Systemic immunological profile of children with B-cell acute lymphoblastic leukemia: performance of cell populations and soluble mediators as serum biomarkers

BackgroundChildren with B-cell acute lymphoblastic leukemia (B-ALL) have an immune imbalance that is marked by remodeling of the hematopoietic compartment, with effects on peripheral blood (PB). Although the bone marrow (BM) is the main maintenance site of malignancy, the frequency with which immune cells and molecules can be monitored is limited, thus the identification of biomarkers in PB becomes an alternative for monitoring the evolution of the disease.MethodsHere, we characterize the systemic immunological profile in children undergoing treatment for B-ALL, and evaluate the performance of cell populations, chemokines and cytokines as potential biomarkers during clinical follow-up. For this purpose, PB samples from 20 patients with B-ALL were collected on diagnosis (D0) and during induction therapy (days 8, 15 and 35). In addition, samples from 28 children were used as a control group (CG). The cellular profile (NK and NKT-cells, Treg, CD3+ T, CD4+ T and CD8+ T cells) and soluble immunological mediators (CXCL8, CCL2, CXCL9, CCL5, CXCL10, IL-6, TNF, IFN-γ, IL-17A, IL- 4, IL-10 and IL-2) were evaluated via flow cytometry immunophenotyping and cytometric bead array assay.ResultsOn D0, B-ALL patients showed reduction in the frequency of cell populations, except for CD4+ T and CD8+ T cells, which together with CCL2, CXCL9, CXCL10, IL-6 and IL-10 were elevated in relation to the patients of the CG. On D8 and D15, the patients presented a transition in the immunological profile. While, on D35, they already presented an opposite profile to D0, with an increase in NKT, CD3+ T, CD4+ T and Treg cells, along with CCL5, and a decrease in the levels of CXCL9, CXCL10 and IL-10, thus demonstrating that B-ALL patients present a complex and dynamic immune network during induction therapy. Furthermore, we identified that many immunological mediators could be used to classify the therapeutic response based on currently used parameters.ConclusionFinally, it is noted that the systemic immunological profile after remission induction still differs significantly when compared to the GC and that multiple immunological mediators performed well as serum biomarkers

Evaluation of the Allergenicity Potential of TcPR-10 Protein from Theobroma cacao

Background: The pathogenesis related protein PR10 (TcPR-10), obtained from the Theobroma cacao-Moniliophthora perniciosa interaction library, presents antifungal activity against M. perniciosa and acts in vitro as a ribonuclease. However, despite its biotechnological potential, the TcPR-10 has the P-loop motif similar to those of some allergenic proteins such as Bet v 1 (Betula verrucosa) and Pru av 1 (Prunus avium). The insertion of mutations in this motif can produce proteins with reduced allergenic power. The objective of the present work was to evaluate the allergenic potential of the wild type and mutant recombinant TcPR-10 using bioinformatics tools and immunological assays. Methodology/Principal Findings: Mutant substitutions (T10P, I30V, H45S) were inserted in the TcPR-10 gene by sitedirected mutagenesis, cloned into pET28a and expressed in Escherichia coli BL21(DE3) cells. Changes in molecular surface caused by the mutant substitutions was evaluated by comparative protein modeling using the three-dimensional structure of the major cherry allergen, Pru av 1 as a template. The immunological assays were carried out in 8-12 week old female BALB/c mice. The mice were sensitized with the proteins (wild type and mutants) via subcutaneous and challenged intranasal for induction of allergic airway inflammation. Conclusions/Significance: We showed that the wild TcPR-10 protein has allergenic potential, whereas the insertion of mutations produced proteins with reduced capacity of IgE production and cellular infiltration in the lungs. On the other hand, in vitro assays show that the TcPR-10 mutants still present antifungal and ribonuclease activity against M. perniciosa RNA. In conclusion, the mutant proteins present less allergenic potential than the wild TcPR-10, without the loss of interesting biotechnological properties. (Résumé d'auteur

Three-dimensional structure of TcPR-10 obtained by homology modeling with Pru av1 (Protein Data Bank, 1e09_A) as template using SWISS-MODEL. A.

<p>The secondary structure elements are colored: alpha-helices in red, anti-parallel beta-sheets in yellow and P-loops in green. <b>B.</b> Molecular surface of TcPR-10 wild with matching regions of contiguous amino acids: 47GDGGVG52 in blue; 59FPEGSHFKY67 in brown; 116TSHYHT121 in gray; 129EEEIKAGK136 in peach. <b>C</b> e <b>E</b> Molecular surface of TcPR-10 wild type with amino acids for mutations highlighted in orangen (Thr10, Ile30, His45). <b>D e F.</b> TcPR-10 mutant type with point mutations in blue (Pro10, Val30, Ser45).</p

Identification of potential allergenic of TcPR-10 protein in the SDAP¹ allergenic proteins database.

1<p>Structural database of allergenic proteins; ²Property distance index; ³Identity for two sequences.</p

Survival of <i>M. perniciosa</i> dikaryotic broken hyphae incubated with different TcPR-10 wild and mutant type protein concentrations (4, 8 and 10 µg/ml).

<p>Survival of <i>M. perniciosa</i> dikaryotic broken hyphae incubated with different TcPR-10 wild and mutant type protein concentrations (4, 8 and 10 µg/ml).</p

Quantification of polyclonal IgE, BAL total cell count and histological illustration of the lung of BALB/c mice.

<p><b>A.</b> Quantification of polyclonal IgE antibody levels in serum of BALB/c mice. <b>B.</b> Cell counting in BAL fluid. The set average values <i>per se</i> quantification of antibodies and showed normal (p<0,05; Shapiro Wilk Test) using the comparison test of means the parametric Tukey Test (α = 0,05). *Significantly high values compared to control. **Significantly reduced values compared to TcPR-10 wt. Horizontal bars represent the mean value of each group. C. Lung were removed twenty-four hours after the last challenge. Lung tissue was fixed, embedded, cut into slices and stained with hematoxylin e eosin (H&E) solution. <b>C:</b> Sections from control; <b>D:</b> wild TcPR-10; <b>E:</b> mutant TcPR-10.</p

Amino acid sequence alignment of TcPR-10 with the allergens from the SDAP database (

<p><a href="http://align.genome.jp/sit-bin/clustalw" target="_blank">http://align.genome.jp/sit-bin/clustalw</a><b>).</b> The three point mutations for TcPR-10 (T10P, I30V, H45S) are marked in sequence alignment and P-loop was underlined. The identical, highly conserved, and conserved amino acids among the sequences are denoted with (*), (:), and (.), respectively. Matching regions of contiguous amino acids are highlighted in black.</p

Template obtained by PSI-Blast algorithm for modeling of proteins structures TcPR-10 wild and mutant.

<p>Template obtained by PSI-Blast algorithm for modeling of proteins structures TcPR-10 wild and mutant.</p

Experimental design of sensitization and challenge with TcPR-10 wild types and mutant.

<p>Experimental design of sensitization and challenge with TcPR-10 wild types and mutant.</p

Ribonuclease activity of recombinant TcPR-10 Wild Types (wt) and Mutant visualized in 1% agarose gel. 1 µg RNA from <i>M. perniciosa</i> was incubated with 1 µg of recombinats proteins at 25°C at different times.

<p>Lane <b>1.</b> RNA without protein; Lane <b>2.</b> RNA with boiled TcPR10 mut 2 h incubation; Lane <b>3.</b> RNA with boiled TcPR10 wt 2 h incubation; Lane <b>4, 6, 8, 10</b> and <b>12</b> RNA incubated with TcPR10 mut by 10 min, 20 min, 1 h, 2 h and 3 h, respectively; Lane <b>5, 7, 9, 11</b> and <b>13</b> RNA incubated with TcPR10 wt by 10 min 20 min, 1 h, 2 h and 3 h, respectively. Arrows indicate RNA bands without degradation.</p