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Tag: Chagas disease

Immune exhaustion in chronic Chagas disease: Pro-inflammatory and immunomodulatory action of IL-27 in vitro

In chronic Chagas disease, Trypanosoma cruzi-specific T-cell function decreases over time, and alterations in the homeostatic IL-7/IL-7R axis are evident, consistent with a process of immune exhaustion. IL-27 is an important immunoregulatory cytokine that shares T-cell signaling with IL-7 and other cytokines of the IL-12 family and might be involved in the transcriptional regulation of T-cell function. Here, we evaluated the expression and function of IL-27R in antigen-experienced T cells from subjects with chronic Chagas disease and assessed whether in vitro treatment with IL-27 and IL-7 might improve T. cruzi-specific polyfunctional T-cell responses. In vitro exposure of PBMCs to T. cruzi induced a downregulation of IL-27R in CD4+ T cells and an upregulation in CD8+ T cells in subjects without heart disease, while IL-27R expression remained unaltered in subjects with more severe clinical stages. The modulation of IL-27R was associated with functional signaling through STAT3 and STAT5 and induction of the downstream genes TBX21, EOMES and CXCL9 in response to IL-27. In vitro treatment of PBMCs with IL-27 and IL-7 improved monofunctional and polyfunctional Th1 responses, accompanied by the induction of IL-10 and Bcl-2 expression in subjects without heart disease but did not improve those in subjects with cardiomyopathy. Our findings support the process of desensitization of the IL-27/IL-27R pathway along with disease severity and that the pro-inflammatory and immunomodulatory mechanisms of IL-27 might be interconnected.

María Ailén Natale, Todd Minning, María Cecilia Albareda, Melisa Daiana Castro Eiro, María Gabriela Álvarez, Bruno Lococo, Gonzalo Cesar, Graciela Bertocchi, María Josefina Elias, María Belén Caputo, Rick Lee Tarleton, Susana Adriana Laucella. PLoS Negl Trop Dis. 2021 Jun 1;15(6):e0009473. doi: 10.1371/journal.pntd.0009473.

High variation in immune responses and parasite phenotypes in naturally acquired Trypanosoma cruzi infection in a captive non-human primate breeding colony in Texas, USA

Trypanosoma cruzi, the causative agent of human Chagas disease, is endemic to the southern region of the United States where it routinely infects many host species. The indoor/outdoor housing configuration used in many non-human primate research and breeding facilities in the southern of the USA provides the opportunity for infection by T. cruzi and thus provides source material for in-depth investigation of host and parasite dynamics in a natural host species under highly controlled and restricted conditions. For cynomolgus macaques housed at such a facility, we used a combination of serial blood quantitative PCR (qPCR) and hemoculture to confirm infection in >92% of seropositive animals, although each method alone failed to detect infection in >20% of cases. Parasite isolates obtained from 43 of the 64 seropositive macaques were of 2 broad genetic types (discrete typing units, (DTU’s) I and IV); both within and between these DTU groupings, isolates displayed a wide variation in growth characteristics and virulence, elicited host immune responses, and susceptibility to drug treatment in a mouse model. Likewise, the macaques displayed a diversity in T cell and antibody response profiles that rarely correlated with parasite DTU type, minimum length of infection, or age of the primate. This study reveals the complexity of infection dynamics, parasite phenotypes, and immune response patterns that can occur in a primate group, despite being housed in a uniform environment at a single location, and the limited time period over which the T. cruzi infections were established.

Padilla AM, Yao PY, Landry TJ, Cooley GM, Mahaney SM, Ribeiro I, VandeBerg JL. Tarleton RL. (2021) High variation in immune responses and parasite phenotypes in naturally acquired Trypanosoma cruzi infection in a captive non-human primate breeding colony in Texas, USA. PLoS Negl Trop Dis 15(3): e0009141.

Deletion of a Golgi protein in Trypanosoma cruzi reveals a critical role for Mn2+ in protein glycosylation needed for host cell invasion and intracellular replication

Trypanosoma cruzi is a protist parasite and the causative agent of American trypanosomiasis or Chagas disease. The parasite life cycle in its mammalian host includes an intracellular stage, and glycosylated proteins play a key role in host-parasite interaction facilitating adhesion, invasion and immune evasion. Here, we report that a Golgi-localized Mn2+-Ca2+/H+ exchanger of T. cruzi (TcGDT1) is required for efficient protein glycosylation, host cell invasion, and intracellular replication. The Golgi localization was determined by immunofluorescence and electron microscopy assays. TcGDT1 was able to complement the growth defect of Saccharomyces cerevisiae null mutants of its ortholog ScGDT1 but ablation of TcGDT1 by CRISPR/Cas9 did not affect the growth of the insect stage of the parasite. The defect in protein glycosylation was rescued by Mn2+ supplementation to the growth medium, underscoring the importance of this transition metal for Golgi glycosylation of proteins.

Ramakrishnan S, Unger LM, Baptista RP, Cruz-Bustos T, Docampo R (2021) Deletion of a Golgi protein in Trypanosoma cruzi reveals a critical role for Mn2+ in protein glycosylation needed for host cell invasion and intracellular replication. PLoS Pathog 17(3): e1009399.

Reduced Trypanosoma cruzi-specific humoral response and enhanced T cell immunity after treatment interruption with benznidazole in chronic Chagas disease

Background: Interruption of benznidazole therapy due to the appearance of adverse effects, which is presumed to lead to treatment failure, is a major drawback in the treatment of chronic Chagas disease.

Methods: Trypanosoma cruzi-specific humoral and T cell responses, T cell phenotype and parasite load were measured to compare the outcome in 33 subjects with chronic Chagas disease treated with an incomplete benznidazole regimen and 58 subjects treated with the complete regimen, during a median follow-up period of 48 months.

Results: Both treatment regimens induced a reduction in the T. cruzi-specific antibody levels and similar rates of treatment failure when evaluated using quantitative PCR. Regardless of the regimen, polyfunctional CD4+ T cells increased in the subjects, with successful treatment outcome defined as a decrease of T. cruzi-specific antibodies. Regardless of the serological outcome, naive and central memory T cells increased after both regimens. A decrease in CD4+ HLA-DR+ T cells was associated with successful treatment in both regimens. The cytokine profiles of subjects with successful treatment showed fewer inflammatory mediators than those of the untreated T. cruzi-infected subjects. High levels of T cells expressing IL-7 receptor and low levels of CD8+ T cells expressing the programmed cell death protein 1 at baseline were associated with successful treatment following benznidazole interruption.

Conclusions: These findings challenge the notion that treatment failure is the sole potential outcome of an incomplete benznidazole regimen and support the need for further assessment of the treatment protocols for chronic Chagas disease.

Melisa D Castro Eiro, María A Natale, María G Alvarez, Huifeng Shen, Rodolfo Viotti, Bruno Lococo, Jacqueline Bua, Myriam Nuñez, Graciela L Bertocchi, María C Albareda, Gonzalo Cesar, Rick L Tarleton, Susana A Laucella. J Antimicrob Chemother. 2021 Mar 7;dkab054. doi: 10.1093/jac/dkab054

Outbreak News Today Interviews Rick Tarleton

Recently, Dr. Rick Tarleton was interviewed by Outbreak News Today about his recently published study in Science Translational Medicine.

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Stronger treatments could cure Chagas disease

3D (left) and single slice (right) light sheet microscopy imaging of the heart of a mouse infected with two strains (red and blue) of Trypanosoma cruzi. (Image credit: Fernando Sanchez-Valdez)

Research shows stronger but less frequent drug doses could be key

Researchers in the University of Georgia’s Center for Tropical and Emerging Global Diseases have found that a more intensive, less frequent drug regimen with currently available therapeutics could cure the infection that causes Chagas disease, a potentially life-threatening illness affecting up to 300,000 people in the United States.

Trypanosoma cruzi is a single-celled parasitic organism that causes Chagas disease. At least 6 million people are infected by T. cruzi, mostly in South America. Current drug therapies have been ineffective in completely clearing the infection and are associated with severe adverse side effects.

A single dose of benznidazole has been shown to be highly effective in killing more than 90% of parasites. However, after a CTEGD team found some of the parasites enter into a dormancy stage, the researchers hypothesized that an intermittent treatment schedule could be effective.

Rick Tarleton
Photo credit: Peter Frey/UGA

“Current human trials are only looking at giving lower doses over a shorter time period, which is the exact opposite of what we show works.”  — Rick Tarleton

“In this system we can see what a single dose of drug does,” said Rick Tarleton, Regents’ Professor in UGA’s department of cellular biology. “Does it make sense to give a drug twice daily when the remaining dormant parasites are insensitive to it?”

The investigators found that giving as little as two-and-a-half times the typical daily dose of benznidazole, once per week for 30 weeks, completely cleared the infection, whereas giving the standard daily dose once a week for a longer period did not.

“Current human trials are only looking at giving lower doses over a shorter time period, which is the exact opposite of what we show works,” said Tarleton.

Since Tarleton’s team worked with a mouse model, how this change in treatment regimen will translate in humans is yet unknown, as are any potential side effects of the higher doses. Adverse reactions already are a problem with current treatments; the hope is that side effects from a less frequent dosage would be more tolerable.

Significant challenge

Assessing the success of treatments in Chagas disease is a significant challenge. Tissue samples from infected organisms might not be representative of the entire organ or animal, since low numbers of persistent, dormant parasites can be difficult to detect. Therefore, Tarleton’s group used light sheet fluorescence microscopy to view intact whole organs from infected mice.

“With light sheet fluorescence microscopy, you have a broad view of potentially any tissue in the mouse that allows for dependable assessment of parasite load and persistence,” said Tarleton. “It gives you an incredible view of the infection.”

Using this technology, they learned something new about the dormant parasites: Some were still susceptible to drug treatment. This provides hope that new drug therapies could be developed to target these parasites.

“Discovery of new drugs should continue,” Tarleton said. “We still need better drugs.”


Co-led by assistant research scientist Juan Bustamante and research professional Fernando Sanchez-Valdez in Tarleton’s research group, the study’s findings appear in Science Translational Medicine.



A modified drug regimen clears active and dormant trypanosomes in mouse models of Chagas disease

A major contributor to treatment failure in Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is that current treatment regimens do not address the drug insensitivity of transiently dormant T. cruzi amastigotes. Here, we demonstrated that use of a currently available drug in a modified treatment regimen of higher individual doses, given less frequently over an extended treatment period, could consistently extinguish T. cruzi infection in three mouse models of Chagas disease. Once per week administration of benznidazole at a dose 2.5 to 5 times the standard daily dose rapidly eliminated actively replicating parasites and ultimately eradicated the residual, transiently dormant parasite population in mice. This outcome was initially confirmed in “difficult to cure” mouse infection models using immunological, parasitological, and molecular biological approaches and ultimately corroborated by whole organ analysis of optically clarified tissues using light sheet fluorescence microscopy (LSFM). This tool was effective for monitoring pathogen load in intact organs, including detection of individual dormant parasites, and for assessing treatment outcomes. LSFM-based analysis also suggested that dormant amastigotes of T. cruzi may not be fully resistant to trypanocidal compounds such as benznidazole. Collectively, these studies provide important information on the phenomenon of dormancy in T. cruzi infection in mice, demonstrate methods to therapeutically override dormancy using a currently available drug, and provide methods to monitor alternative therapeutic approaches for this, and possibly other, low-density infectious agents.

Juan M. Bustamante, Fernando Sanchez-Valdez, Angel M. Padilla, Brooke White, Wei Wang and Rick L. Tarleton. Science Translational Medicine 28 Oct 2020: Vol. 12, Issue 567, eabb7656. DOI: 10.1126/scitranslmed.abb7656

New Scheme of Intermittent Benznidazole Administration in Patients Chronically Infected With Trypanosoma cruzi: Clinical, Parasitological and Serological Assessment After Three Years of Follow-Up

Introduction. In a pilot study, we showed that intermittent administration of benznidazole in chronic Chagas disease patients resulted in a low rate of treatment suspension and therapeutic failure, as assessed by qPCR at the end of treatment. Herein, a three-year post-treatment follow-up study of the same cohort of patients is presented.

Methods. The treatment scheme consisted of 12 doses of benznidazole at 5 mg/kg/day in two daily doses every 5 days. Parasite load, T. cruzi-specific antibodies and serum chemokine levels were measured prior to treatment and after a median follow-up of 36 months post-treatment by kDNA and SatDNA qPCR methods, conventional serological techniques and a Luminex-based assay with recombinant T. cruzi protein, and a cytometric bead array, respectively.

Results. At the end of follow-up, 14 of 17 (82%) patients had negative qPCR findings, whereas three of 17 (18%) had detectable nonquantifiable findings by at least one of the qPCR techniques. A decline in parasite-specific antibodies at 12 months post-treatment was confirmed by conventional serological tests and the Luminex assays. Monocyte chemoattractant protein-1 (MCP-1) levels increased after treatment, whereas monokine induced by gamma interferon (MIG) levels decreased. New post-treatment electrocardiographic abnormalities were observed in only one patient who had cardiomyopathy prior to treatment.

Conclusions. Altogether, these data strengthen our previous findings by showing that the intermittent administration of benznidazole results in a low rate of treatment suspension, with comparable treatment efficacy to that of a daily dose of 5mg/kg for 60 days.

María Gabriela Álvarez, Juan Carlos Ramírez, Graciela Bertocchi, Marisa Fernández, Yolanda Hernández, Bruno Lococo, Constanza Lopez-Albizu, Alejandro Schijman, Carolina Cura, Marcelo Abril, Susana Laucella, Rick L Tarleton, María Ailen Natale, Melisa Castro Eiro, Sergio Sosa-Estani, Rodolfo Viotti. Antimicrob Agents Chemother. 2020 Jun 22;AAC.00439-20. doi: 10.1128/AAC.00439-20.

CRISPR/Cas9 Technology Applied to the Study of Proteins Involved in Calcium Signaling in Trypanosoma cruzi

Chagas disease is a vector-borne tropical disease affecting millions of people worldwide, for which there is no vaccine or satisfactory treatment available. It is caused by the protozoan parasite Trypanosoma cruzi and considered endemic from North to South America. This parasite has unique metabolic and structural characteristics that make it an attractive organism for basic research. The genetic manipulation of T. cruzi has been historically challenging, as compared to other pathogenic protozoans. However, the use of the prokaryotic CRISPR/Cas9 system for genome editing has significantly improved the ability to generate genetically modified T. cruzi cell lines, becoming a powerful tool for the functional study of proteins in different stages of this parasite’s life cycle, including infective trypomastigotes and intracellular amastigotes. Using the CRISPR/Cas9 method that we adapted to T. cruzi, it has been possible to perform knockout, complementation and in situ tagging of T. cruzi genes. In our system we cotransfect T. cruzi epimastigotes with an expression vector containing the Cas9 sequence and a single guide RNA, together with a donor DNA template to promote DNA break repair by homologous recombination. As a result, we have obtained homogeneous populations of mutant epimastigotes using a single resistance marker to modify both alleles of the gene. Mitochondrial Ca2+ transport in trypanosomes is critical for shaping the dynamics of cytosolic Ca2+ increases, for the bioenergetics of the cells, and for viability and infectivity. In this chapter we describe the most effective methods to achieve genome editing in T. cruzi using as example the generation of mutant cell lines to study proteins involved in calcium homeostasis. Specifically, we describe the methods we have used for the study of three proteins involved in the calcium signaling cascade of T. cruzi: the inositol 1,4,5-trisphosphate receptor (TcIP3R), the mitochondrial calcium uniporter (TcMCU) and the calcium-sensitive pyruvate dehydrogenase phosphatase (TcPDP), using CRISPR/Cas9 technology as an approach to establish their role in the regulation of energy metabolism.

Noelia Lander, Miguel A. Chiurillo, Roberto Docampo. Methods Mol Biol. 2020;2116:177-197. doi: 10.1007/978-1-0716-0294-2_13.