreaded parasitic disease after malaria, is currently endemic in 88 countries. Dramatic increases in the rates of infection, drug resistance and non-availability of safe vaccines have highlighted the need for identification of novel and inexpensive anti-leishmanial agents from natural sources. In this study, we showed the leishmanicidal effect of essential oil from Artemisia annua leaves (AALEO against Leishmania donovani in vitro and in vivo. AALEO was extracted by hydrodistillation and characterized by GC-MS, the most abundant compounds were found to be camphor (52.06 % followed by β-caryophyllene (10.95 %. AALEO exhibited significant leishmanicidal activity against L. donovani, with 50 % inhibitory concentration of 14.63 ± 1.49 µg ml-1 and 7.3 ± 1.85 µg ml─1, respectively, against the promastigotes and intracellular amastigotes. The effect was mediated through programmed cell death as confirmed by externalization of phosphatidylserine, DNA nicking by TdT-mediated dUTP nick-end labelling (TUNEL assay, dyskinetoplastidy, cell cycle arrest at sub-G0–G1 phase, loss of mitochondrial membrane potential and reactive oxygen species (ROS generation in promastigotes and nitric oxide (NO generation in ex vivo model. AALEO presented no cytotoxic effects against mammalian macrophages even at 200 µg ml─1. Intra-peritoneal administration of AALEO (200 mg/ kg.b.w. to infected BALB/c mice reduced the parasite burden by almost 90 % in the liver and spleen with significant reduction in weight. There was no hepato- or nephro-toxicity as demonstrated by normal levels of serum enzymes. The promising antileishmanial activity shown by camphor-rich AALEO may provide a new lead in the treatment of VL.

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

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

2013-11-08

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

Evaluation of the leishmanicidal activity of plants used by Peruvian Chayahuita ethnic group.

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Estevez, Y; Castillo, D; Pisango, M Tangoa; Arevalo, J; Rojas, R; Alban, J; Deharo, E; Bourdy, G; Sauvain, M

2007-11-01

A total of 27 ethanolic plant extracts from 27 species were screened for leishmanicidal activity in vitro against Leishmania amazonensis. Most of the selected species (19) are traditionally used by the Chayahuitas, an Amazonian Peruvian ethnic group, to treat skin affections and/or leishmaniasis. A colorimetric method based on the reduction of tetrazolium salt (MTT) was used to measure the viability of Leishmania amazonensis promastigote and amastigote stages. Only the leaves of two species of the Piperaceae family (Piper hispidum Sw., and Piper strigosum Trel.) showed good leishmanicidal activities (IC(50)Cycadaceae) showed low activity against amastigote stage (IC(50) around 50 microg/ml). Of those only Tabernaemontana sananho displayed also good activity on promastigotes (IC(50)<10 microg/ml). Results are discussed herein, in relation with the traditional use of the plants and compared with other data from the relevant literature.

Synthesis and Comparison of In Vitro Leishmanicidal Activity of 5-(Nitroheteroaryl-1, 3, 4-Thiadiazols Containing Cyclic Amine of Piperidin-4-ol at C-2 with Acyclic Amine Analogues against Iranian Strain of Leishmania major (MRHO/IR/75/ER

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Azar Tahghighi

2017-04-01

Full Text Available Background: Cutaneous Leishmaniasis (CL is endemic in many tropical and subtropical regions of the world. Due to the prolonged duration of therapy, adverse effect and resistance to current drugs in the treatment of CL, the dis­covery of novel, efficient, and safe leishmanicidal drugs is required. The aims of the present study was to synthesis of new compounds based on the active compounds of 5-(5-nitrofuran-2-yl- and 5-(5-nitrothiophen-2-yl-1,3,4-thia­diazole bearing the linear amino alcohol of 3-aminopropan-1-ol in the C-2 position of thiadiazole ring and evaluation of their activity against the promastigote and amastigote forms of Leishmania major.Methods: Reaction between the solution of 5-(5-nitro heteroaryl-2-chloro-1, 3, 4-thiadiazole and piperidin-4-ol in absolute ethanol was performed and the resulting products were evaluated against promastigotes form of L. major with MTT assay and amastigote form of L. major in murine peritoneal macrophages. In addition, the toxicity of these compounds was assessed against mouse peritoneal macrophages with MTT assay.Results: New synthetic compounds 5a-b showed moderate in vitro antileishmanial activity against L. major pro­mastigotes with IC50 values of 68.9 and 27 µM, respectively. These compounds have also demonstrated a good antiamastigote activity in terms of amastigote number per macrophage, the percentage of macrophage infectivity and infectivity index.Conclusion: Novel cyclic compounds 5a-b were synthesized and exhibited less antipromastigote and antiamastigote activity compared to linear analogues.

Immunopathology of post kala-azar dermal leishmaniasis (PKDL): T-cell phenotypes and cytokine profile

DEFF Research Database (Denmark)

Ismail, A; El Hassan, A M; Kemp, K

1999-01-01

In Sudan, post kala-azar dermal leishmaniasis (PKDL) caused by Leishmania donovani develops in half of the patients treated for visceral leishmaniasis (kala-azar). In most patients lesions heal spontaneously, but in others symptoms are severe and persist for years. This study examined...

Epidemiology and clinical manifestations of visceral and cutaneous leishmaniasis in Baringo District, Rift Valley, Kenya. A literature review

NARCIS (Netherlands)

Schaefer, K. U.; Kurtzhals, J. A.; Sherwood, J. A.; Githure, J. I.; Kager, P. A.; Muller, A. S.

1994-01-01

Visceral leishmaniasis (VL), caused by Leishmania donovani, is endemic in Baringo District, Kenya. The disease has a focal distribution in the dry, hot areas below 1500 metres. Infections may be characterized as follows: 1) asymptomatic, 2) subclinical and self-limiting (not medically identifiable),

Sensitive Molecular Diagnostics for Cutaneous Leishmaniasis.

Science.gov (United States)

Sagi, Orli; Berkowitz, Anat; Codish, Shlomi; Novack, Victor; Rashti, Aviv; Akad, Fouad; Shemer-Avni, Yonat

2017-01-01

Rapid diagnosis of cutaneous leishmaniasis (CL) and identification of Leishmania species is highly important for the disease management. In Israel, CL is caused mainly by Leishmania major and Leishmania tropica species. We established an easy to handle point of care lesion-swabbing, combined with a highly sensitive multiplex real time PCR (multiplex qPCR) for accurate and rapid diagnosis of Leishmania species. Using three probes: one general for: Leishmania species, and two specific for L major , and L tropica , we screened 1783 clinical samples collected during two years. Leishmania species was found in 1086 individuals, 1008 L major , and 70 L tropica . Eight samples positive for Leishmania species only, were further tested using a second set of multiplex qPCR developed, and were found positive for Leishmania braziliensis and Leishmania infantum/donovani (2 and 6 samples, concomitantly). Taken together, the test enabled diagnostics and better treatment of Leishmania infections from the Old World (1078 samples) and the New World (8 samples), and the subtyping of the dominant strains in the region, as well as in returning travelers'.

Diagnosis of visceral leishmaniasis by the polymerase chain reaction using blood, bone marrow and lymph node samples from patients from the Sudan

DEFF Research Database (Denmark)

Andresen, K; Gasim, S; Elhassan, A M

1997-01-01

We have evaluated the sensitivity of the polymerase chain reaction (PCR) as a diagnostic tool for Leishmania donovani using blood, bone marrow and lymph node samples from Sudanese patients with a confirmed infection. Forty patients were diagnosed by microscopic examination of bone marrow or lymph...

Anti-leishmanial and Anti-cancer Activities of a Pentacyclic ...

African Journals Online (AJOL)

Erah

against promastigotes of Leishmania donovani, and anti-cancer activity on K562 leukaemic cell line. Results: A .... crisis of chronic myeloid leukemia was used for this test. The cells ... containing 1×106 cells/ml, 2 mM L-glutamine and 50 µg/ml ...

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

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Elsy Nalleli Loría-Cervera

2013-04-01

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

Detection of Leishmania RNA virus in Leishmania parasites.

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Haroun Zangger

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

Teste de aglutinação direta no sorodiagnóstico da leishmaniose visceral no estado do Pará

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Lourdes M. Garcez

1996-04-01

Full Text Available Avaliou-se o teste de aglutinação direta (TAD no sorodiagnóstico da leishmaniose visceral (LV humana e de canídeos (cão e raposa Cerdocyon thous, sendo os resultados comparados com aqueles obtidos em imunofluorescência indireta (IFI e ensaio imunoenzimático (ELISA. Utilizaram-se soros: humanos (303: indivíduos com LV confirmada (16, suspeitos de LV (65, em outras condições (102, controles negativos (15, indivíduos em área endêmica (105: de cães (82: de área endêmica (68, Salvaterra/Marajó/PA. (21 parasitologicamente positivos, e controles negativos (14, de Belém; de raposas (9: espécimes capturados na Ilha do Marajó. Antígenos para o TAD foram preparados a partir de promastigotas de Leishmania (Leishmania donovani e L. (L. chagasi. Aqueles utilizados em ELISA e IFI, respectivamente, de promastigotas (antígeno solúvel e amastigotas deL. (L. chagasi. Em humanos, a especificidade e sensibilidade do TAD, utilizando-se antígeno deL. (L. donovani, foram altas (98,4% e 100%, respectivamente e comparáveis às de IFI (97,5% e 100%. ELISA foi menos específico (84,8%, embora igualmente sensível. Em cães, o TAD foi mais específico com antígeno de L. (L. donovani do que com aquele preparado a partir de L. (L. chagasi. Entretanto, ELISA e TAD foram menos sensíveis (ambos 71,4% que IFI (100%. Essa diferença foi reflelida nos resultados de cães de área endêmica, 87% dos quais foram positivos para IFI, mas somente 54% para ELISA e 49% para o TAD. Resultados similares foram observados com raposas, quando todos os 9 soros foram positivos para IFI, 7 de 9 (78% positivos para ELISA, enquanto que o TAD não revelou nenhum resultado positivo. Concluiu-se que o TAD, usando antígeno de L. (L. donovani é um teste útil para LV humana; sua utilização em ampla escala é indicada, desde que monitorada por um laboratório de referência que garanta qualidade dos antígenos. Contudo, não é a melhor opção em inquéritos sorol

Cutaneous leishmaniasis in North Dakota.

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Douvoyiannis, Miltiadis; Khromachou, Tamim; Byers, Norman; Hargreaves, James; Murray, Henry W

2014-09-01

In the United States, autochthonous cutaneous leishmaniasis caused by infection with Leishmania mexicana has been reported from Texas and Oklahoma. Here, we describe a child with 2 new features: cutaneous infection acquired outside of the south-central United States (in North Dakota) and infection caused by Leishmania donovani species complex. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The action of ionizing radiation on the morphology, physiology and growth of Leishmania Amazonensis, with evaluation of their immunogenic power in experimental models

International Nuclear Information System (INIS)

Bonetti, Franco Claudio

2002-01-01

Leishmaniasis is a disease which affects thousands of people in the Tropical regions around the world, is caused by a protozoan of the genus Leishmania spp., with urban and wild mammals acting as reservoirs. In the mammal host, the amastigote form of the parasite infects and multiplies into macrophages. Treatments for leishmaniasis have a high cost and are long lasting, frequently resulting in therapy interruption. This procedure culminates with a selection of resistant parasite strains, inducing tolerance to the therapy. Either the control of vectors or the mammal host are difficult due the social and economic implications. Thus, the search for alternatives treatments against these protozoans have been stimulated. The gamma radiation ( 60 CO) shown to be an efficient toll to kill these parasites maintaining their immunogenicity. Cellular viability, Electronically microscopy and Multiplex-PCR techniques showed that, after irradiation, the parasites had their growth inhibited by cytoplasmatic and nucleic material disorganisation, appointing the gamma radiation as important in terms of immunogens improvement. (author)

Canine Visceral Leishmaniasis in Rio de Janeiro, Brazil: clinical, parasitological, therapeutical and epidemiological findings (1977-1983

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Mauro C. A. Marzochi

1985-09-01

Full Text Available Forty dogs from the periphery of the city of Rio de Janeiro were studied. All dogs where diagnosed as positive for leishmaniasis either parasitologically and/or serologically. Among them, 19 came from areas where only Visceral Leishmaniasis (VL occurs (Realengo, Bangu, Senador Camará. Clinical signs of the disease were seen in 36.8% of the cases, including emaciation - 100%, lymphadenopathy and depilation - 85.7%. The other 21 dogs came from an area (Campo Grande where both diseases (VL, and American Cutaneous Leishmaniasis - ACL occur. Clinical signs of the disease, mainly cutaneous or mucocutaneous ulcers were seen in 76.2% of the cases. Leishmania parasites were found in 39 cases: 22% in viscera, 42.5% in viscera and normal skin and 35% in cutaneous or mucocutaneous ulcers. All the Leishmania stocks isolated from dogs which came from Realengo, Bangu, Senador Camará (VL area, and from Campo Grande (VL + ACL area were characterized as L. donovani (except in one case according to their schizodeme, zymodeme and serodeme. The only stock characterized as L. b. braziliensis, was isolated from the lymph node of a dog from Campo Grande with visceral disease and without skin lesions. Antimony therapy attempted in eight Leishmania donovani positive dogs was unsuccessful.Durante inquéritos caninos realizados na periferia da cidade do Rio de Janeiro, foram estudados clínica e laboratorialmente 40 cães. Todos apresentavam diagnóstico parasitológico e/ou sorológico de leishmaniose. Dentre esses, 19 procediam de áreas de ocorrência de leishmaniose visceral (LV - Realengo, Bangu e Senador Camará. Sinais clínicos sugestivos da infecção foram observados em 36,8% deles (incluindo emagrecimento - 100%, linfadenopatia e depilação - 85,7%. Outros 21 cães procediam da área de Campo Grande onde tanto a LV como a leishmaniose tegumentar americana (LTA ocorrem. Sinais clínicos da infecção por Leishmania, principalmente ulcerações cutâneas e

Natural product inhibitors of fatty acid biosynthesis: synthesis of the marine microbial metabolites pseudopyronines A and B and evaluation of their anti-infective activities

DEFF Research Database (Denmark)

Giddens, Anna C.; Nielsen, Lone; Boshoff, Helena I.

2007-01-01

of pathogenic microorganisms and were found to exhibit good potency (IC50≥0.46 μg/mL) and selectivity towards Leishmania donovani. Several of the compounds inhibited recombinant fatty acid biosynthesis enzymes from both Plasmodium falciparum and Mycobacterium tuberculosis, validating these targets in the search...

Natural infection of bats with Leishmania in Ethiopia.

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

2015-10-01

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

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

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

2017-08-01

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

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

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Sarah Athayde Couto Falcão

2016-08-01

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

High frequency of visceral leishmaniasis in dogs under veterinary clinical care in an intense transmission area in the state of Tocantins, Brazil

OpenAIRE

Santos, Helcileia Dias; Galvão, Samara Rocha; Dias, Francisca Elda Ferreira; Ribeiro, Taiã Mairon Peixoto; Negreiros Filho, Osmar; Sousa, Sebastiana Adriana Pereira; Minharro, Silvia

2017-01-01

ABSTRACT: A direct search for parasites were used as the diagnostic test to determine the frequency of Leishmania spp. infection in dogs ( Canis lupus familiaris ) under veterinary clinical care in the city of Araguaína, Tocantins, Brazil. For this approach, lymph node cell samples were collected using needle aspiration from 649 dogs of different breeds and ages. Two hundred and sixty four (40.7%) dogs tested positive for amastigote forms of Leishmania spp. Furthermore, 202 (76.5%) dogs tha...

Journal of Biosciences | Indian Academy of Sciences

Indian Academy of Sciences (India)

Leishmania donovani requires an exogenous source of heme for growth and transformation. In in vitro culture of the free-living promastigotes, exogenously added hemin enhances cell proliferation. In this investigation, the question of the function of heme with particular reference to protein synthesis and cell proliferation has ...

Molecular identification of Leishmania species in Taybad district, Iran

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Salehi Ghodratollah

2014-09-01

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

Innate Immunity against Leishmania Infections

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Gurung, Prajwal; Kanneganti, Thirumala-Devi

2015-01-01

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

Genes that encodes NAGT, MIF1 and MIF2 are not virulence factors for kala-azar caused by Leishmania infantum

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Bruno Guedes Alcoforado Aguiar

2014-10-01

Full Text Available Introduction Kala-azar is a disease resulting from infection by Leishmania donovani and Leishmania infantum. Most patients with the disease exhibit prolonged fever, wasting, anemia and hepatosplenomegaly without complications. However, some patients develop severe disease with hemorrhagic manifestations, bacterial infections, jaundice, and edema dyspnea, among other symptoms, followed by death. Among the parasite molecules that might influence the disease severity are the macrophage migration inhibitory factor-like proteins (MIF1 and MIF2 and N-acetylglucosamine-1-phosphotransferase (NAGT, which act in the first step of protein N-glycosylation. This study aimed to determine whether MIF1, MIF2 and NAGT are virulence factors for severe kala-azar. Methods To determine the parasite genotype in kala-azar patients from Northeastern Brazil, we sequenced the NAGT genes of L. infantum from 68 patients as well as the MIF1 and MIF2 genes from 76 different subjects with diverse clinical manifestations. After polymerase chain reaction (PCR, the fragments were sequenced, followed by polymorphism identification. Results The nucleotide sequencing of the 144 amplicons revealed the absence of genetic variability of the NAGT, MIF1 and MIF2 genes between the isolates. The conservation of these genes suggests that the clinical variability of kala-azar does not depend upon these genes. Additionally, this conservation suggests that these genes may be critical for parasite survival. Conclusions NAGT, MIF1 and MIF2 do not alter the severity of kala-azar. NAGT, MIF1 and MIF2 are highly conserved among different isolates of identical species and exhibit potential for use in phylogenetic inferences or molecular diagnosis.

Adipose Tissue-Derived Mesenchymal Stem Cells as a New Host Cell in Latent Leishmaniasis

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Allahverdiyev, Adil M.; Bagirova, Melahat; Elcicek, Serhat; Koc, Rabia Cakir; Baydar, Serap Yesilkir; Findikli, Necati; Oztel, Olga N.

2011-01-01

Some protozoan infections such as Toxoplasma, Cryptosporidium, and Plasmodium can be transmitted through stem cell transplantations. To our knowledge, so far, there is no study about transmission of Leishmania parasites in stem cell transplantation and interactions between parasites and stem cells in vitro. Therefore, the aim of this study was to investigate the interaction between different species of Leishmania parasites and adipose tissue-derived mesenchymal stem cells (ADMSCs). ADMSCs have been isolated, cultured, characterized, and infected with different species of Leishmania parasites (L. donovani, L. major, L. tropica, and L. infantum). Infectivity was examined by Giemsa staining, microculture, and polymerase chain reaction methods. As a result, infectivity of ADMSCs by Leishmania parasites has been determined for the first time in this study. According to our findings, it is very important that donors are screened for Leishmania parasites before stem cell transplantations in regions where leishmaniasis is endemic. PMID:21896818

Early and late molecular and morphologic changes that occur during the in vitro transformation of Trypanosoma cruzi metacyclic trypomastigotes to amastigotes

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VÍCTOR T CONTRERAS

2002-01-01

Full Text Available The amastigogenesis primary of T. cruzi occurs naturally when metacyclic trypomastigotes transform into amastigotes within the cells of the mammalian host. The in vitro study of the macromolecular changes that occur over several days during the transformation process should provide significant indications of how the parasite adapts to the mammalian host environment. We show here that metacyclic trypomastigotes pre-incubated at 37° C in a protein-rich medium reach a high degree of transformation to amastigotes when re-incubated in the fresh medium. Giemsa-stained smears show that during the pre-incubation phase, the metacyclic trypomastigotes undergo lengthening at the posterior end and a thinning out of the entire body. SDS-PAGE analysis of polypeptides and glycopeptides or Western blot with stage-specific antisera analyses indicate that the in vitro primary amastigogenesis is associated with abrupt changes in protein, glycoprotein, and stage-specific antigens that occur simultaneously during the first 24 hours of pre-incubation. Since the differentiating system consists of a rich media at 37° C, temperature and medium constitution must trigger a macromolecular differentiation to amastigotes that precedes the morphological transformation by several days. This transformation is associated with the rearrangement of stage-specific antigens and takes place when the culture medium is changed.

Cytotoxicity and Phototoxicity of Chlorophyll a/Hydroxypropyl-γ-cyclodextrin Complex on Leishmania Major Promastigotes

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Azam Jafari Parizi

2011-06-01

Full Text Available Introduction: Cutaneous leishmaniasis (CL is a widespread disease that is epidemic in Iran, too. Photodynamic therapy (PDT is an attractive modality to treat cancer and hyper proliferative diseases based on the use of a photosensitizer in the presence of oxygen and proper wavelength of light. In consideration of lesion location, lack of systemic involvement and inefficiency of current treatments, nowadays this modality is purposed for treating Leishmaniasis. In this paper, efficacy of PDT using a natural dye (chlorophyll a on Leishmania major promastigotes is reported. Material and Methods: The experiments was done on Leishmania major parasites (MRHO/IR/75/ER in the presence of Chlorophyll a /Hydroxypropyl-γ-cyclodextrin(chl a/CD complex as a photosensitizer. At first, dye uptake by promastigotes was evaluated via fluorimetric assessments after different incubation periods. Then dye cytotoxicity was evaluated at different concentration after 24 h incubation. Finally PDT experiments were designed with two doses of light and 10 µM of photosensitizer. Considering all possible controls, the percentage of the parasite survival at 24 hours post treatment was assessed by MTS method. All experiments were repeated at least three times. Results: On the basis of the dye uptake data, 24h was considered for incubating of photosensitizer with promastigotes. IC50 of chl a/CD complex was about 42.6 µM. After parasites irradiation by light at 248 j/cm2, more than 50% of cell death was recorded that is significant in comparing with similar groups without dye, without light, and lower dose of light. In these conditions, ED50 of PDT on promastigotes is determined nearly 246 J/cm2. Discussion and Conclusion: Considering low cytotoxicity in darkness and adequate phototoxicity of chl a/CD complex in comparison with other photosensitizers such as AlPhtalocyanine chloride, it can be introduced as a promising candidate for futher use in PDT experiments on amastigotes

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

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C. Vasconcellos

1999-07-01

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

Clinical and histopathological characteristics of cutaneous Leishmaniasis in Sanliurfa City of Turkey including Syrian refugees.

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Koçarslan, Sezen; Turan, Enver; Ekinci, Turan; Yesilova, Yavuz; Apari, Rabia

2013-01-01

The aim of our study was to investigate the clinical and histopathological characteristics of cutaneous leishmaniasis (CL) in the city of Sanliurfa in Turkey, where Syrian refugees also reside. At the Harran University Hospital outpatient clinics between 2012 and 2013, 54 CL cases, including 24 Syrian patients, underwent punch biopsy of the skin and/or a touch imprint. Patients in whom leishmania parasites were detected were included in the study. The clinical and histopathological data of the patients were obtained by a review of the patients' medical records. All the slides of each patient were re-evaluated histopathologically. Fifty-four cases (mean age; 17 ± 12 years), consisting of 32 males (59.3%) and 22 females (40.7%), were examined. The most common site of involvement was the face (63%). The most common presentation was noduloulcerative lesions (57.4%). Histopathologically, the majority of the cases exhibited hyperkeratosis, follicular plugging of the epidermis, chronic inflammatory infiltration, leishmania amastigotes and non-caseating granulomatous inflammation in the dermis. CL presents with a wide spectrum of expression, both clinically and histologically, and may mimic other inflammatory and neoplastic diseases. The diagnosis of CL relies on the identification of leishmania amastigotes in either a direct smear of the lesion or in a tissue section.

Phylogenomic reconstruction supports supercontinent origins for Leishmania.

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

2016-03-01

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

Screening for Inhibitors of Essential Leishmania Glucose Transporters

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2013-07-01

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

Biomarkers of safety and immune protection for genetically modified live attenuated Leishmania vaccines against visceral leishmaniasis-Discovery and implications

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Sreenivas eGannavaram

2014-05-01

Full Text Available Despite intense efforts there is no safe and efficacious vaccine against visceral leishmaniasis, which is fatal and endemic in many tropical countries. A major shortcoming in the vaccine development against blood borne parasitic agents such as Leishmania is the inadequate predictive power of the early immune responses mounted in the host against the experimental vaccines. Often immune correlates derived from in-bred animal models do not yield immune markers of protection that can be readily extrapolated to humans. The limited efficacy of vaccines based on DNA, sub-unit, heat killed parasites has led to the realization that acquisition of durable immunity against the protozoan parasites requires a controlled infection with a live attenuated organism. Recent success of irradiated malaria parasites as a vaccine candidate further strengthens this approach to vaccination. We developed several gene deletion mutants in L. donovani as potential live attenuated vaccines and reported extensively on the immunogenicity of LdCentrin1 deleted mutant in mice, hamsters and dogs. Additional limited studies using genetically modified live attenuated Leishmania parasites as vaccine candidates have been reported. However, for the live attenuated parasite vaccines, the primary barrier against widespread use remains the absence of clear biomarkers associated with protection and safety. Recent studies in evaluation of vaccines e.g., influenza and yellow fever vaccines, using systems biology tools demonstrated the power of such strategies in understanding the immunological mechanisms that underpin a protective phenotype. Applying similar tools in isolated human tissues such as PBMCs from healthy individuals infected with live attenuated parasites such as LdCen1-/- in vitro followed by human microarray hybridization experiments will enable us to understand how early vaccine-induced gene expression profiles and the associated immune responses are coordinately regulated

Visceral leishmaniasis with cutaneous lesions in a patient infected with human immunodeficiency virus.

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Ara, M; Maillo, C; Peón, G; Clavel, A; Cuesta, J; Grasa, M P; Carapeto, F J

1998-07-01

We report a case of visceral leishmaniasis (VL) with cutaneous lesions in a patient infected with human immunodeficiency virus (HIV). The cutaneous lesions consisted of erythematous papules on the legs. Biopsy of one lesion showed abundant Leishmania amastigotes within epithelial cells of an eccrine sweat gland in the dermis. Leishmania organisms were also found in a blood smear. Rapid and complete clearance of the cutaneous lesions was achieved after antimony therapy. Cutaneous lesions in VL are being reported increasingly frequently in patients with HIV infection and their significance remains in discussion.

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

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

2018-03-15

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

SHORT COMMUNICATION SQUALENE AND AMENTOFLAVONE ...

African Journals Online (AJOL)

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against Leishmania donovani and Trypanosoma cruzi [4]. In addition, strong fungitoxic activity ... leaves of A. laciniatum. Except for the study of the essential oil of this plant [5], which showed activity against strain of ... Squalene is the main constituent of shark liver oil, yeast and many vegetable oils [12] and it has also been ...

Leishmania infections: Molecular targets and diagnosis.

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

2017-10-01

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

Metabolomic Analyses of Leishmania Reveal Multiple Species Differences and Large Differences in Amino Acid Metabolism.

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Gareth D Westrop

Full Text Available Comparative genomic analyses of Leishmania species have revealed relatively minor heterogeneity amongst recognised housekeeping genes and yet the species cause distinct infections and pathogenesis in their mammalian hosts. To gain greater information on the biochemical variation between species, and insights into possible metabolic mechanisms underpinning visceral and cutaneous leishmaniasis, we have undertaken in this study a comparative analysis of the metabolomes of promastigotes of L. donovani, L. major and L. mexicana. The analysis revealed 64 metabolites with confirmed identity differing 3-fold or more between the cell extracts of species, with 161 putatively identified metabolites differing similarly. Analysis of the media from cultures revealed an at least 3-fold difference in use or excretion of 43 metabolites of confirmed identity and 87 putatively identified metabolites that differed to a similar extent. Strikingly large differences were detected in their extent of amino acid use and metabolism, especially for tryptophan, aspartate, arginine and proline. Major pathways of tryptophan and arginine catabolism were shown to be to indole-3-lactate and arginic acid, respectively, which were excreted. The data presented provide clear evidence on the value of global metabolomic analyses in detecting species-specific metabolic features, thus application of this technology should be a major contributor to gaining greater understanding of how pathogens are adapted to infecting their hosts.

Quantification of anti-Leishmania antibodies in saliva of dogs.

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

2017-08-15

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

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

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

2013-01-01

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

Human immunodeficiency virus/Leishmania infantum in the first foci of urban American visceral leishmaniasis: clinical presentation from 1994 to 2010

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Iúri Paz Lima

2013-04-01

Full Text Available INTRODUCTION: Human immunodeficiency virus (HIV coinfection with Leishmania infantum or Leishmania donovani, the agents of visceral leishmaniasis (or kala-azar, has become a fatal public health problem in the tropics where kala-azar is endemic. METHODS: The clinical presentation of patients with HIV and L. infantum coinfection is described using two unique databases that together produce the largest case series of patients with kala-azar infected with HIV in South America. First, a retrospective study paired the list of all patients with kala-azar from 1994 to 2004 with another of all patients with HIV/AIDS from the reference hospital for both diseases in the City of Teresina, State of Piauí, Brazil. Beginning in 2005 through to 2010 this information was prospectively collected at the moment of hospitalization. RESULTS: During the study, 256 admissions related to 224 patients with HIV/L. infantum coinfection were registered and most of them were males between 20-40 years of age. Most of the 224 patients were males between 20-40 years of age. HIV contraction was principally sexual. The most common symptoms and signs were pallor, fever, asthenia and hepatosplenomegaly. 16.8% of the cohort died. The primary risk factors associated to death were kidney or respiratory failure, somnolence, hemorrhagic manifestations and a syndrome of systemic inflammation. The diagnosis of HIV and kala-azar was made simultaneously in 124 patients. CONCLUSIONS: The urban association between HIV and kala-azar coinfection in South America is worrisome due to difficulty in establishing the diagnosis and higher mortality among the coinfected then those with either disease independently. HIV/L. infantum coinfection exhibits some singular characteristics and due to its higher mortality it requires immediate assistance to patients and greater research on appropriate combination therapy.

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

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

2012-06-08

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

Strategic evaluation of vaccine candidate antigens for the prevention of Visceral Leishmaniasis.

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Duthie, Malcolm S; Favila, Michelle; Hofmeyer, Kimberley A; Tutterrow, Yeung L; Reed, Steven J; Laurance, John D; Picone, Alessandro; Guderian, Jeffrey; Bailor, H Remy; Vallur, Aarthy C; Liang, Hong; Mohamath, Raodoh; Vergara, Julie; Howard, Randall F; Coler, Rhea N; Reed, Steven G

2016-05-27

Infection with Leishmania parasites results in a range of clinical manifestations and outcomes, the most severe of which is visceral leishmaniasis (VL). Vaccination will likely provide the most effective long-term control strategy, as the large number of vectors and potential infectious reservoirs renders sustained interruption of Leishmania parasite transmission extremely difficult. Selection of the best vaccine is complicated because, although several vaccine antigen candidates have been proposed, they have emerged following production in different platforms. To consolidate the information that has been generated into a single vaccine platform, we expressed seven candidates as recombinant proteins in E. coli. After verifying that each recombinant protein could be recognized by VL patients, we evaluated their protective efficacy against experimental L. donovani infection of mice. Administration in formulation with the Th1-potentiating adjuvant GLA-SE indicated that each antigen could elicit antigen-specific Th1 responses that were protective. Considering the ability to reduce parasite burden along with additional factors such as sequence identity across Leishmania species, we then generated a chimeric fusion protein comprising a combination of the 8E, p21 and SMT proteins. This E. coli -expressed fusion protein was also demonstrated to protect against L. donovani infection. These data indicate a novel recombinant vaccine antigen with the potential for use in VL control programs. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Antileishmanial activity of 12-methoxycarnosic acid from Salvia repens Burch. ex Benth. (Lamiaceae)

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Mokoka, TA

2014-01-01

Full Text Available .5 ml/min, and the injection volume was 35 µl (350 µg extract in DMSO). Thirty-five one-92 minute fractions were collected in 96 deep well plates during the run (Screenmates 96 well, 93 Matrix Technology, Hudson, USA) and dried in a GeneVac EZ-2 Plus...) at two different concentrations (5 µg/ml) and (0.8 102 6 µg/ml) against L. donovani amastigotes (MHOM/ET/67/L82) in 96-well microtiter plate. The 103 plate was read in a spectramax Gemini XS microplate fluorometer (Molecular Devices 104 Cooperation...

Leishmanicidal activity of Piper nigrum bioactive fractions is interceded via apoptosis in vitro and substantiated by Th1 immunostimulatory potential in vivo

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Garima eChouhan

2015-12-01

Full Text Available VL is a life-threatening protozoal infection chiefly impinging the rural and poor population in the tropical and sub-tropical countries. The deadly affliction is rapidly expanding after its association with AIDS, swiftly defying its status of a neglected disease. Despite successful formulation of vaccine against canine leishmaniasis, no licensed vaccine is yet available for human VL, chemotherapy is in appalling state, and the development of new candidate drugs has been painfully slow. In face of lack of proper incentives, immunostimulatory plant preparations owing antileishmanial efficacy bear potential to rejuvenate awful antileishmanial chemotherapy. We have earlier reported profound leishmanicidal activity of P. nigrum seeds hexane (PNH and P. nigrum ethanolic (PNE fractions derived from P. nigrum seeds against Leishmania donovani promastigotes and amastigotes. In the present study, we illustrate that the remarkable anti-promastigote activity exhibited by PNH and PNE is mediated via apoptosis as evidenced by phosphatidylserine externalization, DNA fragmentation, arrest in sub G0/G1 phase, loss of mitochondrial membrane potential and generation of reactive oxygen species. Further, P. nigrum bioactive fractions rendered significant protection to L. donovani infected BALB/c mice in comparison to piperine, a known compound present in Piper species. The substantial therapeutic potential of PNH and PNE was accompanied by elicitation of cell-mediated immune response. The bioactive fractions elevated the secretion of Th1 (INF-γ, TNF-α and IL-2 cytokines and declined IL-4 and IL-10. PNH and PNE enhanced the production of IgG2a, upregulated the expression of co-stimulatory molecules CD80 and CD86, augmented splenic CD4+ and CD8+ T cell population, induced strong lymphoproliferative and DTH responses and partially stimulated NO production. PNH and PNE were devoid of any hepatic or renal toxicity. These encouraging findings merit further exploration

Medicinal plants from the Yanesha (Peru): evaluation of the leishmanicidal and antimalarial activity of selected extracts.

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Valadeau, Céline; Pabon, Adriana; Deharo, Eric; Albán-Castillo, Joaquina; Estevez, Yannick; Lores, Fransis Augusto; Rojas, Rosario; Gamboa, Dionicia; Sauvain, Michel; Castillo, Denis; Bourdy, Geneviève

2009-06-25

Ninety-four ethanolic extracts of plants used medicinally by the Yanesha, an Amazonian Peruvian ethnic group, for affections related to leishmaniasis and malaria were screened in vitro against Leishmania amazonensis amastigotes and against a Plasmodium falciparum chloroquine resistant strain. The viability of Leishmania amazonensis amastigote stages was assessed by the reduction of tetrazolium salt (MTT) while the impact on Plasmodium falciparum was determined by measuring the incorporation of radio-labelled hypoxanthine. Six plant species displayed good activity against Plasmodium falciparum chloroquine resistant strain (IC(50) Piper aduncum L. and Piper sp.) and the leaves of Jacaranda copaia (Aubl.) D. Don (Bignoniaceae). Eight species displayed interesting leishmanicidal activities (IC50 < 10 microg/ml): Carica papaya L. (Caricaceae), Piper dennisii Trel (Piperaceae), Hedychium coronarium J. König (Zingiberaceae), Cestrum racemosum Ruiz & Pav. (Solanaceae), Renealmia alpinia (Rottb.) Zingiberaceae, Lantana sp. (Verbenaceae), Hyptis lacustris A. St.-Hil. ex Benth. (Lamiaceae) and Calea montana Klat. (Asteraceae). Most of them are used against skin affections by Yanesha people. Results are discussed herein, according to the traditional use of the plants and compared with data obtained from the literature.

Understanding serine proteases implications on Leishmania spp lifecycle.

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

2018-01-01

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

Improved proteolytic stability and potent activity against Leishmania infantum trypanothione reductase of α/β-peptide foldamers conjugated to cell-penetrating peptides.

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de Lucio, Héctor; Gamo, Ana María; Ruiz-Santaquiteria, Marta; de Castro, Sonia; Sánchez-Murcia, Pedro A; Toro, Miguel A; Gutiérrez, Kilian Jesús; Gago, Federico; Jiménez-Ruiz, Antonio; Camarasa, María-José; Velázquez, Sonsoles

2017-11-10

The objective of the current study was to enhance the proteolytic stability of peptide-based inhibitors that target critical protein-protein interactions at the dimerization interface of Leishmania infantum trypanothione reductase (Li-TryR) using a backbone modification strategy. To achieve this goal we carried out the synthesis, proteolytic stability studies and biological evaluation of a small library of α/β 3 -peptide foldamers of different length (from 9-mers to 13-mers) and different α→β substitution patterns related to prototype linear α-peptides. We show that several 13-residue α/β 3 -peptide foldamers retain inhibitory potency against the enzyme (in both activity and dimerization assays) while they are far less susceptible to proteolytic degradation than an analogous α-peptide. The strong dependence of the binding affinities for Li-TryR on the length of the α,β-peptides is supported by theoretical calculations on conformational ensembles of the resulting complexes. The conjugation of the most proteolytically stable α/β-peptide with oligoarginines results in a molecule with potent activity against L. infantum promastigotes and amastigotes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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

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Elizaide L. A. Yoshida

1993-09-01

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

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

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

2016-11-01

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

Clinical and histopathological characteristics of cutaneous Leishmaniasis in Sanliurfa City of Turkey including Syrian refugees

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Sezen Koçarslan

2013-01-01

Full Text Available Background: The aim of our study was to investigate the clinical and histopathological characteristics of cutaneous leishmaniasis (CL in the city of Sanliurfa in Turkey, where Syrian refugees also reside. Materials and Methods: At the Harran University Hospital outpatient clinics between 2012 and 2013, 54 CL cases, including 24 Syrian patients, underwent punch biopsy of the skin and/or a touch imprint. Patients in whom leishmania parasites were detected were included in the study. The clinical and histopathological data of the patients were obtained by a review of the patients′ medical records. All the slides of each patient were re-evaluated histopathologically. Results: Fifty-four cases (mean age; 17 ± 12 years, consisting of 32 males (59.3% and 22 females (40.7%, were examined. The most common site of involvement was the face (63%. The most common presentation was noduloulcerative lesions (57.4%. Histopathologically, the majority of the cases exhibited hyperkeratosis, follicular plugging of the epidermis, chronic inflammatory infiltration, leishmania amastigotes and non-caseating granulomatous inflammation in the dermis. Conclusion: CL presents with a wide spectrum of expression, both clinically and histologically, and may mimic other inflammatory and neoplastic diseases. The diagnosis of CL relies on the identification of leishmania amastigotes in either a direct smear of the lesion or in a tissue section.

Leishmania Hijacks Myeloid Cells for Immune Escape

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María Martínez-López

2018-05-01

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

Visceral leishmaniasis in Teresina, state of Piauí, Brazil: preliminary observations on the detection and transmissibility of canine and sandfly infections

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J. A. Vexenat

1994-06-01

Full Text Available A Leishmania donovani-complex specific DNA probe was usedto confirm the widespread dissemination of amastigotes in apparently normal skinof dogs with canine visceral leishmaniasis. When Lutzomyia longipalpis were fed on abnormal skin of five naturally infected dogs 57 of 163 (35 per cent fliesbecame infected: four of 65 flies (6 per cent became infected when fed on apparently normal skin. The bite of a single sandfly that had fed seven days previouslyon a naturally infected dog transmitted the infection to a young dog from a non-endemic area. Within 22 days a lesion had developed at the site of the infectivebite (inner ear: 98 days after infection organisms had not disseminated throughout the skin, bone marrow, spleen or liver and the animal was still serologically negative by indirect immunofluorescence and dot-enzyme-linked immunosorbent assay. When fed Lu. longipalpis were captured from a kennel with a sick dog known to be infected, 33 out of 49 (67 per cent of flies contained promastigotes. In contrast only two infections were detected among more than 200 sandflies captured in houses. These observations confirm the ease of transmissibility of L.chagasi from dog to sandfly to dog in Teresina. It is likely that canine VL is the major source of human VL by the transmission route dog-sandfly-human. the Lmet2 DNA probe was a useful epidemiological tool for detecting L. chagasi in sandflies.

Arginase activity of Leishmania isolated from patients with cutaneous leishmaniasis.

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

2017-09-01

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

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

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2013-02-07

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

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

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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

Occurrence of Leishmania infantum in the central nervous system of naturally infected dogs: Parasite load, viability, co-infections and histological alterations.

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Oliveira, Valéria da Costa; Boechat, Viviane Cardoso; Mendes Junior, Artur Augusto Velho; Madeira, Maria de Fátima; Ferreira, Luiz Claudio; Figueiredo, Fabiano Borges; Campos, Monique Paiva; de Carvalho Rodrigues, Francisco das Chagas; Carvalhaes de Oliveira, Raquel de Vasconcellos; Amendoeira, Maria Regina Reis; Menezes, Rodrigo Caldas

2017-01-01

Zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and little is known about the occurrence and pathogenesis of this parasite in the CNS. The aims of this study were to evaluate the occurrence, viability and load of L. infantum in the CNS, and to identify the neurological histological alterations associated with this protozoan and its co-infections in naturally infected dogs. Forty-eight Leishmania-seropositive dogs from which L. infantum was isolated after necropsy were examined. Cerebrospinal fluid (CSF) samples were analyzed by parasitological culture, quantitative real-time PCR (qPCR) and the rapid immunochromatographic Dual Path Platform test. Brain, spinal cord and spleen samples were submitted to parasitological culture, qPCR, and histological techniques. Additionally, anti-Toxoplasma gondii and anti-Ehrlichia canis antibodies in serum and distemper virus antigens in CSF were investigated. None of the dogs showed neurological signs. All dogs tested positive for L. infantum in the CNS. Viable forms of L. infantum were isolated from CSF, brain and spinal cord in 25% of the dogs. Anti-L. infantum antibodies were detected in CSF in 61% of 36 dogs. Inflammatory histological alterations were observed in the CNS of 31% of the animals; of these, 66% were seropositive for E. canis and/or T. gondii. Amastigote forms were associated with granulomatous non-suppurative encephalomyelitis in a dog without evidence of co-infections. The highest frequency of L. infantum DNA was observed in the brain (98%), followed by the spinal cord (96%), spleen (95%), and CSF (50%). The highest L. infantum load in CNS was found in the spinal cord. These results demonstrate that L. infantum can cross the blood-brain barrier, spread through CSF, and cause active infection in the entire CNS of dogs. Additionally, L. infantum can cause inflammation in the CNS that can lead to neurological signs with progression of the disease.

Occurrence of Leishmania infantum in the central nervous system of naturally infected dogs: Parasite load, viability, co-infections and histological alterations.

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Valéria da Costa Oliveira

Full Text Available Zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and little is known about the occurrence and pathogenesis of this parasite in the CNS. The aims of this study were to evaluate the occurrence, viability and load of L. infantum in the CNS, and to identify the neurological histological alterations associated with this protozoan and its co-infections in naturally infected dogs. Forty-eight Leishmania-seropositive dogs from which L. infantum was isolated after necropsy were examined. Cerebrospinal fluid (CSF samples were analyzed by parasitological culture, quantitative real-time PCR (qPCR and the rapid immunochromatographic Dual Path Platform test. Brain, spinal cord and spleen samples were submitted to parasitological culture, qPCR, and histological techniques. Additionally, anti-Toxoplasma gondii and anti-Ehrlichia canis antibodies in serum and distemper virus antigens in CSF were investigated. None of the dogs showed neurological signs. All dogs tested positive for L. infantum in the CNS. Viable forms of L. infantum were isolated from CSF, brain and spinal cord in 25% of the dogs. Anti-L. infantum antibodies were detected in CSF in 61% of 36 dogs. Inflammatory histological alterations were observed in the CNS of 31% of the animals; of these, 66% were seropositive for E. canis and/or T. gondii. Amastigote forms were associated with granulomatous non-suppurative encephalomyelitis in a dog without evidence of co-infections. The highest frequency of L. infantum DNA was observed in the brain (98%, followed by the spinal cord (96%, spleen (95%, and CSF (50%. The highest L. infantum load in CNS was found in the spinal cord. These results demonstrate that L. infantum can cross the blood-brain barrier, spread through CSF, and cause active infection in the entire CNS of dogs. Additionally, L. infantum can cause inflammation in the CNS that can lead to neurological signs with progression of the disease.

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

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Louise Basmaciyan

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

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

Science.gov (United States)

Basmaciyan, Louise; Azas, Nadine; Casanova, Magali

2017-01-01

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

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

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Marcelo Rosandiski LYRA

2015-10-01

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

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

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Camilo Andrés Morales

2014-09-01

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

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

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

2010-01-01

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

Investigation of Antileishmanial Activities of Acridines Derivatives against Promastigotes and Amastigotes Form of Parasites Using Quantitative Structure Activity Relationship Analysis

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Samir Chtita

2016-01-01

Full Text Available In a search of newer and potent antileishmanial (against promastigotes and amastigotes form of parasites drug, a series of 60 variously substituted acridines derivatives were subjected to a quantitative structure activity relationship (QSAR analysis for studying, interpreting, and predicting activities and designing new compounds by using multiple linear regression and artificial neural network (ANN methods. The used descriptors were computed with Gaussian 03, ACD/ChemSketch, Marvin Sketch, and ChemOffice programs. The QSAR models developed were validated according to the principles set up by the Organisation for Economic Co-operation and Development (OECD. The principal component analysis (PCA has been used to select descriptors that show a high correlation with activities. The univariate partitioning (UP method was used to divide the dataset into training and test sets. The multiple linear regression (MLR method showed a correlation coefficient of 0.850 and 0.814 for antileishmanial activities against promastigotes and amastigotes forms of parasites, respectively. Internal and external validations were used to determine the statistical quality of QSAR of the two MLR models. The artificial neural network (ANN method, considering the relevant descriptors obtained from the MLR, showed a correlation coefficient of 0.933 and 0.918 with 7-3-1 and 6-3-1 ANN models architecture for antileishmanial activities against promastigotes and amastigotes forms of parasites, respectively. The applicability domain of MLR models was investigated using simple and leverage approaches to detect outliers and outsides compounds. The effects of different descriptors in the activities were described and used to study and design new compounds with higher activities compared to the existing ones.

Erythema exsudativum multiforme after a Leishmania skin test

NARCIS (Netherlands)

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

2014-01-01

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

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

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

2017-01-01

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

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

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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.

Identification and characterization of surface antigens in parasites, using radiolabelling techniques

International Nuclear Information System (INIS)

Ramasamy, R.

1982-04-01

Surface proteins of Schistosoma sp and Leishmania sp were studied using 125-Iodine as tracer. The surface proteins were labelled by the Lactoperoxidase method and the proteins then separated using SDS PAG electrophoresis and autoradiography. The possible immunogens were then separated using immunoprecipitation and Fluorescent Antibody techniques using sera from patients or from artificially immunized rabbits. Four common antigens were identified from the surfaces of male and female adult worms, cercariae and schistosomulae of S.mansoni. These antigens, which had molecular weights of 150,000, 78,000, 45,000, and 22,000 were also isolated from the surfaces of S.haematobium adults. The surface antigens on promastigotes of a Kenyan strain of Leishmania donovani were separated into three protein antigens with molecular weights of 66,000, 59,000 and 43,000 respectively. The 59,000 molecular weight antigen was a glycoprotein and was common to promastigotes of an American and Indian strain of L.donovani and to L.braziliensis mexicana. None of the isolated antigens have been shown to have a protective effect when vaccinated into mice, but the study illustrates the value of radionuclide tracers in the unravelling of the mosaic of antigens which parasites possess

Regulation and spatial organization of PCNA in Trypanosoma brucei

International Nuclear Information System (INIS)

Kaufmann, Doris; Gassen, Alwine; Maiser, Andreas; Leonhardt, Heinrich; Janzen, Christian J.

2012-01-01

Highlights: ► Characterization of the proliferating cell nuclear antigen in Trypanosoma brucei (TbPCNA). ► TbPCNA is a suitable marker to detect replication in T. brucei. ► TbPCNA distribution and regulation is different compared to closely related parasites T. cruzi and Leishmania donovani. -- Abstract: As in most eukaryotic cells, replication is regulated by a conserved group of proteins in the early-diverged parasite Trypanosoma brucei. Only a few components of the replication machinery have been described in this parasite and regulation, sub-nuclear localization and timing of replication are not well understood. We characterized the proliferating cell nuclear antigen in T. brucei (TbPCNA) to establish a spatial and temporal marker for replication. Interestingly, PCNA distribution and regulation is different compared to the closely related parasites Trypanosoma cruzi and Leishmania donovani. TbPCNA foci are clearly detectable during S phase of the cell cycle but in contrast to T. cruzi they are not preferentially located at the nuclear periphery. Furthermore, PCNA seems to be degraded when cells enter G2 phase in T. brucei suggesting different modes of replication regulation or functions of PCNA in these closely related eukaryotes.

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

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Kelly Ben L

2010-02-01

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

[Parasitological, immunohistochemical and histopathological study for Leishmania chagasi detection in splenic tissues of dogs with visceral leishmaniasis].

Science.gov (United States)

Tasca, Karen Ingrid; Buzetti, Wilma Aparecida Starke; Tenorio, Michely da Silva; Paulan, Silvana de Cássia; Lima, Flávia Luna; de Queiroz, Nina Mari Gual Pimenta; Machado, Rosângela Zacarias; Oliveira, Tricia Maria Ferreira de Souza; Neves, Maria Francisca; de Noronha, Antonio Carlos Faconti; de Assis, Juliana

2009-01-01

The purpose of this work was a Canine Visceral Leishmaniasis--CVL study by parasitological direct examination of Leishmania (L.) chagasi (imprinting and histological), immunohistochemical test and histopathological analysis using spleen tissues from 34 dogs euthanized by the Zoonotic Disease Control Centre from Ilha Solteira, SP, Brazil. According to the clinical signs, the dogs were divided in three groups: asymptomatics (8 dogs), oligosymptomatics (17 dogs) and symptomatics (9 dogs). After the accomplishment of all diagnostic tests, 22 dogs were considered positives (64.7%) and 12 (35.3%) were negatives to CVL. From these positive dogs, 1/22 (4.5%) was asymptomatic, 12/22(54.5%) were oligosymptomatics and 8/22 (40.1%) were symptomatics. The histopathological study in spleen tissues from positive, especially symptomatic dogs, showed a diffuse chronic inflammation with thickness of capsular and trabecular regions and there was extensive morphologic alteration of the red and white pulp by the presence of abundant macrophages full with amastigotes, the granulomatous inflammatory reaction and haemorrhagic areas. The data of this work from histopathologic examination and direct microscopic visualization of L. (L.) chagasi showed that the spleen was an useful organ to collect sample tissues for CVL diagnosis. The immunostaining detected the highest number of positive dogs and were considered an important and conclusive method to be used in addition to parasitological methods for CVL, particularly in asymptomatic or oligosymptomatic dogs.

Leishmanicidal Activity of Nine Novel Flavonoids from Delphinium staphisagria

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Inmaculada Ramírez-Macías

2012-01-01

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

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

Indian Academy of Sciences (India)

NII

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

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

Science.gov (United States)

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

2010-05-01

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

Isolation and identification of an antiparasitic triterpenoid estersaponin from the stem bark of Pittosporum mannii (Pittosporaceae

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Kennedy D Nyongbela

2013-10-01

Full Text Available Objective: To screen for antiparasitic properties of Pittosporum mannii Hook (Pittosporaceae through in vitro bioassay tests and to identify the bioactive compound(s. Methods: The stem bark of Pittosporum mannii was harvested in Bali Nyonga in January 2007. The CH 2Cl2 and MeOH extracts were tested in vitro for antiparasitic activity. NF54 (an airport strain of unknown origin and sensitive to all known drugs and K1 (a clone originating from Thailand and resistant to chloroquine/pyrimethamine strains were used for the antiplasmodial screening while Leishmania donovani MHOM-ET-67/L82 was used for antileishmanial testing. 1H and 13C NMR spectra were recorded on a Bruker AMX-500 spectrometer using CDCl3 as solvent. EIMS were recorded on a double-focusing mass spectrometer (Varian MAT 311A while HREIMS were recorded on a JEOL HX 110 mass spectrometer. Results: The MeOH extract was active on both the chloroquine-resistant (K1 strain (IC50=4.3 μg/ mL and on the macrophages of Leishmania donovani (IC50=8.6 μg/mL. The CH2Cl2 extract was considered inactive on both parasites (IC50>5.0 μg/mL and 21.7 μg/mL respectively. Compound 1, a constituent that precipitated from the MeOH extract, showed pronounced activity on both Plasmodium falciparum and Leishmania donovani parasites (IC 50=1.02 and 1.80 μg/mL respectively with artemisinin and miltefosine included as reference drugs. Its structure was identified as 1-O-[apha-L-(Rhamnopyranosyl]-23-acetoxyimberbic acid 29-methyl ester, a pentacyclic triterpenoid estersaponin. Conclusions: The present study constitutes the first report on the antiparasitic activity of this plant and provides some support for the traditional use of the plant in the treatment of malaria. The plant has therefore been identified as a potential source for the discovery of antiparasitic lead compounds.

Autochthonous case of Canine Visceral Leishmaniasis in a non-endemic area in Minas Gerais, Brazil

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Mariana Teixeira de Faria

Full Text Available ABSTRACT: Visceral Leishmaniasis by Leishmania infantum chagasi is an endemic zoonosis present in many areas of Brazil. This parasite needs reservoirs for maintenance of the infection and the presence of dogs in urban areas is a key factor for the spread of canine visceral leishmaniasis (CVL. The aim of this study was to report the first autochthonous case of CVL in the municipality of Iguatama, in west central region of Minas Gerais State. Dog infection by Leishmania infantum chagasi was confirmed in the municipality, previously considered as non-endemic area to CVL. The canine infection by Leishmania was confirmed by three immunological tests for antibodies: indirect immunofluorescence assay (IFA, rapid Dual Path Platform (DPP® CVL immunochromatographic test, enzyme-linked immunosorbent assay (ELISA, and microscopic demonstration of Leishmania amastigotes in imprints of spleen and bone marrow stained by Giemsa. The species Leishmania infantum chagasi was confirmed by molecular diagnosis (PCR. Studies are being carried out, aiming to describe the importance and the prevalence of this disease in the region and factors associated with its transmission.

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

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

2017-05-16

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

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

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

2016-03-01

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

Leishmaniosis and generalized demodicosis in three dogs: a clinicopathological and immunohistochemical study.

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Mozos, E; Pérez, J; Day, M J; Lucena, R; Ginel, P J

1999-04-01

This paper describes the clinicopathological and immunohistochemical aspects of the skin lesions in three dogs with leishmaniosis and generalized demodicosis. Diffuse alopecia, crusts, folliculitis and furunculosis, as commonly seen in generalized demodicosis, were prominent in all the dogs. MicroIscopically, there was a diffuse and perifollicular superficial and deep granulomatous dermatitis and, in two dogs, both Copyright Demodex canis mites and Leishmania spp. amastigotes were observed in the same lesions. Numerous Mac387(+)macrophages were observed in the inflammatory infiltrates, but macrophages loaded with amastigotes were Mac387(-). In all cases, immunoreactive CD3 lymphocytes were sparse, both in the granulomatous and perifollicular infiltrates. There were numerous IgG+, IgG4(+)-secreting plasma cells in areas of folliculitis and furunculosis and fewer IgG2(+), IgG3(+), IgA+and IgM+-secreting plasma cells in the inflammatory infiltrate. In all cases, MHC Class II was expressed by the majority of dermal macrophages and dendritic cells, as well as by lymphocytes and fibroblasts. The paucity of CD3(+)lymphocytes, usually abundant in D. canis lesions, points to leishmania-induced cell-mediated immunosuppression as a predisposing factor for generalized demodicosis. 1999 W.B. Saunders and Company Ltd.

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

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Kárita Cláudia Freitas-Lidani

2014-07-01

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

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

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

2015-01-01

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

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

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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.

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

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2013-01-01

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

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

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

2016-06-01

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

Leishmania infantum ecto-nucleoside triphosphate diphosphohydrolase-2 is an apyrase involved in macrophage infection and expressed in infected dogs.

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Raphael De Souza Vasconcellos

2014-11-01

Full Text Available Visceral leishmaniasis is an important tropical disease, and Leishmania infantum chagasi (synonym of Leishmania infantum is the main pathogenic agent of visceral leishmaniasis in the New World. Recently, ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases were identified as enablers of infection and virulence factors in many pathogens. Two putative E-NTPDases (∼70 kDa and ∼45 kDa have been found in the L. infantum genome. Here, we studied the ∼45 kDa E-NTPDase from L. infantum chagasi to describe its natural occurrence, biochemical characteristics and influence on macrophage infection.We used live L. infantum chagasi to demonstrate its natural ecto-nucleotidase activity. We then isolated, cloned and expressed recombinant rLicNTPDase-2 in bacterial system. The recombinant rLicNTPDase-2 hydrolyzed a wide variety of triphosphate and diphosphate nucleotides (GTP> GDP  =  UDP> ADP> UTP  =  ATP in the presence of calcium or magnesium. In addition, rLicNTPDase-2 showed stable activity over a pH range of 6.0 to 9.0 and was partially inhibited by ARL67156 and suramin. Microscopic analyses revealed the presence of this protein on cell surfaces, vesicles, flagellae, flagellar pockets, kinetoplasts, mitochondria and nuclei. The blockade of E-NTPDases using antibodies and competition led to lower levels of parasite adhesion and infection of macrophages. Furthermore, immunohistochemistry showed the expression of E-NTPDases in amastigotes in the lymph nodes of naturally infected dogs from an area of endemic visceral leishmaniasis.In this work, we cloned, expressed and characterized the NTPDase-2 from L. infantum chagasi and demonstrated that it functions as a genuine enzyme from the E-NTPDase/CD39 family. We showed that E-NTPDases are present on the surface of promastigotes and in other intracellular locations. We showed, for the first time, the broad expression of LicNTPDases in naturally infected dogs. Additionally, the blockade of

Leishmania infantum Ecto-Nucleoside Triphosphate Diphosphohydrolase-2 is an Apyrase Involved in Macrophage Infection and Expressed in Infected Dogs

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Vasconcellos, Raphael De Souza; Mariotini-Moura, Christiane; Gomes, Rodrigo Saar; Serafim, Tiago Donatelli; Firmino, Rafaela de Cássia; Silva e Bastos, Matheus; de Castro, Felipe Freitas; de Oliveira, Claudia Miranda; Borges-Pereira, Lucas; de Souza, Anna Cláudia Alves; de Souza, Ronny Francisco; Gómez, Gabriel Andres Tafur; Pinheiro, Aimara da Costa; Maciel, Talles Eduardo Ferreira; Silva-Júnior, Abelardo; Bressan, Gustavo Costa; Almeida, Márcia Rogéria; Baqui, Munira Muhammad Abdel; Afonso, Luís Carlos Crocco; Fietto, Juliana Lopes Rangel

2014-01-01

Background Visceral leishmaniasis is an important tropical disease, and Leishmania infantum chagasi (synonym of Leishmania infantum) is the main pathogenic agent of visceral leishmaniasis in the New World. Recently, ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) were identified as enablers of infection and virulence factors in many pathogens. Two putative E-NTPDases (∼70 kDa and ∼45 kDa) have been found in the L. infantum genome. Here, we studied the ∼45 kDa E-NTPDase from L. infantum chagasi to describe its natural occurrence, biochemical characteristics and influence on macrophage infection. Methodology/Principal Findings We used live L. infantum chagasi to demonstrate its natural ecto-nucleotidase activity. We then isolated, cloned and expressed recombinant rLicNTPDase-2 in bacterial system. The recombinant rLicNTPDase-2 hydrolyzed a wide variety of triphosphate and diphosphate nucleotides (GTP> GDP  =  UDP> ADP> UTP  =  ATP) in the presence of calcium or magnesium. In addition, rLicNTPDase-2 showed stable activity over a pH range of 6.0 to 9.0 and was partially inhibited by ARL67156 and suramin. Microscopic analyses revealed the presence of this protein on cell surfaces, vesicles, flagellae, flagellar pockets, kinetoplasts, mitochondria and nuclei. The blockade of E-NTPDases using antibodies and competition led to lower levels of parasite adhesion and infection of macrophages. Furthermore, immunohistochemistry showed the expression of E-NTPDases in amastigotes in the lymph nodes of naturally infected dogs from an area of endemic visceral leishmaniasis. Conclusions/Significance In this work, we cloned, expressed and characterized the NTPDase-2 from L. infantum chagasi and demonstrated that it functions as a genuine enzyme from the E-NTPDase/CD39 family. We showed that E-NTPDases are present on the surface of promastigotes and in other intracellular locations. We showed, for the first time, the broad expression of LicNTPDases in

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