Journal: Immunology

Article Title: The cytosolic tryparedoxin peroxidase from Trypanosoma cruzi induces a pro‐inflammatory Th1 immune response in a peroxidatic cysteine‐dependent manner

doi: 10.1111/imm.13302

Figure Lengend Snippet: c‐TXNPx induces CD4+ and CD8+ T‐cell proliferation with IFN‐γ production. T‐cell proliferation and cytokine production by splenocytes from wt and mutated c‐TXNPx‐treated mice. Mice were i.p. injected with c‐TXNPx, c‐TXNPxC52S or mock (control) in PBS 6 times every two days. Spleens were removed, labelled with CFSE and cultured in the presence of different stimuli, to evaluate CD4 and CD8 T‐cell proliferation and cytokine production in culture supernatants by specific ELISA. (A) Splenocytes primed with c‐TXNPx or c‐TXNPxC52S and in vitro stimulated with the corresponding immunogens, c‐TXNPx (c‐TXNPx,/c‐TXNPx) or c‐TXNPxC52S (c‐TXNPx,/c‐TXNPxC52S). (B) Splenocytes primed with c‐TXNPx and in vitro stimulated with c‐TXNPx (c‐TXNPx/c‐TXNPx) or c‐TXNPxC52S (c‐TXNPx/c‐TXNPxC52S). (C) Splenocytes primed with c‐TXNPxC52S and in vitro stimulated with c‐TXNPx (c‐TXNPxC52S/c‐TXNPx) or c‐TXNPxC52S (c‐TXNPxC52S/c‐TXNPxC52S). Controls consisted in in vivo priming with mock preparation. The proliferation index was calculated with respect to cells incubated in the presence of medium alone on CD4 or CD8‐gated cells. IFN‐γ and IL‐4 were determined on culture supernatants by ELISA. Data shown are the mean of three independent experiments ± SEM, n = 6 animals per group. Asterisks indicate statistically significant differences (*P < 0·05).

Article Snippet: INF‐γ secretion was measured by ELISA (OptEIA™ Human IFN‐γ ELISA set, BD Pharmingen, San Diego, CA, USA) according to the manufacturer´s instructions.

Techniques: Injection, Cell Culture, Enzyme-linked Immunosorbent Assay, In Vitro, In Vivo, Incubation

Journal: Immunology

Article Title: The cytosolic tryparedoxin peroxidase from Trypanosoma cruzi induces a pro‐inflammatory Th1 immune response in a peroxidatic cysteine‐dependent manner

doi: 10.1111/imm.13302

Figure Lengend Snippet: Cellular and humoral immune response from chronic Chagas disease patients triggered by c‐TXNPx and c‐TXNPxC52S. PBMC from chronic Chagas patients without demonstrable cardiac involvement (G0, n = 7), with Chagas cardiomyopathy (G1, n = 8), and non‐infected donors (NI, n = 5) were incubated with recombinant c‐TXNPx or c‐TXNPxC52S, mock and no antigen (culture medium only as controls) for 5 days in complete RPMI medium. Each condition was performed by triplicate. (A) Cells were pulsed with [3H]‐thymidine for the last 18 h of incubation, and PBMC proliferation was determined by [3H]‐thymidine uptake. (B) Culture supernatants were collected after 5 days of stimulation with c‐TXNPx, c‐TXNPxC52S or mock, and IFN‐γ secretion was quantified by ELISA. Box‐and‐whisker plots (min to max) show the stimulation index (SI) that was calculated for each readout as the mean value for triplicate stimulated cultures of each individual divided by the mean value of triplicate non‐stimulated cultures (medium only). In cases for which the level of secreted IFN‐γ was undetectable, the obtained value from the stimulated cultures was divided by the lowest detectable value (2·3 pg/ml). (C) Anti‐c‐TXNPx (dots) and anti‐c‐TXNPxC52S (squares) antibody titration was carried out in sera from chronic Chagas disease patients without demonstrable cardiac manifestations (G0, n = 8) and with Chagas cardiomyopathy (G1, n = 8), and non‐infected donors (NI, n = 7) by ELISA. The titre of antibodies from each individual was expressed as the serum dilution factor that corresponds to the IC50 value of the adjusted curve, which was calculated by non‐linear regression analysis. Each dot corresponds to the value from a single individual, and the horizontal line shows the median value for each group. Statistical analysis was performed using a linear mixed‐effects model (LMM) fitted by maximum likelihood and Tukey's honest significant difference contrasts for post hoc comparisons. Asterisks indicate statistically significant differences (***P < 0·001; **P < 0·01; *P < 0·05).

Article Snippet: INF‐γ secretion was measured by ELISA (OptEIA™ Human IFN‐γ ELISA set, BD Pharmingen, San Diego, CA, USA) according to the manufacturer´s instructions.

Techniques: Infection, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Whisker Assay, Titration