Thymopoiesis and regulatory T cells in healthy children and adolescents

OBJECTIVES: The purpose of this study was to investigate the association between T cell receptor excision circle levels in peripheral blood mononuclear cells and regulatory T cells that co-express CD25 and Foxp3 in healthy children and adolescents of different ages. MATERIALS AND METHODS: The quantification of signal-joint T-cell receptor excision circle levels in the genomic DNA of peripheral blood mononuclear cells was performed using real-time quantitative PCR. The analysis of CD4, CD8, CD25, and Foxp3 expression was performed using flow cytometry. RESULTS: Ninety-five healthy controls (46 females and 49 males) ranging in age from 1 to 18 years were analyzed. The mean T-cell receptor excision circle count in all individuals was 89.095 +/- 36.790 T-cell receptor excision circles per microgram of DNA. There was an inverse correlation between T-cell receptor excision circles counts and age (r = -0.846; p < 0.001) as well as between the proportion of CD4(+)CD25(+)Foxp3(+) T cells and age (r = -0.467; p = 0.04). In addition, we observed a positive correlation between the amount of CD4(+)CD25(+)Foxp3(+) T cells and the amount of T-cell receptor excision circles per microgram of DNA in individuals of all ages (r = -0.529; p = 0.02). CONCLUSIONS: In this study, we observed a decrease in the thymic function with age based on the fact that the level of T-cell receptor excision circles in the peripheral blood positively correlated with the proportion of regulatory T cells in healthy children and adolescents. These findings indicate that although T-cell receptor excision circles and regulatory T cells levels decrease with age, homeostasis of the immune system and relative regulatory T cells population levels are maintained in the peripheral blood.

The Impact of Regulatory Heterogeneity on Agri-food Trade

We estimate the impact of regulatory heterogeneity on agri-food trade using a gravity analysis that relies on detailed data on non-tariff measures (NTMs) collected by the NTM-Impact project. The data cover a broad range of import requirements for agricultural and food products for the EU and nine of its major trade partners. We find that trade is significantly reduced when importing countries have stricter maximum residue limits (MRLs) for plant products than exporting countries. For most other measures, due to their qualitative nature, we were unable to infer whether the importer has stricter standards relative to the exporter, and we do not find a robust relationship between these measures and trade. Our findings suggest that, at least for some import standards, harmonising regulations will increase trade. We also conclude that tariff reductions remain an effective means to increase trade even when NTMs abound.

Regulatory variation in a TBX5 enhancer leads to isolated congenital heart disease

Recent studies have identified the genetic underpinnings of a growing number of diseases through targeted exome sequencing. However, this strategy ignores the large component of the genome that does not code for proteins, but is nonetheless biologically functional. To address the possible involvement of regulatory variation in congenital heart diseases (CHDs), we searched for regulatory mutations impacting the activity of TBX5, a dosage-dependent transcription factor with well-defined roles in the heart and limb development that has been associated with the HoltOram syndrome (hearthand syndrome), a condition that affects 1/100 000 newborns. Using a combination of genomics, bioinformatics and mouse genetic engineering, we scanned approximate to 700 kb of the TBX5 locus in search of cis-regulatory elements. We uncovered three enhancers that collectively recapitulate the endogenous expression pattern of TBX5 in the developing heart. We re-sequenced these enhancer elements in a cohort of non-syndromic patients with isolated atrial and/or ventricular septal defects, the predominant cardiac defects of the HoltOram syndrome, and identified a patient with a homozygous mutation in an enhancer approximate to 90 kb downstream of TBX5. Notably, we demonstrate that this single-base-pair mutation abrogates the ability of the enhancer to drive expression within the heart in vivo using both mouse and zebrafish transgenic models. Given the population-wide frequency of this variant, we estimate that 1/100 000 individuals would be homozygous for this variant, highlighting that a significant number of CHD associated with TBX5 dysfunction might arise from non-coding mutations in TBX5 heart enhancers, effectively decoupling the heart and hand phenotypes of the HoltOram syndrome.

CD25(+) T cell depletion impairs murine squamous cell carcinoma development via modulation of antitumor immune responses

Squamous cell carcinoma (SCC) constitutes a microenvironment that could modulate the antitumor immune response. Also, tumor-infiltrating lymphocytes are believed to play complex regulatory roles in antitumor immunity against SCC. The presence of regulatory T cells (Tregs) has been associated with the suppression of tumor-reactive T cells. However, the underlying mechanism for this T cell dysfunction is not clear. We used a multistage model of SCC to examine the role of Treg cells during tumor development. 7,12-dimethylbenz[a]-anthracene/phorbol 12-myristate 13-acetate treatment and systemic depletion of Treg cells using an anti-CD25 monoclonal antibody (PC61) resulted in a decrease in the number and incidence of papilloma. Furthermore, CD25 depletion increased the proportion of CD8(+) and CD4(+) T cells that were isolated from tumor lesions. The levels of interleukin (IL)-1 beta, IL-10, IL-12, IL-13, interferon-gamma, transforming growth factor-beta and tumor necrosis factor-alpha, but not IL-17, were increased in the tumor microenvironment after Treg depletion. Therefore, our results indicated involvement of CD25(+) T cells in SCC development and in the suppression of the inflammatory immune response.

In silico identification of conserved intercoding sequences in Leishmania genomes: Unraveling putative cis-regulatory elements

In silico analyses of Leishmania spp. genome data are a powerful resource to improve the understanding of these pathogens' biology. Trypanosomatids such as Leishmania spp. have their protein-coding genes grouped in long polycistronic units of functionally unrelated genes. The control of gene expression happens by a variety of posttranscriptional mechanisms. The high degree of synteny among Leishmania species is accompanied by highly conserved coding sequences (CDS) and poorly conserved intercoding untranslated sequences. To identify the elements involved in the control of gene expression, we conducted an in silico investigation to find conserved intercoding sequences (CICS) in the genomes of L major, L infantum, and L braziliensis. We used a combination of computational tools, such as Linux-Shell, PERL and R languages, BLAST, MSPcrunch, SSAKE, and Pred-A-Term algorithms to construct a pipeline which was able to: (i) search for conservation in target-regions, (ii) eliminate CICS redundancy and mask repeat elements, (iii) predict the mRNA's extremities, (iv) analyze the distribution of orthologous genes within the generated LeishCICS-clusters, (v) assign GO terms to the LeishCICS-clusters. and (vi) provide statistical support for the gene-enrichment annotation. We associated the LeishCICS-cluster data, generated at the end of the pipeline, with the expression profile oft. donovani genes during promastigote-amastigote differentiation, as previously evaluated by others (GEO accession: GSE21936). A Pearson's correlation coefficient greater than 0.5 was observed for 730 LeishCICS-clusters containing from 2 to 17 genes. The designed computational pipeline is a useful tool and its application identified potential regulatory cis elements and putative regulons in Leishmania. (C) 2012 Elsevier B.V. All rights reserved.

Air accident investigation among regulatory agencies

The aim of this manuscript is to describe and compare regulatory aviation agencies according to their subordination and attributions to investigate air accidents. Possible consequences in identifying the contributory factors are also presented. Distinct procedures investigating air accidents among agencies lead to a lack of standardization of the statistics, making it difficult to analyze the data globally. Separately the information does not configure the entire scenario of what occurred, affecting the analysis and subsequent interventions. We recommend a joint and collaborative work between the different committees that operate in the investigation of air accidents.

Increased Expression of Regulatory T Cells and Down-Regulatory Molecules in Lepromatous Leprosy

T regulatory cells (Tregs) play an important role in the mechanism of host's failure to control pathogen dissemination in severe forms of different chronic granulomatous diseases, but their role in leprosy has not yet been elucidated; 28 newly diagnosed patients (16 patients with lepromatous leprosy and 12 patients with tuberculoid leprosy) and 6 healthy Mycobacterium leprae-exposed individuals (contacts) were studied. Tregs were quantified by flow cytometry (CD4+ CD25+ Foxp3+) in peripheral blood mononuclear cells stimulated in vitro with a M. leprae antigenic preparation and phytohemagglutinin as well as in skin lesions by immunohistochemistry. The lymphoproliferative (LPR), interleukin-10 (IL-10), and interferon-gamma (IFN-gamma) responses of the in vitro-stimulated peripheral blood mononuclear cells and the in situ expression of IL-10, transforming growth factor-beta (TGF-beta), and cytotoxic T-lymphocyte antigen 4 (CTLA-4) were also determined. We show that M. leprae antigens induced significantly lower LPR but significantly higher Treg numbers in lepromatous than tuberculoid patients and contacts. Mitogen-induced LPR and Treg frequencies were not significantly different among the three groups. Tregs were also more frequent in situ in lepromatous patients, and this finding was paralleled by increased expression of the antiinflammatory molecules IL-10 and CTLA-4 but not TGF-beta. In lepromatous patients, Tregs were intermingled with vacuolized hystiocyte infiltrates all over the lesion, whereas in tuberculoid patients, Tregs were rare. Our results suggest that Tregs are present in increased numbers, and they may have a pathogenic role in leprosy patients harboring uncontrolled bacillary multiplication but not in those individuals capable of limiting M. leprae growth.

Identifying regulational alterations in gene regulatory networks by state space representation of vector autoregressive models and variational annealing

Background: In the analysis of effects by cell treatment such as drug dosing, identifying changes on gene network structures between normal and treated cells is a key task. A possible way for identifying the changes is to compare structures of networks estimated from data on normal and treated cells separately. However, this approach usually fails to estimate accurate gene networks due to the limited length of time series data and measurement noise. Thus, approaches that identify changes on regulations by using time series data on both conditions in an efficient manner are demanded. Methods: We propose a new statistical approach that is based on the state space representation of the vector autoregressive model and estimates gene networks on two different conditions in order to identify changes on regulations between the conditions. In the mathematical model of our approach, hidden binary variables are newly introduced to indicate the presence of regulations on each condition. The use of the hidden binary variables enables an efficient data usage; data on both conditions are used for commonly existing regulations, while for condition specific regulations corresponding data are only applied. Also, the similarity of networks on two conditions is automatically considered from the design of the potential function for the hidden binary variables. For the estimation of the hidden binary variables, we derive a new variational annealing method that searches the configuration of the binary variables maximizing the marginal likelihood. Results: For the performance evaluation, we use time series data from two topologically similar synthetic networks, and confirm that our proposed approach estimates commonly existing regulations as well as changes on regulations with higher coverage and precision than other existing approaches in almost all the experimental settings. For a real data application, our proposed approach is applied to time series data from normal Human lung cells and Human lung cells treated by stimulating EGF-receptors and dosing an anticancer drug termed Gefitinib. In the treated lung cells, a cancer cell condition is simulated by the stimulation of EGF-receptors, but the effect would be counteracted due to the selective inhibition of EGF-receptors by Gefitinib. However, gene expression profiles are actually different between the conditions, and the genes related to the identified changes are considered as possible off-targets of Gefitinib. Conclusions: From the synthetically generated time series data, our proposed approach can identify changes on regulations more accurately than existing methods. By applying the proposed approach to the time series data on normal and treated Human lung cells, candidates of off-target genes of Gefitinib are found. According to the published clinical information, one of the genes can be related to a factor of interstitial pneumonia, which is known as a side effect of Gefitinib.

Chronic cold stress in mice induces a regulatory phenotype in macrophages: Correlation with increased 11 beta-hydroxysteroid dehydrogenase expression

Susceptibility to infections, autoimmune disorders and tumor progression is strongly influenced by the activity of the endocrine and nervous systems in response to a stressful stimulus. When the adaptive system is switched on and off efficiently, the body is able to recover from the stress imposed. However, when the system is activated repeatedly or the activity is sustained, as during chronic or excessive stress, an allostatic load is generated, which can lead to disease over long periods of time. We investigated the effects of chronic cold stress in BALB/c mice (4 degrees C/4 h daily for 7 days) on functions of macrophages. We found that chronic cold stress induced a regulatory phenotype in macrophages, characterized by diminished phagocytic ability, decreased TNF-alpha and IL-6 and increased IL-10 production. In addition, resting macrophages from mice exposed to cold stress stimulated spleen cells to produce regulatory cytokines, and an immunosuppressive state that impaired stressed mice to control Trypanosoma cruzi proliferation. These regulatory effects correlated with an increase in macrophage expression of 11 beta-hydroxysteroid dehydrogenase, an enzyme that converts inactive glucocorticoid into its active form. As stress is a common aspect of modern life and plays a role in the etiology of many diseases, the results of this study are important for improving knowledge regarding the neuro-immune-endocrine interactions that occur during stress and to highlight the role of macrophages in the immunosuppression induced by chronic stress. (C) 2011 Elsevier Inc. All rights reserved.

Exploring the Role of Soluble Factors Associated with Immune Regulatory Properties of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are characterized as multipotent stromal cells with the capacity for both self-renewal and differentiation into mesodermal cell lineages. MSCs also have a fibroblast-like phenotype and can be isolated from several tissues. In recent years, researchers have found that MSCs secrete several soluble factors that exert immunosuppressive effects by modulating both innate (macrophages, dendritic and NK cells) and adaptive (B cells and CD4+ and CD8+ T cells) immune responses. This review summarizes the principal trophic factors that are related to immune regulation and secreted by MSCs under both autoimmune and inflammatory conditions. The understanding of mechanisms that regulate immunity in MSCs field is important for their future use as a novel cellular-based immunotherapy with clinical applications in several diseases.

The SUCEST-FUN regulatory network database: Designing an energy grass

Modern sugarcane cultivars are complex hybrids resulting from crosses among several Saccharum species. Traditional breeding methods have been employed extensively in different countries over the past decades to develop varieties with increased sucrose yield and resistance to pests and diseases. Conventional variety improvement, however, may be limited by the narrow pool of suitable genes. Thus, molecular genetics is seen as a promising tool to assist in the process of developing improved varieties. The SUCEST-FUN Project (http://sucest-fun.org) aims to associate function with sugarcane genes using a variety of tools, in particular those that enable the study of the sugarcane transcriptome. An extensive analysis has been conducted to characterise, phenotypically, sugarcane genotypes with regard to their sucrose content, biomass and drought responses. Through the analysis of different cultivars, genes associated with sucrose content, yield, lignin and drought have been identified. Currently, tools are being developed to determine signalling and regulatory networks in grasses, and to sequence the sugarcane genome, as well as to identify sugarcane promoters. This is being implemented through the SUCEST-FUN (http://sucest-fun.org) and GRASSIUS databases (http://grassius.org), the cloning of sugarcane promoters, the identification of cis-regulatory elements (CRE) using Chromatin Immunoprecipitation-sequencing (ChIP-Seq) and the generation of a comprehensive Signal Transduction and Transcription gene catalogue (SUCAST Catalogue).

Relationship between B-Cell-specific moloney murine leukemia virus integration site 1 (BMI-1) and homologous recombination regulatory genes in invasive ductal breast carcinomas

B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) is a Polycomb group protein that is able to induce telomerase activity, enabling the immortalization of epithelial cells. Immortalized cells are more susceptible to double-strand breaks (DSB), which are subsequently repaired by homologous recombination (HR). BRCA1 is among the HR regulatory genes involved in the response to DNA damage associated with the RAD51 protein, which accumulates in DNA damage foci after signaling H2AX, another important marker of DNA damage. Topoisomerase III beta (topoIII beta) removes HR intermediates before chromosomal segregation, preventing damage to cellular DNA structure. In breast carcinomas positive for BMI-1 the role of proteins involved in HR remains to be investigated. The aim of this study was to evaluate the association between BMI-1 and homologous recombination proteins. Using tissue microarrays containing 239 cases of primary breast tumors, the expression of Bmi-1, BRCA-1, H2AX, Rad51, p53, Ki-67, topoIII beta, estrogen receptors (ER), progesterone receptors (PR), and HER-2 was analyzed by immunohistochemistry. We observed high Bmi-1 expression in 66 cases (27.6%). Immunohistochemical overexpression of BMI-1 was related to ER (p=0.004), PR (p<0.001), Ki-67 (p<0.001), p53 (p=0.003), BRCA1 (p=0.003), H2AX (p=0.024) and topoIII beta (p<0,001). Our results show a relationship between the expression of BMI-1 and HR regulatory genes, suggesting that Bmi-1 overexpression might be an important event in HR regulation. However, further studies are necessary to understand the mechanisms in which Bmi-1 could regulate HR pathways in invasive ductal breast carcinomas.

Effects of aerobic exercise on chronic allergic airway inflammation and remodeling in guinea pigs

We evaluated the effects of aerobic exercise (AE) on airway inflammation, exhaled nitric oxide levels (ENO), airway remodeling, and the expression of Thl, Th2 and regulatory cytokines in a guinea pig asthma model. Animals were divided into 4 groups: non-trained and non-sensitized (C), non-sensitized and AE (AE), ovalbumin-sensitized and non-trained (OVA), and OVA-sensitized and AE (OVA + AE). OVA inhalation was performed for 8 weeks, and AE was conducted for 6 weeks beginning in the 3rd week of OVA sensitization. Compared to the other groups, the OVA + AE group had a reduced density of eosinophils and lymphocytes, reduced expression of interleukin (IL)-4 and IL-13 and an increase in epithelium thickness (p < 0.05). AE did not modify airway remodeling or ENO in the sensitized groups (p > 0.05). Neither OVA nor AE resulted in differences in the expression of IL-2, IFN-gamma, IL-10 or IL1-ra. Our results show that AE reduces the expression of Th2 cytokines and allergic airway inflammation and induces epithelium remodeling in sensitized guinea pigs. (c) 2012 Elsevier B.V. All rights reserved.

DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes

Recurrent chromosomal translocations underlie both haematopoietic and solid tumours. Their origin has been ascribed to selection of random rearrangements, targeted DNA damage, or frequent nuclear interactions between translocation partners; however, the relative contribution of each of these elements has not been measured directly or on a large scale. Here we examine the role of nuclear architecture and frequency of DNA damage in the genesis of chromosomal translocations by measuring these parameters simultaneously in cultured mouse B lymphocytes. In the absence of recurrent DNA damage, translocations between Igh or Myc and all other genes are directly related to their contact frequency. Conversely, translocations associated with recurrent site-directed DNA damage are proportional to the rate of DNA break formation, as measured by replication protein A accumulation at the site of damage. Thus, non-targeted rearrangements reflect nuclear organization whereas DNA break formation governs the location and frequency of recurrent translocations, including those driving B-cell malignancies.

Regulatory T Cells Accumulate in the Lung Allergic Inflammation and Efficiently Suppress T-Cell Proliferation but Not Th2 Cytokine Production

Foxp3(+)CD25(+)CD4(+) regulatory T cells are vital for peripheral tolerance and control of tissue inflammation. In this study, we characterized the phenotype and monitored the migration and activity of regulatory T cells present in the airways of allergic or tolerant mice after allergen challenge. To induce lung allergic inflammation, mice were sensitized twice with ovalbumin/aluminum hydroxide gel and challenged twice with intranasal ovalbumin. Tolerance was induced by oral administration of ovalbumin for 5 consecutive days prior to OVA sensitization and challenge. We detected regulatory T cells (Foxp3(+)CD25(+)CD4(+) T cells) in the airways of allergic and tolerant mice; however, the number of regulatory T cells was more than 40-fold higher in allergic mice than in tolerant mice. Lung regulatory T cells expressed an effector/memory phenotype (CCR4(high)CD62L(low)CD44(high)CD54(high)CD69(+)) that distinguished them from naive regulatory T cells (CCR4(int)CD62L(high)CD44(int)CD54(int)CD69(-)). These regulatory T cells efficiently suppressed pulmonary T-cell proliferation but not Th2 cytokine production.