Cytokine Production but Lack of Proliferation in Peripheral Blood Mononuclear Cells from Chronic Chagas' Disease Cardiomyopathy Patients in Response to T. cruzi Ribosomal P Proteins

Background:Trypanosoma cruzi ribosomal P proteins, P2β and P0, induce high levels of antibodies in patients with chronic Chagas' disease Cardiomyopathy (CCC). It is well known that these antibodies alter the beating rate of cardiomyocytes and provoke apoptosis by their interaction with β1-adrenergic and M2-muscarinic cardiac receptors. Based on these findings, we decided to study the cellular immune response to these proteins in CCC patients compared to non-infected individuals.Methodology/Principal findings:We evaluated proliferation, presence of surface activation markers and cytokine production in peripheral blood mononuclear cells (PBMC) stimulated with P2β, the C-terminal portion of P0 (CP0) proteins and T. cruzi lysate from CCC patients predominantly infected with TcVI lineage. PBMC from CCC patients cultured with P2β or CP0 proteins, failed to proliferate and express CD25 and HLA-DR on T cell populations. However, multiplex cytokine assays showed that these antigens triggered higher secretion of IL-10, TNF-α and GM-CSF by PBMC as well as both CD4+ and CD8+ T cells subsets of CCC subjects. Upon T. cruzi lysate stimulation, PBMC from CCC patients not only proliferated but also became activated within the context of Th1 response. Interestingly, T. cruzi lysate was also able to induce the secretion of GM-CSF by CD4+ or CD8+ T cells.Conclusions/Significance:Our results showed that although the lack of PBMC proliferation in CCC patients in response to ribosomal P proteins, the detection of IL-10, TNF-α and GM-CSF suggests that specific T cells could have both immunoregulatory and pro-inflammatory potential, which might modulate the immune response in Chagas' disease. Furthermore, it was possible to demonstrate for the first time that GM-CSF was produced by PBMC of CCC patients in response not only to recombinant ribosomal P proteins but also to parasite lysate, suggesting the value of this cytokine to evaluate T cells responses in T. cruzi infection.Fil: Longhi, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Atienza, Augusto. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Perez Prados, Graciela. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; ArgentinaFil: Buying, Alcinette. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Balouz, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Buscaglia, Carlos Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Santos, Radleigh. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Tasso, Laura Mónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Bonato, Ricardo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Chiale, Pablo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Pinilla, Clemencia. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Judkowski, Valeria A.. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Gomez, Karina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

An α-Gal antigenic surrogate as a biomarker of treatment evaluation in Trypanosoma cruzi-infected children. A retrospective cohort study.

BackgroundProper evaluation of therapeutic responses in Chagas disease is hampered by the prolonged persistence of antibodies to Trypanosoma cruzi measured by conventional serological tests and by the lack of sensitivity of parasitological tests. Previous studies indicated that tGPI-mucins, an α-Gal (α-d-Galp(1→3)-β-d-Galp(1→4)-d-GlcNAc)-rich fraction obtained from T. cruzi trypomastigotes surface coat, elicit a strong and protective antibody response in infected individuals, which disappears soon after successful treatment. The cost and technical difficulties associated with tGPI-mucins preparation, however, preclude its routine implementation in clinical settings.Methods/principle findingsWe herein developed a neoglycoprotein consisting of a BSA scaffold decorated with several units of a synthetic α-Gal antigenic surrogate (α-d-Galp(1→3)-β-d-Galp(1→4)-β-d-Glcp). Serological responses to this reagent, termed NGP-Tri, were monitored by means of an in-house enzyme-linked immunosorbent assay (α-Gal-ELISA) in a cohort of 82 T. cruzi-infected and Benznidazole- or Nifurtimox-treated children (3 days to 16 years-old). This cohort was split into 3 groups based on the age of patients at the time of treatment initiation: Group 1 comprised 24 babies (3 days to 5 months-old; median = 26 days-old), Group 2 comprised 31 children (7 months to 3 years-old; median = 1.0-year-old) and Group 3 comprised 26 patients (3 to 16 years-old; median = 8.4 years-old). A second, control cohort (Group 4) included 39 non-infected infants (3 days to 5 months-old; median = 31 days-old) born to T. cruzi-infected mothers. Despite its suboptimal seroprevalence (58.4%), α-Gal-ELISA yielded shorter median time values of negativization (23 months [IC 95% 7 to 36 months] vs 60 months [IC 95% 15 to 83 months]; p = 0.0016) and higher rate of patient negative seroconversion (89.2% vs 43.2%, p Conclusions/significanceThe tools evaluated here provide the cornerstone for the development of an efficacious, reliable, and straightforward post-therapeutic marker for pediatric Chagas disease

Serological regression analyses.

Serological profiles for tELISA and α-Gal-ELISA for patients from Group 1 (A), Group 2 (B), Group 3 (C) and the whole cohort of T. cruzi-infected children (D). Reactivity values are expressed as % of the first (pre-treatment, P) sample and regression curves are indicated in red and green lines, respectively. Mean reactivity and SD values for each time point are shown in red (tELISA) and green (α-Gal-ELISA) dots. Slope (95% CI) and R2 values are indicated for each data set. ANCOVA analyses were performed to compare slopes. (TIF)</p

Serological responses in patients with efficacious chemotherapy.

A) Diagram showing direct comparison of α-Gal-ELISA and tELISA results in the fraction (n = 37) of α-Gal reactive patients. B) Kaplan-Meier curves showing the survival time for antibodies determined by tELISA (red; n = 59) and α-Gal-ELISA (green; n = 37) in T. cruzi-infected children. Median negativization values are indicated for each data set. Censored cases are indicated with filled circles. Log-rank (Mantel-Cox) analyses were performed to compare median time of negative seroconversion.</p

Serological results for patients #783, #554 and #1598.

A-C) tELISA (red), α-Gal-ELISA (green) and qPCR results (+, positive; -, negative; N.D., non-determined) for patient #554 (A), #783 (B) and #1598 (C). Treatment was initiated at time 0 (expressed in months, panels A and B) or at 19 months after birth (arrow, panel C). The cutoff determined for tELISA and α-Gal-ELISA are indicated with color-matched dotted lines. m.p.t., months post-treatment.</p

α-Gal antibodies in <i>T</i>. <i>cruzi</i> infected-mother/newborn binomials.

α-Gal antibodies in T. cruzi infected-mother/newborn binomials.</p

Clinical and diagnostic features of patients from Group 4.

Clinical and diagnostic features of patients from Group 4.</p

Study population, inclusion criteria and group composition.

Study population, inclusion criteria and group composition.</p

Clinical and diagnostic features of patients from Groups 1 to 3.

Clinical and diagnostic features of patients from Groups 1 to 3.</p

Kaplan-Meier curves of patients from different groups.

Survival time of antibodies detected by tELISA (red lines) and α-Gal-ELISA (green lines) for patients from Group 1 (A), Group 2 (B), Group 3 (C) and Group 4 (D). Median values are indicated for each data set. Censored cases are indicated with filled circles. Log-rank (Mantel-Cox) analyses were performed to compare median time of negative seroconversion.</p