The induction of Trypanosoma cruzi trypomastigote to amastigote transformation by low pH

Parasitology ◽  
1995 ◽  
Vol 110 (5) ◽  
pp. 547-554 ◽  
Author(s):  
S. Tomlinson ◽  
F. Vandekerckhove ◽  
U. Frevert ◽  
V. Nussenzweig
Keyword(s):  
Trypanosoma Cruzi ◽  
Molecular Mechanisms ◽  
Low Ph ◽  
Critical Stage ◽  
Intracellular Amastigotes ◽  
Electron Microscopical Analysis ◽  
Physiological Relevance ◽  
Electron Microscopical ◽  
Extracellular Amastigotes

SUMMARYFollowing cell invasion, Trypanosoma cruzi trypomastigotes transform into amastigotes, which are the mammalian replicative forms of the parasite. Although amastigotes represent a critical stage in the life-cycle of T. cruzi, little is known of the factors controlling trypomastigote to amastigote transformation. Kanbera et al. (1990) observed that exposure of trypomastigotes to acidic pH induced their transformation into rounded forms resembling amastigotes. We confirm their observation and, using two strains of T. cruzi, establish that these transformants are ultrastructurally and biochemically indistinguishable from natural amastigotes. Incubation of trypomastigotes in medium at pH 5·0 for 2 h was sufficient to trigger their transformation into forms resembling amastigotes. Electron microscopical analysis confirmed that the kinetoplast structure, and general morphological features of the acid-induced, extracellular amastigotes were indistinguishable from those of intracellular-derived amastigotes. The extracellular transformation was accompanied by the acquisition of the stage-specific surface antigen of the naturally transformed amastigotes (Ssp-4), and loss of a stagespecific trypomastigote antigen (Ssp-3). Trypomastigotes incubated at neutral pH did not transform into amastigotes, and did not acquire the Ssp-4 epitope or lose the Ssp-3 epitope. Finally, acid-induced amastigotes subsequently incorporated [3H]thymidine into their DNA, indicating that the important replicative property of intracellular amastigotes is also exhibited by these in vitro transformants. This effect of low pH appears to be of physiological relevance, and acid-induced extracellular transformation appears to represent a valid experimental technique for studies of the molecular mechanisms involved in the differentiation process.

scholarly journals Trypanosoma cruzi Infection Induces Differential Modulation of Costimulatory Molecules and Cytokines by Monocytes and T Cells from Patients with Indeterminate and Cardiac Chagas' Disease

2007 ◽  
Vol 75 (4) ◽  
pp. 1886-1894 ◽  
Author(s):  
Paulo E. A. Souza ◽  
Manoel O. C. Rocha ◽  
Cristiane A. S. Menezes ◽  
Janete S. Coelho ◽  
Andréa C. L. Chaves ◽  
...  
Keyword(s):  
T Cells ◽  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Molecular Mechanisms ◽  
Costimulatory Molecule ◽  
Costimulatory Molecules ◽  
Necrosis Factor Alpha ◽  
Pathogenic Potential ◽  
Indeterminate Form

ABSTRACT Interactions between macrophages and lymphocytes through costimulatory molecules and cytokines are essential for mounting an efficient immune response and controlling its pathogenic potential. Here we demonstrate the immunomodulatory capacity of Trypanosoma cruzi, the causative agent of Chagas' disease, through its ability to induce differential expression of costimulatory molecules and cytokines by monocytes and T cells. Costimulatory molecule and cytokine modulation was evaluated using cells from noninfected individuals and from patients with the asymptomatic indeterminate form and those with the severe cardiac clinical form of Chagas' disease. Our results show that while exposure of monocytes to live T. cruzi leads to an increase in the frequency of CD80+ monocytes in all groups, it decreases both the frequency and intensity of CD86 expression by monocytes from patients with the cardiac form but not from those with the indeterminate form. Conversely, exposure of lymphocytes to monocytes infected with T. cruzi increased the surface expression of cytotoxic-T-lymphocyte-associated antigen 4 (CTLA-4) by T cells from indeterminate but not from cardiac patients, compared to that from control patients. These data suggest that T. cruzi induces a potentially down-regulatory environment in indeterminate subjects, which is associated with higher CD80 and CTLA-4 expression. To test the functional importance of this modulation, we evaluated the expression of cytokines after in vitro infection. Although exposure of lymphocytes to parasite-infected monocytes induced high expression of inflammatory and anti-inflammatory cytokines by T cells in all groups, indeterminate patients displayed a higher ratio of monocytes expressing interleukin 10 than tumor necrosis factor alpha following infection than did controls. These data show the ability of T. cruzi to actively change the expression of costimulatory molecules and cytokines, suggesting molecular mechanisms for the differential clinical evolution of human Chagas' disease.

scholarly journals A Tetrahydrophthalazinone Inhibitor of Phosphodiesterase with In Vitro Activity Against Intracellular Trypanosomatids

2020 ◽  
Author(s):  
Julianna Siciliano de Araújo ◽  
Raiza Brandão Peres ◽  
Patrícia Bernardino da Silva ◽  
Marcos Meuser Batista ◽  
Geert Jan Sterk ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Phosphodiesterase Inhibitor ◽  
Drug Candidate ◽  
In Vitro Activity ◽  
Target Validation ◽  
Activity Profile ◽  
Intracellular Amastigotes ◽  
Potential Activity ◽  
Camp Levels

The phosphodiesterase inhibitor tetrahydrophthalazinone NPD-008 was explored by phenotypic in vitro screening, target validation and ultrastructural approaches against Trypanosoma cruzi. NPD-008 displayed activity against different forms and strains of T. cruzi (EC50 = 6.6 – 39.5 μM), increased cAMP levels of T. cruzi and its combination with Bz gave synergistic interaction. It was also moderately active against intracellular amastigotes of Leishmania amazonensis and L. infantum, confirming a potential activity profile as an antitrypanosomatid drug candidate.

scholarly journals Evaluation of phthalazinone phosphodiesterase inhibitors with improved activity and selectivity against Trypanosoma cruzi

2019 ◽  
Vol 75 (4) ◽  
pp. 958-967 ◽  
Author(s):  
Julianna Siciliano De Araújo ◽  
Patrícia Bernardino da Silva ◽  
Marcos Meuser Batista ◽  
Raiza Brandão Peres ◽  
Camila Cardoso-Santos ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Phosphodiesterase Inhibitors ◽  
Protozoan Parasite ◽  
Human Infection ◽  
Intracellular Camp ◽  
Mrna Levels ◽  
Camp Content ◽  
Intracellular Amastigotes ◽  
Pde Inhibitors

Abstract Background Chagas’ disease, caused by the protozoan parasite Trypanosoma cruzi, needs urgent alternative therapeutic options as the treatments currently available display severe limitations, mainly related to efficacy and toxicity. Objectives As phosphodiesterases (PDEs) have been claimed as novel targets against T. cruzi, our aim was to evaluate the biological aspects of 12 new phthalazinone PDE inhibitors against different T. cruzi strains and parasite forms relevant for human infection. Methods In vitro trypanocidal activity of the inhibitors was assessed alone and in combination with benznidazole. Their effects on parasite ultrastructural and cAMP levels were determined. PDE mRNA levels from the different T. cruzi forms were measured by quantitative reverse transcription PCR. Results Five TcrPDEs were found to be expressed in all parasite stages. Four compounds displayed strong effects against intracellular amastigotes. Against bloodstream trypomastigotes (BTs), three were at least as potent as benznidazole. In vitro combination therapy with one of the most active inhibitors on both parasite forms (NPD-040) plus benznidazole demonstrated a quite synergistic profile (xΣ FICI = 0.58) against intracellular amastigotes but no interaction (xΣ FICI = 1.27) when BTs were assayed. BTs treated with NPD-040 presented disrupted Golgi apparatus, a swollen flagellar pocket and signs of autophagy. cAMP measurements of untreated parasites showed that amastigotes have higher ability to efflux this second messenger than BTs. NPD-001 and NPD-040 increase the intracellular cAMP content in both BTs and amastigotes, which is also released into the extracellular milieu. Conclusions The findings demonstrate the potential of PDE inhibitors as anti-T. cruzi drug candidates.

scholarly journals Synergic Effect of Allopurinol in Combination with Nitroheterocyclic Compounds against Trypanosoma cruzi

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Ana Lia Mazzeti ◽  
Lívia de F. Diniz ◽  
Karolina R. Gonçalves ◽  
Ruan Schott WonDollinger ◽  
Tassiane Assíria ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Parasite Load ◽  
H9c2 Cells ◽  
Cure Rate ◽  
Positive Interaction ◽  
Content Type ◽  
Intracellular Amastigotes ◽  
Subsequent Relapse

ABSTRACT Combination therapy has gained attention as a possible strategy for overcoming the limitations of the present therapeutic arsenal for Chagas disease. The aim of this study was to evaluate the effect of allopurinol in association with nitroheterocyclic compounds on infection with the Y strain of Trypanosoma cruzi. The in vitro effect of allopurinol plus benznidazole or nifurtimox on intracellular amastigotes in infected H9c2 cells was assessed in a 72-h assay. The interactions were classified as synergic for both allopurinol-nifurtimox (sums of fractional inhibitory concentrations [∑FICs] = 0.49 ± 0.08) and allopurinol-benznidazole (∑FICs = 0.48 ± 0.09). In the next step, infected Swiss mice were treated with allopurinol at 30, 60, and 90 mg/kg of body weight and with benznidazole at 25, 50, and 75 mg/kg in monotherapy and in combination at the same doses; as a reference treatment, another group of animals received benznidazole at 100 mg/kg. Allopurinol in monotherapy led to a smaller or nil effect in the reduction of parasite load and mortality rate. Treatment with benznidazole at suboptimal doses induced a transient suppression of parasitaemia with subsequent relapse in all animals treated with 25 and 50 mg/kg and in 80% of those that received 75 mg/kg. Administration of the drugs in combination significantly increased the cure rate to 60 to 100% among mice treated with benznidazole at 75 mg/kg plus 30, 60, or 90 mg/kg of allopurinol. These results show a positive interaction between allopurinol and benznidazole, and since both drugs are commercially available, their use in combination may be considered for the assessment in the treatment of Chagas disease patients.

The development of Trypanosoma cruzi in macrophages in vitro. Interaction with lysosomes and host cell fate

Parasitology ◽  
1980 ◽  
Vol 80 (1) ◽  
pp. 139-145 ◽  
Author(s):  
Regina Milder ◽  
Judith Kloetzel
Keyword(s):  
Trypanosoma Cruzi ◽  
Host Cell ◽  
Cell Fate ◽  
Peritoneal Macrophages ◽  
Blue Dye ◽  
Cell Monolayers ◽  
Secondary Lysosomes ◽  
Electron Microscopical ◽  
In Vitro Interaction

SummaryThe interaction between mouse peritoneal macrophages and ‘Y’ strain Trypanosoma cruzi bloodstream forms was studied at optical and electron microscopical levels. The method of marking lysosomes with Thorotrast, either before or after infection of cell monolayers with parasites, revealed that secondary lysosomes fused with phagosomes shortly after trypanosome interiorization. In spite of this, 24 h later most parasites were no longer in a vacuole but lay free within the host cell cytoplasm, multiplying actively. At this time, and up to shortly before 96 h when parasites escaped to the external milieu, most parasitized cells were not lethally injured, as revealed by the Trypan blue dye-exclusion test. Only when parasites were released into the external medium was this situation reversed and infected macrophages took up the dye.

scholarly journals Analogues of Marine Guanidine Alkaloids Are in Vitro Effective against Trypanosoma cruzi and Selectively Eliminate Leishmania (L.) infantum Intracellular Amastigotes

2016 ◽  
Vol 79 (9) ◽  
pp. 2202-2210 ◽  
Author(s):  
Ligia F. Martins ◽  
Juliana T. Mesquita ◽  
Erika G. Pinto ◽  
Thais A. Costa-Silva ◽  
Samanta E. T. Borborema ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Intracellular Amastigotes ◽  
Guanidine Alkaloids

scholarly journals Antiparasitic Effect of Stilbene and Terphenyl Compounds against Trypanosoma cruzi Parasites

2021 ◽  
Vol 14 (11) ◽  
pp. 1199
Author(s):  
Federica Bruno ◽  
Germano Castelli ◽  
Fabrizio Vitale ◽  
Simone Catanzaro ◽  
Valeria Vitale Badaco ◽  
...  
Keyword(s):  
Natural Products ◽  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Flow Cytometric Analysis ◽  
New Drugs ◽  
Normal Cells ◽  
Antiparasitic Activity ◽  
Intracellular Amastigotes ◽  
Caspase 1

Background: Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi. No progress in the treatment of this pathology has been made since Nifurtimox was introduced more than fifty years ago, and this drug is considered very aggressive and may cause several adverse effects. This drug currently has severe limitations, including a high frequency of undesirable side effects and limited efficacy and availability, so research to discover new drugs for the treatment of Chagas disease is imperative. Many drugs available on the market are natural products as found in nature or compounds designed based on the structure and activity of these natural products. Methods: This study evaluated the in vitro antiparasitic activity of a series of previously synthesized stilbene and terphenyl compounds in T. cruzi epimastigotes and intracellular amastigotes. The action of the most selective compounds was investigated by flow cytometric analysis to evaluate the mechanism of cell death. The ability to induce apoptosis or caspase-1 inflammasomes was assayed in macrophages infected with T. cruzi after treatment, comparing it with that of Nifurtimox. Results: The stilbene ST18 was the most potent compound of the series. It was slightly less active than Nifurtimox in epimastigotes but most active in intracellular amastigotes. Compared to Nifurtimox, it was markedly less cytotoxic when tested in vitro on normal cells. ST18 was able to induce a marked increase in parasites positive for Annexin V and monodansylcadaverine. Moreover, ST18 induced the activation, in infected macrophages, of caspase-1, a conserved enzyme that plays a major role in controlling parasitemia, host survival and the onset of the adaptive immune response in Trypanosoma infection. Conclusions: The antiparasitic activity of ST18 together with its ability to activate caspase-1 in infected macrophages and its low toxicity toward normal cells makes this compound interesting for further clinical investigation.

scholarly journals Antiparasitic effect of stilbene and terphenyl compounds against Trypanosoma cruzi parasites

2021 ◽  
Author(s):  
Federica Bruno ◽  
Germano Castelli ◽  
Fabrizio Vitale ◽  
Simone Catanzaro ◽  
Valeria Vitale Badaco ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Annexin V ◽  
New Drugs ◽  
Main Role ◽  
Normal Cells ◽  
Antiparasitic Activity ◽  
Intracellular Amastigotes ◽  
Caspase 1

AbstractBackgroundChagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi. No progress in the treatment of this pathology has been made since Nifurtimox was introduced more than fifty years ago and is considered very aggressive and may cause several adverse effects. Currently, this drug has severe limitations, including high frequency of undesirable side effects and limited efficacy and availability and the research to discover new drugs for the treatment of Chagas disease is imperative. Many drugs available in the market are natural products as found in nature or compounds designed based on the structure and activity of these natural products.Methodology/Principal FindingsThis study evaluated the in vitro antiparasitic activity in T. cruzi epimastigotes and intracellular amastigotes of a series of stilbene and terphenyl compounds previously synthesized. The action of the most selective compounds has been investigated by flow cytometry analysis to evaluate the mechanism of cell death. The ability to induce apoptosis or caspase-1 inflammasome were assayed in macrophages infected with T. cruzi after treatment comparing with Nifurtimox.Conclusions/SignificanceThe stilbene ST18 was the most potent compound of the series. It was slightly less active than Nifurtimox in epimastigotes but most active in intracellular amastigotes. Compared to Nifurtimox, it was markedly less cytotoxic when tested in vitro on normal cells. ST18 was able to induce a marked increase of parasites positive to Annexin V and monodansylcadaverine. Moreover, ST18 induced the activation in infected macrophages of caspase-1, a conserved enzyme which plays a main role in controlling parasitemia, host survival, and the onset of adaptive immune response in Trypanosoma infection. The antiparasitic activity of ST18 together to its ability to activate caspase-1 in infected macrophages and its low toxicity on normal cells makes this compound interesting for further clinical investigations.Author SummaryChagas disease is a pathology caused by the protozoan parasite Trypanosoma cruzi. No progress in the treatment of this pathology has been made since benznidazole and Nifurtimox were introduced more than fifty years ago. However, these drugs have severe limitations and the research to discover new drugs for the treatment of Chagas disease is imperative. We evaluated the in vitro antiparasitic activity in T. cruzi epimastigotes of a series of stilbene and terphenyl compounds previously synthesized. The stilbene ST18 was the most potent compound of the series. It was slightly less active than nifurtimox in epimastigotes but most active in intracellular amastigotes. Compared to Nifurtimox, it was markedly less cytotoxic when tested in vitro on normal cells. ST18 was able to induce a marked increase of parasites positive to Annexin V and monodansylcadaverine. Moreover, this compound induced the activation in infected macrophages of caspase-1, an evolutionarily conserved enzyme which plays a main role in controlling parasitemia, host survival, and the onset of adaptive immune response in T. cruzi infection. The antiparasitic activity of ST18 together to its ability to activate caspase-1 in infected macrophages and its low toxicity on normal cells makes this compound interesting for further clinical investigations.

High resolution mapping of nucleic acid containing complexes in vitro and in situ

1996 ◽  
Vol 54 ◽  
pp. 48-49
Author(s):  
D. P. Bazett-Jones ◽  
M. J. Hendzel
Keyword(s):  
Nucleic Acid ◽  
High Resolution ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Heavy Atom ◽  
Regulation Of Transcription ◽  
High Exposure ◽  
Resolution Mapping ◽  
Tata Sequence

Structural analysis of combinations of nucleosomes and transcription factors on promoter and enhancer elements is necessary in order to understand the molecular mechanisms responsible for the regulation of transcription initiation. Such complexes are often not amenable to study by high resolution crystallographic techniques. We have been applying electron spectroscopic imaging (ESI) to specific problems in molecular biology related to transcription regulation. There are several advantages that this technique offers in studies of nucleoprotein complexes. First, an intermediate level of spatial resolution can be achieved because heavy atom contrast agents are not necessary. Second, mass and stoichiometric relationships of protein and nucleic acid can be estimated by phosphorus detection, an element in much higher proportions in nucleic acid than protein. Third, wrapping or bending of the DNA by the protein constituents can be observed by phosphorus mapping of the complexes. Even when ESI is used with high exposure of electrons to the specimen, important macromolecular information may be provided. For example, an image of the TATA binding protein (TBP) bound to DNA is shown in the Figure (top panel). It can be seen that the protein distorts the DNA away from itself and much of its mass sits off the DNA helix axis. Moreover, phosphorus and mass estimates demonstrate whether one or two TBP molecules interact with this particular promoter TATA sequence.

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