Fibrocytes are increased in lung and peripheral blood of patients with idiopathic pulmonary fibrosis

Background: Fibrocytes are implicated in Idiopathic Pulmonary Fibrosis (IPF) pathogenesis and increased proportions in the circulation are associated with poor prognosis. Upon tissue injury, fibrocytes migrate to the affected organ. In IPF patients, circulating fibrocytes are increased especially during exacerbations, however fibrocytes in the lungs have not been examined. Therefore, we sought to evaluate if fibrocytes can be detected in IPF lungs and we compare percentages and phenotypic characteristics of lung fibrocytes with circulating fibrocytes in IPF. Methods: First we optimized flow cytometric detection circulating fibrocytes using a unique combination of intra- and extra-cellular markers to establish a solid gating strategy. Next we analyzed lung fibrocytes in single cell suspensions of explanted IPF and control lungs and compared characteristics and numbers with circulating fibrocytes of IPF. Results: Using a gating strategy for both circulating and lung fibrocytes, which excludes potentially contaminating cell populations (e.g. neutrophils and different leukocyte subsets), we show that patients with IPF have increased proportions of fibrocytes, not only in the circulation, but also in explanted end-stage IPF lungs. These lung fibrocytes have increased surface expression of HLA-DR, increased intracellular collagen-1 expression, and also altered forward and side scatter characteristics compared with their circulating counterparts. Conclusions: These findings demonstrate that lung fibrocytes in IPF patients can be quantified and characterized by flow cytometry. Lung fibrocytes have different characteristics than circulating fibrocytes and represent an intermediate cell population between circulating fibrocytes and lung fibroblast. Therefore, more insight in their phenotype might lead to specific therapeutic targeting in fibrotic lung diseases

Enhanced Bruton's tyrosine kinase in B-cells and autoreactive IgA in patients with idiopathic pulmonary fibrosis

RATIONALE: Idiopathic Pulmonary Fibrosis (IPF) is thought to be triggered by repeated alveolar epithelial cell injury. Current evidence suggests that aberrant immune activation may contribute. However, the role of B-cell activation remains unclear. We determined the phenotype and activation status of B-cell subsets and evaluated the contribution of activated B-cells to the development of lung fibrosis both in humans and in mice. METHODS: B-cells in blood, mediastinal lymph node, and lung single-cell suspensions of IPF patients and healthy controls (HC) were characterized using 14-color flow cytometry. Mice were exposed to bleomycin to provoke pulmonary fibrosis. RESULTS: More IgA+ memory B-cells and plasmablasts were found in blood (n = 27) and lungs (n = 11) of IPF patients compared to HC (n = 21) and control lungs (n = 9). IPF patients had higher levels of autoreactive IgA in plasma, which correlated with an enhanced decline of forced vital capacity (p = 0.002, r = - 0.50). Bruton's tyrosine kinase expression was higher in circulating IPF B-cells compared to HC, indicating enhanced B-cell activation. Bleomycin-exposed mice had increased pulmonary IgA+ germinal center and plasma cell proportions compared to control mice. The degree of lung fibrosis correlated with pulmonary germinal center B-cell proportions (p = 0.010, r = 0.88). CONCLUSION: Our study demonstrates that IPF patients have more circulating activated B-cells and autoreactive IgA, which correlate with disease progression. These B-cell alterations were also observed in the widely used mouse model of experimental pulmonary fibrosis. Autoreactive IgA could be useful as a biomarker for disease progression in IPF

A methodology for exploring biomarker – phenotype associations: application to flow cytometry data and systemic sclerosis clinical manifestations

BACKGROUND: This work seeks to develop a methodology for identifying reliable biomarkers of disease activity, progression and outcome through the identification of significant associations between high-throughput flow cytometry (FC) data and interstitial lung disease (ILD) - a systemic sclerosis (SSc, or scleroderma) clinical phenotype which is the leading cause of morbidity and mortality in SSc. A specific aim of the work involves developing a clinically useful screening tool that could yield accurate assessments of disease state such as the risk or presence of SSc-ILD, the activity of lung involvement and the likelihood to respond to therapeutic intervention. Ultimately this instrument could facilitate a refined stratification of SSc patients into clinically relevant subsets at the time of diagnosis and subsequently during the course of the disease and thus help in preventing bad outcomes from disease progression or unnecessary treatment side effects. The methods utilized in the work involve: (1) clinical and peripheral blood flow cytometry data (Immune Response In Scleroderma, IRIS) from consented patients followed at the Johns Hopkins Scleroderma Center. (2) machine learning (Conditional Random Forests - CRF) coupled with Gene Set Enrichment Analysis (GSEA) to identify subsets of FC variables that are highly effective in classifying ILD patients; and (3) stochastic simulation to design, train and validate ILD risk screening tools. RESULTS: Our hybrid analysis approach (CRF-GSEA) proved successful in predicting SSc patient ILD status with a high degree of success (>82 % correct classification in validation; 79 patients in the training data set, 40 patients in the validation data set). CONCLUSIONS: IRIS flow cytometry data provides useful information in assessing the ILD status of SSc patients. Our new approach combining Conditional Random Forests and Gene Set Enrichment Analysis was successful in identifying a subset of flow cytometry variables to create a screening tool that proved effective in correctly identifying ILD patients in the training and validation data sets. From a somewhat broader perspective, the identification of subsets of flow cytometry variables that exhibit coordinated movement (i.e., multi-variable up or down regulation) may lead to insights into possible effector pathways and thereby improve the state of knowledge of systemic sclerosis pathogenesis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12859-015-0722-x) contains supplementary material, which is available to authorized users

Discrimination for genotoxic and non-genotoxic carcinogens by gene expression profiling in primary mouse hepatocytes improves with exposure time

Assessing the potential carcinogenicity of chemicals for humans represents an ongoing challenge. Chronic rodent bioassays predict human cancer risk at only limited reliability and are simultaneously expensive and long-lasting. In order to seek for alternatives, the ability of a transcriptomics-based primary mouse hepatocyte model to classify carcinogens by their modes of action was evaluated. As it is obvious that exposure will induce a cascade of gene expression modifications, in particular, the influence of exposure time in vitro on discriminating genotoxic (GTX) carcinogens from non-genotoxic (NGTX) carcinogens class discrimination was investigated. Primary mouse hepatocytes from male C57Bl6 mice were treated for 12, 24, 36 and 48 h with two GTX and two NGTX carcinogens. For validation, two additional GTX compounds were studied at 24 and 48 h. Immunostaining of gammaH2AX foci was applied in order to phenotypically verify DNA damage. It confirmed significant induction of DNA damage after treatment with GTX compounds but not with NGTX compounds. Whole genome gene expression modifications were analyzed by means of Affymetrix microarrays. When using differentially expressed genes from datasets normalized by RMA, the two classes and various compounds were better separated from each other by hierarchical clustering when increasing the treatment period. Discrimination of GTX and NGTX carcinogens by Prediction Analysis of Microarray improved with time, and resulted in correct classification of the validation compounds. The present study shows that gene expression profiling in primary mouse hepatocytes is promising for discriminating GTX from NGTX compounds and that this discrimination improves with increasing treatment period

Molecular characterization of trimellitic anhydride-induced respiratory allergy in Brown Norway rats

To contribute to the hazard identification of low molecular weight (LMW) respiratory allergens, respiratory allergy induced by trimellitic anhydride (TMA) was characterized by whole genome analysis of lung tissue and blood proteomics in Brown Norway rats. Dermal sensitization (50% and 25% w/v) with TMA and an inhalation challenge of 15 mg/m(3) TMA-induced apneas, laryngeal inflammation, increased numbers of eosinophils, neutrophils and macrophages in bronchoalveolar lavage (BAL), and increased immunoglobulin E levels in serum and lung tissue. Whole genome analysis of lung, sampled 24 hours after challenge, showed expression changes of not only genes belonging to several Gene Ontology groups with up-regulation of inflammatory-associated genes and those associated with lung remodeling but also genes involved in downsizing these processes. Blood proteomics reflected activation of inflammation-inhibiting pathways. Unsensitized animals challenged with TMA exhibited also an increased number of macrophages in BAL, but gene expression in the above-mentioned gene pathways was unchanged or down-regulated. The authors conclude that parameters for lung remodeling can be a valuable tool in hazard identification of LMW respiratory allergens

Teor de ácido cianídrico em variedades de mandioca cultivadas em quintais do estado de São Paulo Cyanide contents in cassava cultivars used for "in natura" consumption in the state of Sao Paulo, Brazil

No Estado de São Paulo, além das culturas comerciais que destinam sua produção às indústrias de transformação ou aos mercados hortifrutigranjeiros, a mandioca (Manihot esculenta Crantz) é muito difundida em culturas denominadas de "fundo de quintal". Nesse caso, muitas variedades são cultivadas e utilizadas precipuamente para consumo doméstico, tendo o presente trabalho por objetivo avaliar a amplitude de variação do seu teor de ácido cianídrico (HCN). Foram analisadas raízes de 206 variedades originárias de uma coleta sistemática realizada em 126 municípios paulistas, utilizando-se o método de Liebig, com maceração por 24 horas. Os resultados mostraram que a amplitude máxima de variação do ácido cianídrico foi de 16 a 482 mg.kg-1 na polpa crua das raízes. A maioria das variedades (67,0%) apresentou até 100 mg.kg-1 de HCN, que, apesar de alto em relação aos citados na literatura, sugere que possa ser considerado o limite superior de segurança para variedades de mesa.<br>Cassava (Manihot esculenta Crantz) is widely cultivated in the State of Sao Paulo, Brazil, mostly as raw material for industrial purposes (production of cassava flour, starch, etc.). A small proportion of cassava production is destinated to "in natura" consumption, obtained essentially from backyard plantations. In this case, many varieties are used, with unknown cyanide contents, which can cause severe human intoxication. The main aim of this research was to evaluate the cyanide content range of these varieties. Roots of 206 varieties, collected at 126 sites in the State of São Paulo, Brazil, were analyzed as to their cyanide contents, using the Liebig method, with maceration for a 24-hour period. Results showed a cyanide content variation from 16 to 482 mg.kg-1 of HCN in the tuber root fresh pulps. On the other hand, most of the varieties (67%) under testing presented root cyanide contents below 100 mg.kg-1. So, this cyanide content may be considered as the uppermost level to be used with security on the selection of new genetic materials with lower HCN contents