Chagasic Thymic Atrophy Does Not Affect Negative Selection but Results in the Export of Activated CD4+CD8+ T Cells in Severe Forms of Human Disease

Extrathymic CD4+CD8+ double-positive (DP) T cells are increased in some pathophysiological conditions, including infectious diseases. In the murine model of Chagas disease, it has been shown that the protozoan parasite Trypanosoma cruzi is able to target the thymus and induce alterations of the thymic microenvironment and the lymphoid compartment. In the acute phase, this results in a severe atrophy of the organ and early release of DP cells into the periphery. To date, the effect of the changes promoted by the parasite infection on thymic central tolerance has remained elusive. Herein we show that the intrathymic key elements that are necessary to promote the negative selection of thymocytes undergoing maturation during the thymopoiesis remains functional during the acute chagasic thymic atrophy. Intrathymic expression of the autoimmune regulator factor (Aire) and tissue-restricted antigen (TRA) genes is normal. In addition, the expression of the proapoptotic Bim protein in thymocytes was not changed, revealing that the parasite infection-induced thymus atrophy has no effect on these marker genes necessary to promote clonal deletion of T cells. In a chicken egg ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic system, the administration of OVA peptide into infected mice with thymic atrophy promoted OVA-specific thymocyte apoptosis, further indicating normal negative selection process during the infection. Yet, although the intrathymic checkpoints necessary for thymic negative selection are present in the acute phase of Chagas disease, we found that the DP cells released into the periphery acquire an activated phenotype similar to what is described for activated effector or memory single-positive T cells. Most interestingly, we also demonstrate that increased percentages of peripheral blood subset of DP cells exhibiting an activated HLA-DR+ phenotype are associated with severe cardiac forms of human chronic Chagas disease. These cells may contribute to the immunopathological events seen in the Chagas disease

The concurrent occurrence of Leishmania chagasi infection and childhood acute leukemia in Brazil

AbstractObjectiveThis study investigated the co-existence of Leishmania chagasi infection and childhood leukemia in patients naïve to treatment; this has serious clinical and epidemiological implications.MethodsThe seroprevalence of L. chagasi antibodies prior to any treatment was investigated in children with clinical features of acute leukemia. Serological tests were performed in 470 samples drawn from under 14-year-old children from different regions of Brazil with clinical suspicion of acute leukemia. Acute leukemia subtypes were characterized by immunophenotyping using flow cytometry. Morphological analyses of bone marrow aspirates were systematically performed to visualize blast cells and/or the formation of L. chagasi amastigotes. Data analysis used a standard univariate procedure and the Pearson's chi-square test.ResultsThe plasma of 437 children (93%) displayed antibodies against L. chagasi by indirect immunofluorescence assay and enzyme-linked immunosorbent assay tests. Of the 437 patients diagnosed from 2002 to 2006, 254 had acute lymphoblastic leukemia, 92 had acute myeloid leukemia, and 91 did not have acute leukemia. The seroprevalence of L. chagasi antibodies according to the indirect immunofluorescence assay test (22.5%) was similar in children with or without acute leukemia (p-value=0.76). The co-existence of visceral leishmanasis and acute leukemia was confirmed in 24 children. The overall survival of these children was poor with a high death rate during the first year of leukemia treatment.ConclusionIn the differential diagnosis of childhood leukemia, visceral leishmanasis should be considered as a potential concurrent disease in regions where L. chagasi is endemic

Multiparametric flow cytometry directing the evaluation of CRLF2 rearrangements and JAK2 status in pediatric B cell precursor acute lymphoblastic leukemia

Asbtract: Introduction: This study aimed to determine whether cytokine receptor-like factor 2 (CRLF2) antigen expression evaluated using multiparametric flow cytometry (MFC) could predict the genotype of CRLF2 and Janus kinase 2 (JAK2) status for application in the diagnosis of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Methods: A total of 321 BCP-ALL bone marrow samples were collected, 291 at diagnosis and 13 at first relapse, while 17 samples were excluded due to low cellular viability. The CRLF2 antigen expression was evaluated using flow cytometry (percentage of positivity and median fluorescence intensity [MFI]). The CRLF2 transcript levels were assessed via quantitative reverse transcription polymerase chain reaction using SYBR Green. The CRLF2 rearrangements (CRLF2-r) were identified using the CRLF2 break-apart probe via fluorescence in situ hybridization. Sanger sequencing was performed to identify the JAK2 exon 16 mutations. Results: We observed that 60 of the 291 cases (20.6%) presented CRLF2 antigen positivity, whereas the CRLF2 transcript overexpression was found in 19 of 113 cases (16.8%). The JAK2 mutation was found in four out of 116 cases (3.4%), all of which had CRLF2 ≥10% of positive cells and intermediate or high MFI (p < 0.0001). In addition, in the 13 cases with the CRLF2-r, a positive correlation was found with the CRLF2 antigen intermediate (61.5%) MFI (p = 0.017). Finally, the CRLF2-positive antigen was identified in the BCP-ALL subclones. Conclusion: The identification of the CRLF2 antigen using the MFC, based on the percentage of positivity and MFI values, is a useful tool for predicting JAK2 mutations and CRLF2-r

Pediatric Acute Promyelocytic Leukemia: Epidemiology, Molecular Features, and Importance of GST-Theta 1 in Chemotherapy Response and Outcome.

Previous studies have suggested a variation in the incidence of acute promyelocytic leukemia (APL) among the geographic regions with relatively higher percentages in the Latin American population. We aimed to explore the population burden of pediatric APL, gathering information from the population-based cancer registry (PBCR) and the diagnosis of APL obtained through incident cases from a hospital-based cohort. The homozygous deletion in glutathione S-transferases (GSTs) leads to a loss of enzyme detoxification activity, possibly affecting the treatment response. Mutations in the RAS pathway genes are also considered to be a key component of the disease both in the pathogenesis and in the outcomes. We have assessed mutations in a RAS-MAP kinase pathway (FLT3, PTPN11, and K-/NRAS) and GST variant predisposition risk in the outcome. Out of the 805 children and adolescents with acute myeloid leukemia (AML) who are registered in the PBCR, 35 (4.3%) were APL cases. The age-adjusted incidence rate (AAIR) was 0.03 per 100,000 person-years. One-hundred and sixty-three patients with APL were studied out of 931 AML cases (17.5%) from a hospital-based cohort. Mutations in FLT3, KRAS, and NRAS accounted for 52.1% of the cases. Patients with APL presented a 5-year probability of the overall survival (OS) of 67.3 ± 5.8%. A GST-theta 1 (GSTT1) null genotype conferred adverse prognosis, with an estimated hazard ratio of 2.8, 95% confidence interval (CI) 1.2-6.9. We speculate that the GSTT1 polymorphism is associated with therapeutics and would allow better OS of patients with APL with a GSTT1 null genotype

Intrathymic tissue-restricted antigen expression levels in atrophic thymus during <i>T cruzi</i> infection.

<p><b>(a)</b> Thymuses were collected from normal or <i>T. cruzi</i> infected BALB/c (day 15 post-infection) and expression levels of Aire and TRA genes were analyzed by real-time PCR. The expression of Aire as well as TRA genes was comparable in infected and uninfected thymus. Data are mean ± standard error. of triplicate measurements in one of two representative experiments using five mice per group. <b>(b)</b> Thymuses were stained with anti-Aire antibody and analyzed by immunofluorescence. The histological profiles show the presence of Aire-positive cells (arrows) in <b>(upper left)</b> normal thymus and <b>(upper right)</b> atrophic thymus at day 15 post-infection. The respective staining controls without the primary antibodies are represented in <b>(lower left)</b> and <b>(lower right)</b>. Inserts represent higher magnifications of Aire-positive cells (brown).</p

Thymic atrophy in BALB/c acutely infected with <i>T. cruzi</i>.

<p>The histological profiles show the <b>(upper left)</b> normal thymic architecture (C, cortex; M, medulla) and <b>(upper right)</b> marked cortical and medullary atrophy in the thymus of an acutely-infected mouse. <b>(lower left)</b> The numerous metallophilic macrophages are present in the cortico-medullary zone (arrows) of normal thymus. However, following <i>T. cruzi</i> acute infection <b>(lower right)</b> the number and distribution of metallophilic macrophages are changed with the cells dispersed throughout not only the cortico-medullar area but also the cortical region. Infected mice were evaluated herein at day 15 post-infection. These data are representative of two independent experiments using four mice per group.</p

Activation markers in lymphocytes from thymus and subcutaneous lymph nodes of <i>T. cruzi</i> infected mice.

<p>Mice were infected with <i>T. cruzi</i> and 15 days later lymphocytes were isolated from thymus and subcutaneous lymph nodes. <b>(a)</b> Thymocytes were stained with CD4-PE, CD8-APC and marker-specific fluorescein isothiocyanate-labeled antibodies prior to flow cytometry analysis. The histograms represent the expression of the CD44, CD69 and CD62L markers in total thymocytes (upper panel) and CD4⁺CD8⁺ T cells (lower panel) from chagasic and normal mice. <b>(b)</b> Comparison of the CD62L expression level from normal or chagasic CD4⁺CD8⁺ T cells <i>versus</i> normal single-positive CD4⁺ and CD8⁺ T cells undergoing intrathymic maturation. Panel <b>(c)</b> depicts representative histograms of CD4⁺CD8⁺ from <i>T. cruzi</i> infected subcutaneous lymph nodes (solid lines), compared with naïve CD4⁺ and CD8⁺ T cells obtained from non-infected mice. <b>(d)</b> Representative histograms of CD44 and CD69 expression in CD4⁺CD8⁺ T cells, activated single-positive CD4⁺ and CD8⁺ T cells from subcutaneous lymph nodes obtained from <i>T. cruzi</i> infected mice. Animals were sacrificed on day 15 post-infection. The data are representative of three independent experiments. The values on the upper right corner indicate the mean fluorescent intensity of the expression of the markers from each histogram. Differences between chagasic DP and activated T cells <i>versus</i> naïve T cells are significant (<i>P</i><0.05). These data are representative of three independent experiments using five mice per group.</p

Intrathymic expression of Bim in the course of acute <i>T. cruzi</i> infection.

<p><b>(a)</b> Thymuses were collected from normal or <i>T cruzi</i> infected BALB/c mice (day 15 post-infection). All numbers are given in 10⁶, and each value represents the mean of total thymocyte numbers in 4-8 mice per group. <b>(b)</b> From individual groups of the same experiments, DP cells isolated by cell sorting from thymus of infected mice at the indicated time-points or normal mice as controls were lysed and proteins were solubilized by detergent lysis and immunoprecipitated with anti-Bim antibody for Western blotting analysis probing with the same antibody. The upper blot shows kinetics of the Bim expression from day 10 to 16 post-infection, during the thymic atrophy, whereas the lower panel reveals a blot was probed with actin, applied as a loading control. <b>(c)</b> Optical densitometry of the western blots using NIH Image software, where Bim expression was normalized with the actin expression. The data are representative of three independent experiments. *Differences between control <i>versus</i> infected mice are significant (<i>P</i><0.05). These data are representative of two independent experiments using five mice per group.</p

CD4⁺CD8⁺ T cell numbers in thymus of <i>T. cruzi</i> infected OVA TCR transgenic mice.

<p>OVA-TCR transgenic mice noninfected or infected with <i>T. cruzi</i> (9 days post-infection) were injected <i>i.v.</i> with 0.5 ml of 450 mM solution of OVAp323–339 daily for 3 days and thymocytes were isolated on the fourth day for quantification of double-positive CD4⁺CD8⁺ T cells by flow cytometry. For control, mice were injected with 0.5 ml of PBS instead of the cognate antigen as indicated in the histogram. All numbers are given in 10⁶. Each value represents the mean of total DP cell numbers in 4-8 mice per group. Data are expressed as mean ± standard error of triplicate measurements of one out of two representative experiments. *Differences between groups are significant (<i>P</i><0.05). These data are representative of two independent experiments using five mice per group.</p