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1.
J Pharm Pharmacol ; 71(12): 1784-1791, 2019 Dec.
Article in English | MEDLINE | ID: mdl-31579947

ABSTRACT

The alkylaminoalkanethiosulfuric acids (AAATs) are amphipathic compounds effective against experimental schistosomiasis, of low toxicity, elevated bioavailability after a single oral dose and prompt tissue absorption. OBJECTIVES: To explore the in-vitro antileishmanial potential of AAATs using five compounds of this series against Leishmania (Viannia) braziliensis. METHODS: Their effects on promastigotes and axenic amastigotes, and cytotoxicity to macrophages were tested by the MTT method, and on Leishmania-infected macrophages by Giemsa stain. Effects on the mitochondrial membrane potential of promastigotes and axenic amastigotes and DNA of intracellular amastigotes were tested using JC-1 and TUNEL assays, respectively. KEY FINDINGS: The 2-(isopropylamino)-1-octanethiosulfuric acid (I) and 2-(sec-butylamino)-1-octanethiosulfuric acid (II) exhibit activity against both promastigotes and intracellular amastigotes (IC50 25-35 µm), being more toxic to intracellular parasites than to the host cell. Compound I induced a loss of viability of axenic amastigotes, significantly reduced (30%) the mitochondrial membrane potential of both promastigotes and axenic amastigotes and promoted selective DNA fragmentation of the nucleus and kinetoplast of intracellular amastigotes. CONCLUSIONS: In this previously unpublished study of trypanosomatids, it is shown that AAATs could also exhibit selective antileishmanial activity, a new possibility to be investigated in oral treatment of leishmaniasis.


Subject(s)
Antiprotozoal Agents/pharmacology , Leishmania braziliensis/isolation & purification , Leishmaniasis/drug therapy , Sulfuric Acids/pharmacology , Administration, Oral , Animals , Antiprotozoal Agents/administration & dosage , Antiprotozoal Agents/chemistry , Inhibitory Concentration 50 , Leishmania braziliensis/drug effects , Membrane Potential, Mitochondrial/drug effects , Mice , Mice, Inbred BALB C , Structure-Activity Relationship , Sulfuric Acids/administration & dosage , Sulfuric Acids/chemistry
2.
Vet Parasitol ; 271: 38-44, 2019 Jul.
Article in English | MEDLINE | ID: mdl-31303201

ABSTRACT

A nucleoside triphosphate diphosphohydrolase-1 (NTPDase 1) was identified on the surface, flagellum and kinetoplast from L. infantum promastigotes by immunocytochemistry and confocal laser scanning microscopy, using immune sera that recognized specifically the B domain of NTPDase 1 and produced against synthetic peptides (LbB1LJ and LbB2LJ) derived from this domain. The polyclonal antibodies had effective antileishmanial effect, reducing significantly in vitro promastigotes growth (21-25%), an antiproliferative effect also demonstrated by immune sera produced against recombinant r-pot B domain, and two other synthetic peptides (potB1LJ and potB2LJ). In addition, using these biomolecules in ELISA technique, IgG1 and IgG2 subclasses reactivities of either healthy dogs or infected by L. infantum and classified clinically as asymptomatic, oligosymptomatic and symptomatic were tested. Analysis of distinct IgG1 and IgG2 seropositivities patterns suggested antibody subclasses binding epitopes along B domain for protection against infection, indicating this domain as a new tool for prophylactic and immunotherapeutic investigations.


Subject(s)
Antibodies, Protozoan/immunology , Dog Diseases/immunology , Immunoglobulin G/immunology , Leishmania infantum/enzymology , Leishmania infantum/immunology , Leishmaniasis, Visceral/veterinary , Nucleoside-Triphosphatase/immunology , Animals , Antibodies, Protozoan/metabolism , Dog Diseases/parasitology , Dogs , Leishmaniasis, Visceral/immunology , Leishmaniasis, Visceral/parasitology , Protein Domains/immunology
3.
Exp Parasitol ; 200: 1-6, 2019 May.
Article in English | MEDLINE | ID: mdl-30904692

ABSTRACT

Nucleoside triphosphate diphosphohydrolase (NTPDase) 1 from intracellular amastigotes of Leishmania infantum-infected macrophage was identified by immunocytochemistry and confocal laser scanning microscopy using antibodies that specifically recognize its B-domain. This enzyme was previously characterized in Leishmania promastigote form, and here it is shown to be susceptible to pentamidine isethionate (PEN). In initial assays, this antileishmanial compound (100 µM) reduced 60% phosphohydrolytic activity of promastigotes preparation. An active NTPDase 1 was then isolated by non-denaturing gel electrophoresis, and PEN (10 µM) inhibited 74% and 35% of the ATPase and ADPase activities, respectively, of this pure protein. In addition, PEN 0.1-1 µM inhibited 56% potato apyrase activity, a plant protein that shares high identity with Leishmania NTPDase 1. In contrast, amphotericin B, fluconazole, ketoconazole or allopurinol did not significantly affect phosphohydrolytic activity of either promastigotes preparation or potato apyrase. This work suggests amastigote NTPDase 1 as a new molecular target, and inhibition of its catalytic activity by pentamidine can be part of the mode of action of this drug contributing with the knowledge of its antileishmanial effect.


Subject(s)
Antiprotozoal Agents/pharmacology , Apyrase/antagonists & inhibitors , Leishmania infantum/drug effects , Leishmania infantum/enzymology , Pentamidine/pharmacology , Animals , Antigens, CD , Immunohistochemistry , Macrophages/parasitology , Male , Mice , Mice, Inbred BALB C , Microscopy, Confocal
4.
Parasitol Int ; 62(1): 44-52, 2013 Feb.
Article in English | MEDLINE | ID: mdl-22995148

ABSTRACT

We identified a shared B domain within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, an NTPDase activity not affected by inhibitors of adenylate kinase and ATPases was detected in Leishmania infantum promastigotes. By non-denaturing gel electrophoresis of detergent-homogenized promastigote preparation, an active band hydrolyzing nucleosides di- and triphosphate was visualized and, following SDS-PAGE and silver staining was identified as a single polypeptide of 50kDa. By Western blots, it was recognized by immune sera raised against potato apyrase (SA), r-pot B domain (SB), a recombinant polypeptide derived from the potato apyrase, and LbB1LJ (SC) or LbB2LJ (SD), synthetic peptides derived from the Leishmania NTPDase 1, and by serum samples from dogs with visceral leishmaniasis, identifying the antigenic L. infantum NTPDase 1 and, also, its conserved B domain (r83-122). By immunoprecipitation assays and Western blots, immune sera SA and SB identified the catalytically active NTPDase 1 in promastigote preparation. In addition, the immune sera SB (44%) and SC or SD (87-99%) inhibited its activity, suggesting a direct effect on the B domain. By ELISA, 37%, 45% or 50% of 38 infected dogs were seropositive for r-pot B domain, LbB1LJ and LbB2LJ, respectively, confirming the B domain antigenicity.


Subject(s)
Antigens, CD/chemistry , Antigens, CD/immunology , Antigens, Protozoan/metabolism , Apyrase/chemistry , Apyrase/immunology , Leishmania infantum/enzymology , Leishmania infantum/immunology , Amino Acid Sequence , Animals , Antigens, CD/isolation & purification , Antigens, CD/metabolism , Antigens, Protozoan/chemistry , Antigens, Protozoan/immunology , Apyrase/isolation & purification , Apyrase/metabolism , Dogs , Leishmania infantum/genetics , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Sequence Alignment
5.
Exp Parasitol ; 132(2): 293-9, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22921497

ABSTRACT

Nucleoside triphosphate diphosphohydrolase (NTPDase) activity was recently characterized in Leishmania (Viannia) braziliensis promastigotes (Lb), and an antigenic conserved domain (r82-121) from the specific NTPDase 1 isoform was identified. In this work, mouse polyclonal antibodies produced against two synthetic peptides derived from this domain (LbB1LJ, r82-103; LbB2LJ, r102-121) were used. The anti-LbB1LJ or anti-LbB2LJ antibodies were immobilized on protein A-sepharose and immunoprecipitated the NTPDase 1 of 48 kDa and depleted approximately 40% of the phosphohydrolytic activity from detergent-homogenized Lb preparation. Ultrastructural immunocytochemical microscopy identified the NTPDase 1 on the parasite surface and in its subcellular cytoplasmic vesicles, mitochondria, kinetoplast and nucleus. The ATPase and ADPase activities of detergent-homogenized Lb preparation were partially inhibited by anti-LbB1LJ antibody (43-79%), which was more effective than that inhibition (18-47%) by anti-LbB2LJ antibody. In addition, the immune serum anti-LbB1LJ (67%) or anti-LbB2LJ (33%) was cytotoxic, significantly reducing the promastigotes growth in vitro. The results appoint the conserved domain from the L. braziliensis NTPDase as an important target for inhibitor design and the potential application of these biomolecules in experimental protocols of disease control.


Subject(s)
Antibodies, Protozoan/immunology , Antigens, CD/analysis , Apyrase/analysis , Leishmania braziliensis/enzymology , Animals , Antibodies, Immobilized/immunology , Antigens, CD/immunology , Apyrase/antagonists & inhibitors , Apyrase/immunology , Blotting, Western , Immunoprecipitation , Isoenzymes/analysis , Isoenzymes/immunology , Leishmania braziliensis/growth & development , Leishmania braziliensis/immunology , Mice , Mice, Inbred BALB C , Microscopy, Immunoelectron , Rabbits
6.
Mem Inst Oswaldo Cruz ; 106(7): 808-13, 2011 Nov.
Article in English | MEDLINE | ID: mdl-22124552

ABSTRACT

A peptide (SmB2LJ; r175-194) that belongs to a conserved domain from Schistosoma mansoni SmATPDase 2 and is shared with potato apyrase, as predicted by in silico analysis as antigenic, was synthesised and its immunostimulatory property was analysed. When inoculated in BALB/c mice, this peptide induced high levels of SmB2LJ-specific IgG1 and IgG2a subtypes, as detected by enzyme linked immunosorbent assay. In addition, dot blots were found to be positive for immune sera against potato apyrase and SmB2LJ. These results suggest that the conserved domain r175-194 from the S. mansoni SmATPDase 2 is antigenic. Western blots were performed and the anti-SmB2LJ antibody recognised in adult worm (soluble worm antigen preparation) or soluble egg antigen antigenic preparations two bands of approximately 63 and 55 kDa, molecular masses similar to those predicted for adult worm SmATPDase 2. This finding strongly suggests the expression of this same isoform in S. mansoni eggs. To assess localisation of SmATPDase 2, confocal fluorescence microscopy was performed using cryostat sections of infected mouse liver and polyclonal antiserum against SmB2LJ. Positive reactions were identified on the external surface from the miracidium in von Lichtenberg's envelope and, in the outer side of the egg-shell, showing that this soluble isoform is secreted from the S. mansoni eggs.


Subject(s)
Antibodies, Helminth/immunology , Antigens, Helminth/immunology , Apyrase/immunology , Schistosoma mansoni/immunology , Animals , Blotting, Western , Cross Reactions , Egg Proteins/immunology , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Immunohistochemistry , Male , Mice , Schistosoma mansoni/enzymology
7.
Mem. Inst. Oswaldo Cruz ; 106(7): 808-813, Nov. 2011. ilus, graf
Article in English | LILACS | ID: lil-606643

ABSTRACT

A peptide (SmB2LJ; r175-194) that belongs to a conserved domain from Schistosoma mansoni SmATPDase 2 and is shared with potato apyrase, as predicted by in silico analysis as antigenic, was synthesised and its immunostimulatory property was analysed. When inoculated in BALB/c mice, this peptide induced high levels of SmB2LJ-specific IgG1 and IgG2a subtypes, as detected by enzyme linked immunosorbent assay. In addition, dot blots were found to be positive for immune sera against potato apyrase and SmB2LJ. These results suggest that the conserved domain r175-194 from the S. mansoni SmATPDase 2 is antigenic. Western blots were performed and the anti-SmB2LJ antibody recognised in adult worm (soluble worm antigen preparation) or soluble egg antigen antigenic preparations two bands of approximately 63 and 55 kDa, molecular masses similar to those predicted for adult worm SmATPDase 2. This finding strongly suggests the expression of this same isoform in S. mansoni eggs. To assess localisation of SmATPDase 2, confocal fluorescence microscopy was performed using cryostat sections of infected mouse liver and polyclonal antiserum against SmB2LJ. Positive reactions were identified on the external surface from the miracidium in von Lichtenberg's envelope and, in the outer side of the egg-shell, showing that this soluble isoform is secreted from the S. mansoni eggs.


Subject(s)
Animals , Male , Mice , Antibodies, Helminth/immunology , Antigens, Helminth/immunology , Apyrase/immunology , Schistosoma mansoni/immunology , Blotting, Western , Cross Reactions , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Egg Proteins/immunology , Immunohistochemistry , Schistosoma mansoni/enzymology
8.
Dev Comp Immunol ; 35(10): 1059-67, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21527274

ABSTRACT

A polypeptide (r78-117) belonging to the potato apyrase was identified as a conserved domain shared with apyrase-like proteins from distinct pathogenic organisms, and was obtained as a 6xHis tag polypeptide (r-Domain B). By ELISA, high IgG, and IgG1 and IgG2a subtypes levels were detected in BALB/c mice pre-inoculated with r-Domain B. In Schistosoma mansoni adult worm or Leishmania (V.) braziliensis promastigote preparation, anti-r-Domain B antibodies inhibit 22-72% of the phosphohydrolytic activities and when immobilized on Protein A-Sepharose immunoprecipitate 42-91% of them. Western blots of the immunoprecipitated resin-antibody-antigen complexes identified bands of mw similar to those predicted for parasite proteins. Total IgG and subclasses of patients with leishmaniasis or schistosomiasis exhibited cross-immunoreactivity with r-Domain B. Therefore, the domain B within both S. mansoni SmATPDase 2 (r156-195) and L. (V.) braziliensis NDPase (r83-122) are potentially involved in the host immune response, and also seem to be conserved during host and parasites co-evolution.


Subject(s)
Antibodies, Helminth/immunology , Antibodies, Protozoan/immunology , Apyrase/immunology , Conserved Sequence/immunology , Leishmania braziliensis , Schistosoma mansoni , Trypanosoma cruzi , Adult , Amino Acid Sequence , Animals , Apyrase/antagonists & inhibitors , Case-Control Studies , Cross Reactions/immunology , Humans , Immunoprecipitation , Leishmania braziliensis/enzymology , Leishmania braziliensis/immunology , Leishmaniasis/blood , Leishmaniasis/immunology , Mice , Mice, Inbred BALB C , Middle Aged , Schistosoma mansoni/enzymology , Schistosoma mansoni/immunology , Schistosomiasis/blood , Schistosomiasis/immunology , Sequence Homology, Amino Acid , Trypanosoma cruzi/enzymology , Trypanosoma cruzi/immunology
9.
Mem Inst Oswaldo Cruz ; 105(4): 370-3, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20721477

ABSTRACT

In this paper, we showed for the first time that the conserved domains within Schistosoma mansoni ATP diphosphohydrolase isoforms, shared with potato apyrase, possess epitopes for the IgG1 and IgG4 subtypes, as 24 (80%) of the 30 schistosomiasis patients were seropositive for this vegetable protein. The analyses for each patient cured (n = 14) after treatment (AT) with praziquantel revealed variable IgG1 and IgG4 reactivity against potato apyrase. Different antigenic epitopes shared between the vegetable and parasite proteins could be involved in susceptibility or resistance to S. mansoni AT with praziquantel and these possibilities should be explored.


Subject(s)
Antibodies, Helminth/immunology , Apyrase/immunology , Immunoglobulin G/immunology , Schistosoma mansoni/immunology , Schistosomiasis mansoni/immunology , Solanum tuberosum/enzymology , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Anthelmintics/therapeutic use , Child , Child, Preschool , Cross Reactions , Humans , Middle Aged , Praziquantel/therapeutic use , Schistosomiasis mansoni/drug therapy , Young Adult
10.
Mem. Inst. Oswaldo Cruz ; 105(4): 370-373, July 2010. tab, ilus
Article in English | LILACS | ID: lil-554799

ABSTRACT

In this paper, we showed for the first time that the conserved domains within Schistosoma mansoni ATP diphosphohydrolase isoforms, shared with potato apyrase, possess epitopes for the IgG1 and IgG4 subtypes, as 24 (80 percent) of the 30 schistosomiasis patients were seropositive for this vegetable protein. The analyses for each patient cured (n = 14) after treatment (AT) with praziquantel revealed variable IgG1 and IgG4 reactivity against potato apyrase. Different antigenic epitopes shared between the vegetable and parasite proteins could be involved in susceptibility or resistance to S. mansoni AT with praziquantel and these possibilities should be explored.


Subject(s)
Adolescent , Adult , Aged , Aged, 80 and over , Animals , Child , Child, Preschool , Humans , Middle Aged , Young Adult , Antibodies, Helminth/immunology , Apyrase/immunology , Immunoglobulin G/immunology , Schistosoma mansoni/immunology , Schistosomiasis mansoni/immunology , Solanum tuberosum/enzymology , Anthelmintics , Cross Reactions , Praziquantel , Schistosomiasis mansoni
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