scholarly journals A Novel High-Content Screening-Based Method for Anti-Trypanosoma cruzi Drug Discovery Using Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Diogo Crispim Nascimento Portella ◽  
Erik Aranha Rossi ◽  
Bruno Diaz Paredes ◽  
Tanira Matutino Bastos ◽  
Cássio Santana Meira ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Imaging System ◽  
Rapid Assessment ◽  
Chronic Phase ◽  
Induced Pluripotent Stem Cell ◽  
High Content Screening ◽  
Reference Drug ◽  
Induced Pluripotent

Chagas disease is caused by Trypanosoma cruzi infection and remains a relevant cause of chronic heart failure in Latin America. The pharmacological arsenal for Chagas disease is limited, and the available anti-T. cruzi drugs are not effective when administered during the chronic phase. Cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) have the potential to accelerate the process of drug discovery for Chagas disease, through predictive preclinical assays in target human cells. Here, we aimed to establish a novel high-content screening- (HCS-) based method using hiPSC-CMs to simultaneously evaluate anti-T. cruzi activity and cardiotoxicity of chemical compounds. To provide proof-of-concept data, the reference drug benznidazole and three compounds with known anti-T. cruzi activity (a betulinic acid derivative named BA5 and two thiazolidinone compounds named GT5A and GT5B) were evaluated in the assay. hiPSC-CMs were infected with T. cruzi and incubated for 48 h with serial dilutions of the compounds for determination of EC50 and CC50 values. Automated multiparametric analyses were performed using an automated high-content imaging system. Sublethal toxicity measurements were evaluated through morphological measurements related to the integrity of the cytoskeleton by phalloidin staining, nuclear score by Hoechst 33342 staining, mitochondria score following MitoTracker staining, and quantification of NT-pro-BNP, a peptide released upon mechanical myocardial stress. The compounds showed EC50 values for anti-T. cruzi activity similar to those previously described for other cell types, and GT5B showed a pronounced trypanocidal activity in hiPSC-CMs. Sublethal changes in cytoskeletal and nucleus scores correlated with NT-pro-BNP levels in the culture supernatant. Mitochondrial score changes were associated with increased cytotoxicity. The assay was feasible and allowed rapid assessment of anti-T. cruzi action of the compounds, in addition to cardiotoxicity parameters. The utilization of hiPSC-CMs in the drug development workflow for Chagas disease may help in the identification of novel compounds.

Dynamics of T Cells Repertoire During Trypanosoma cruzi Infection and its Post-Treatment Modulation

2019 ◽  
Vol 26 (36) ◽  
pp. 6519-6543 ◽  
Author(s):  
Adriana Egui ◽  
Paola Lasso ◽  
Elena Pérez-Antón ◽  
M. Carmen Thomas ◽  
Manuel Carlos López
Keyword(s):  
Immune Response ◽  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Cardiac Tissue ◽  
Acute Infection ◽  
Clinical Status ◽  
Chronic Phase ◽  
Parasite Load ◽  
Chronic Patients ◽  
Cruzi Infection

Chagas disease courses with different clinical phases and has a variable clinical presentation and progression. The acute infection phase mostly exhibits a non-specific symptomatology. In the absence of treatment, the acute phase is followed by a chronic phase, which is initially asymptomatic. This chronic asymptomatic phase of the disease is characterized by a fragile balance between the host’s immune response and the parasite replication. The loss of this balance is crucial for the progression of the sickness. The virulence and tropism of the T. cruzi infecting strain together to the inflammation processes in the cardiac tissue are the main factors for the establishment and severity of the cardiomyopathy. The efficacy of treatment in chronic Chagas disease patients is controversial. However, several studies carried out in chronic patients demonstrated that antiparasitic treatment reduces parasite load in the bloodstream and leads to an improvement in the immune response against the Trypanosoma cruzi parasite. The present review is mainly focused on the cellular patterns associated to the clinical status and the evolution of the disease in chronic patients, as well as the effectiveness of the treatment related to T. cruzi infection control. Therefore, an emphasis is placed on the dynamics of specific-antigens T cell subpopulations, their memory and activation phenotypes, their functionality and their contribution to pathogenesis or disease control, as well as their association with risk of congenital transmission of the parasite.

scholarly journals Emerging hiPSC Models for Drug Discovery in Neurodegenerative Diseases

2021 ◽  
Vol 22 (15) ◽  
pp. 8196
Author(s):  
Dorit Trudler ◽  
Swagata Ghatak ◽  
Stuart A. Lipton
Keyword(s):  
Drug Discovery ◽  
Animal Models ◽  
Neurodegenerative Diseases ◽  
Disease Modeling ◽  
Induced Pluripotent Stem Cell ◽  
Neural Function ◽  
Neural Cells ◽  
Human Samples ◽  
Healthy Donors ◽  
Induced Pluripotent

Neurodegenerative diseases affect millions of people worldwide and are characterized by the chronic and progressive deterioration of neural function. Neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington’s disease (HD), represent a huge social and economic burden due to increasing prevalence in our aging society, severity of symptoms, and lack of effective disease-modifying therapies. This lack of effective treatments is partly due to a lack of reliable models. Modeling neurodegenerative diseases is difficult because of poor access to human samples (restricted in general to postmortem tissue) and limited knowledge of disease mechanisms in a human context. Animal models play an instrumental role in understanding these diseases but fail to comprehensively represent the full extent of disease due to critical differences between humans and other mammals. The advent of human-induced pluripotent stem cell (hiPSC) technology presents an advantageous system that complements animal models of neurodegenerative diseases. Coupled with advances in gene-editing technologies, hiPSC-derived neural cells from patients and healthy donors now allow disease modeling using human samples that can be used for drug discovery.

scholarly journals Immunoblotting analyses using two-dimensional gel electrophoresis of Trypanosoma cruzi excreted-secreted antigens

2004 ◽  
Vol 37 (6) ◽  
pp. 454-459
Author(s):  
Adriano Gomes Silva ◽  
Elisangela Paula Silveira-Lacerda ◽  
Jair Pereira Cunha-Júnior ◽  
Maria Aparecida de Souza ◽  
Silvio Favoreto Junior
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Gel Electrophoresis ◽  
Complex Mixture ◽  
Chronic Phase ◽  
Disease Diagnosis ◽  
Basic Knowledge ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Secreted Antigens

Trypanosoma cruzi trypomastigotes excrete-secrete a complex mixture of antigenic molecules. This antigenic mixture denominated trypomastigote excreted-secreted antigens contains a 150-160 kDa band that shows excellent performance in Chagas' disease diagnosis by immunoblotting. The present study partially characterized by two-dimensional gel electrophoresis the immunoreactivity against the 150-160kDa protein using sera samples from chagasic patients in different phases of the disease. Trypomastigote excreted-secreted antigen preparations were subjected to high-resolution two-dimensional (2D) gel electrophoresis followed by immunoblotting with sera from chagasic and non-chagasic patients. The 150-160kDa protein presented four isoforms with isoelectric focusing ranging from 6.2 to 6.7. The four isoforms were recognized by IgM from acute phase and IgG from chronic phase sera of chagasic patients. The 150-160kDa isoform with IF of approximately 6.4 became the immunodominant spot with the progression of the disease. No cross-reactivity was observed with non-chagasic or patients infected with Leishmania sp. In this study we provide basic knowledge that supports the validation of trypomastigote excreted-secreted antigens for serological diagnosis of Chagas' disease.

scholarly journals Analysis of cloned Trypanosoma cruzi proteins that are antigenic during the acute and chronic phase of Chagas' disease

1988 ◽  
Vol 83 (suppl 1) ◽  
pp. 345-346 ◽  
Author(s):  
Alberto C. C. Frasch ◽  
Jose L. Affranchino ◽  
Carlos F. Ibañez ◽  
Maria B. Reyes ◽  
Roberto A. Macina ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Chronic Phase

scholarly journals Using High-Content Screening Technology as a Tool to Generate Single-Cell Patient-Derived Gene-Corrected Isogenic iPS Clones for Parkinson’s Disease Research

2020 ◽  
Author(s):  
Peter A. Barbuti ◽  
Paul M. Antony ◽  
Bruno F.R. Santos ◽  
François Massart ◽  
Gérald Cruciani ◽  
...  
Keyword(s):  
Single Cell ◽  
Clonal Selection ◽  
Induced Pluripotent Stem Cell ◽  
Alpha Synuclein ◽  
Screening Strategy ◽  
High Content Screening ◽  
Selection Strategy ◽  
Microtiter Plates ◽  
Automated Platform ◽  
Induced Pluripotent

The generation of isogenic induced pluripotent stem cell (iPSC) lines using CRISPR-Cas9 technology is a technically challenging, time-consuming process with variable efficiency. Here we use fluorescence-activated cell sorting (FACS) to sort biallelic CRISPR-Cas9 edited single-cell iPS clones into high-throughput 96-well microtiter plates. We used high-content screening (HCS) technology and generated an in-house developed algorithm to select the correctly edited isogenic clones for continued expansion and validation. In our model we have gene-corrected the iPSCs of a Parkinson’s disease (PD) patient carrying the autosomal dominantly inherited heterozygous c.88G>C mutation in the SNCA gene, which leads to the pathogenic p.A30P form of the alpha-synuclein protein. Undertaking a PCR restriction-digest mediated clonal selection strategy prior to sequencing, we were able to post-sort validate each isogenic clone using a quadruple screening strategy. Subsequent transfection with mRNA encoding excision-only transposase allows for the generation of footprint-free isogenic iPSC lines. These monoclonal isogenic iPSC lines retain a normal molecular genotype, express pluripotency markers and have the ability to differentiate into the three germ layers. This combinatory approach of FACS, HCS and post-sorted restriction digestion facilitates the generation of isogenic cell lines for disease modelling to be scaled-up on an automated platform.

scholarly journals Accelerated Maturation of Non‐cycling Human Induced Pluripotent Stem Cell‐derived neurons for High Content Screening

2021 ◽  
Vol 35 (S1) ◽  
Author(s):  
Elizabeth Sharlow ◽  
Anna Mendelson ◽  
Danielle Llaneza ◽  
Veronica Porterfield ◽  
George Bloom ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
High Content Screening ◽  
Induced Pluripotent

scholarly journals Experimental Chemotherapy for Chagas Disease: A Morphological, Biochemical, and Proteomic Overview of Potential Trypanosoma cruzi Targets of Amidines Derivatives and Naphthoquinones

2011 ◽  
Vol 2011 ◽  
pp. 1-13 ◽  
Author(s):  
Solange L. de Castro ◽  
Denise G. J. Batista ◽  
Marcos M. Batista ◽  
Wanderson Batista ◽  
Anissa Daliry ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Chronic Phase ◽  
Public Health Problem ◽  
Term Therapy ◽  
High Morbidity ◽  
Synthetic Compounds ◽  
Natural And Synthetic

Chagas disease (CD), caused by Trypanosoma cruzi, affects approximately eight million individuals in Latin America and is emerging in nonendemic areas due to the globalisation of immigration and nonvectorial transmission routes. Although CD represents an important public health problem, resulting in high morbidity and considerable mortality rates, few investments have been allocated towards developing novel anti-T. cruzi agents. The available therapy for CD is based on two nitro derivatives (benznidazole (Bz) and nifurtimox (Nf)) developed more than four decades ago. Both are far from ideal due to substantial secondary side effects, limited efficacy against different parasite isolates, long-term therapy, and their well-known poor activity in the late chronic phase. These drawbacks justify the urgent need to identify better drugs to treat chagasic patients. Although several classes of natural and synthetic compounds have been reported to act in vitro and in vivo on T. cruzi, since the introduction of Bz and Nf, only a few drugs, such as allopurinol and a few sterol inhibitors, have moved to clinical trials. This reflects, at least in part, the absence of well-established universal protocols to screen and compare drug activity. In addition, a large number of in vitro studies have been conducted using only epimastigotes and trypomastigotes instead of evaluating compounds' activities against intracellular amastigotes, which are the reproductive forms in the vertebrate host and are thus an important determinant in the selection and identification of effective compounds for further in vivo analysis. In addition, due to pharmacokinetics and absorption, distribution, metabolism, and excretion characteristics, several compounds that were promising in vitro have not been as effective as Nf or Bz in animal models of T. cruzi infection. In the last two decades, our team has collaborated with different medicinal chemistry groups to develop preclinical studies for CD and investigate the in vitro and in vivo efficacy, toxicity, selectivity, and parasite targets of different classes of natural and synthetic compounds. Some of these results will be briefly presented, focusing primarily on diamidines and related compounds and naphthoquinone derivatives that showed the most promising efficacy against T. cruzi.

Chagas Disease Drug Discovery: Multiparametric Lead Optimization against Trypanosoma cruzi in Acylaminobenzothiazole Series

2019 ◽  
Vol 62 (22) ◽  
pp. 10362-10375 ◽  
Author(s):  
Charlotte Fleau ◽  
Angel Padilla ◽  
Juan Miguel-Siles ◽  
Maria T. Quesada-Campos ◽  
Isabel Saiz-Nicolas ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Lead Optimization

scholarly journals Effects of betamethasone on the course of experimentai. Infection with Trypanosoma cruzi

1986 ◽  
Vol 19 (3) ◽  
pp. 161-164 ◽  
Author(s):  
Frederico G.C. Abath ◽  
Yara M. Gomes ◽  
Eridan M. Coutinho ◽  
Silvia M.L. Montenegro ◽  
Maria E.B. Melo ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Acute Phase ◽  
Bacterial Infections ◽  
Acute Infection ◽  
Chronic Phase ◽  
Control Group ◽  
Cumulative Mortality ◽  
Histopathological Findings ◽  
Experimental Group

In this experiment, the effect of betamethasone administered in the early post- acute infection of mice by Trypanosoma cruzi was studied. This drug was administered during 30 days after the 42nd day of infection in a dose of 0.15 mg/day. The betamethasone treatment did not cause fresh outbreaks of parasitemia and the histopathological findings in the chronic phase were not different from those in the control group. The higher cumulative mortality after treatment in the experimental group was due to superimposed bacterial infections. Outbred albino mice infected with low numbers ofY strain Trypanosoma cruzi trypomastigotes were not suitable models for Chagas' disease, since after 7 months of observation only mild histological lesions developed in all the animais. Prolonged betamethasone treatment of mice infected with low numbers o/Trypanosoma cruzi of the Y strain, during the post-acute phase did not aggravate the course of infection.

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