MMF treatment decreases the autoimmune phenotype in <i>gld.apoE−/−</i> mice.

<p><i>A,</i> Spleen from mice, treated with 200 mg/kg/day MMF or untreated, was harvested and weighed (*, <i>p</i><0.05). <i>B,</i> The lymph nodes were also harvested and weighed. <i>C,</i> ANA titer was determined from serum samples using HEp-2-coated slides and scored using the value of the last positive dilution (**, <i>p = </i>0.001). Data are expressed as means ± SEM.</p

Renal disease is ameliorated in <i>gld.apoE−/−</i> mice after MMF treatment.

<p>The mice were treated with MMF or control for 12 wk. <i>A,</i> Representative H&E-stained sections of kidney in both groups. Glomerular tuft size (<i>B</i>) (*, <i>p = </i>0.002) and cell count (<i>C</i>) (**, <i>p = </i>0.004) were measured by computer-assisted pixel counting. Values shown are the mean ± SEM.</p

Decreased atherosclerosis in <i>gld.apoE−/−</i> mice treated with MMF.

<p>Aortic root atherosclerotic lesion area in MMF-treated or control mice. <i>A,</i> Representative photographs of aortic root stained with Oil Red O from mice maintained on Western diet for 12 wk and treated with 200 mg/kg/day MMF or untreated. <i>B,</i> Atherosclerotic lesion area of Oil Red O-stained aortae was quantified in both groups (*, <i>p = </i>0.02). <i>C</i>, Total serum cholesterol was quantified in control mice and mice treated with 200 mg/kg/day MMF.</p

Isolation of neutrophils and characterization of gene expression patterns.

<p><b>A.</b> Neutrophils were isolated from bone marrow (BM) and blood (BL) of untreated mice, from the peritoneal cavity of mice administered thioglycollate (TG) or uric acid (UA) intraperitoneally, and from the synovial fluid (SF) of mice with autoantibody-induced arthritis, on the basis of scatter patterns (which differed among conditions, left panels) and staining for CD11b and Ly6G (right panels). The population in the upper left corner of the TG plot did not express CD11b or Ly6G. <b>B.</b> Comparison of global gene expression patterns in neutrophils (labeled) to all of the other populations in ImmGen, using axes determined by principal components analysis (PCA). Populations in red on the right side of the diagram represent stromal cell populations; other colors represent various lymphoid and myeloid populations. To convert ImmGen nomenclature to the abbreviations used in this paper: Thio.PC = TG; UrAc.PC = UA; Arth.SynF = SF; GN.Bl = BL; GN.BM = BM = bone-marrow neutrophils from normal mice; Arth.BM = bone-marrow neutrophils from arthritic mice, note similarity to GN.BM. <b>C.</b> Expression of genes for components of neutrophil primary granules (top), secondary granules (middle), and 15 genes showing greater expression in neutrophils than non-neutrophils in ImmGen [mean expression among 5 neutrophil populations (BM, BL, SF, UA, and TG) being greater than 4 times the maximum expression among 198 non-neutrophil populations](bottom), during neutrophil development and activation. CMP = common myeloid precursor; GMP = granulocyte/monocyte precursor. Note that expression patterns in the “neutrophil-specific” genes as identified in this study resembled those of secondary but not primary granule components. <b>D.</b> Expression of groups of genes related to translation (per Gene Ontology = GO) in neutrophils (blue) and other leukocytes (red). Each bar represents mean expression among 5 neutrophil or 198 non-neutrophil populations, and error bars show standard errors.</p

Regulatory genes implicated in neutrophil activation, with further focus on IRF family members.

<p><b>A.</b> Genes were placed into 25 clusters (1–128 genes each, shown as the column headings; A and B are used to identify clusters that have the same numbers of genes) based on patterns of expression in individual samples of neutrophils from blood, SF, UA, and TG, as shown in the heatmap at the top. Clusters that clearly represented up-regulated (U) or down-regulated (D) genes (relative to blood) were pooled and were used to generate a list of predicted regulatory genes (rows) showing enrichment based on the ImmGen regulatory model. Association of each of the 64 regulators with each of the 25 gene clusters was then quantified (P-value of chi-square test), and this matrix of P-values was subjected to hierarchical clustering in order to identify related regulators (rows) and related gene clusters (columns). The lower heatmap indicates these P-values (darker = lower), and the dendrogram and colored bars on the right show groups of regulators with similar patterns of association with various gene clusters. The presence of patterns in the top heatmap (e.g., clustering of clusters characterized by up-regulation in TG, SF, or UA, or by down-regulation in SF), which shows normalized average expression in the 4 neutrophil populations in each cluster, validates this method. The group of regulators shown in light blue was associated with gene clusters indicated in bold; inspection of genes in these clusters led to implication of the type 1 interferon pathway and Irf9. <b>B.</b> Up-regulation of genes induced by type 1 interferons via Irf9, in TG and/or UA but not SF neutrophils. The heatmap shows mean expression in blood (BL), SF, UA, and TG neutrophils. Mean expression across all four conditions was placed at the center of the gradient (white) for each gene. The full color gradient for each gene represents an 8-fold difference in expression. The list of genes of interest and the pathway diagram were generated using the KEGG and Ingenuity databases. Only genes showing at least 1.5-fold differences in expression comparing conditions are shown in the heatmap. In the pathway diagram, genes showing statistically significant (Q<0.05 by ANOVA) differences that varied 2-fold in at least one pairwise comparison of conditions are shown in red, and genes showing fold differences of 1.5–2 and/or not meeting statistical significance are shown in pink. <b>C.</b><i>Irf5</i> is required for production of several cytokines and chemokines by mouse neutrophils stimulated in vitro with the TLR9 ligand CpG-B, but not for production induced by the TLR2 ligand Pam3Cys nor the TLR4 ligand LPS. The panels show the mean ± SEM of 3 independent experiments using FACS-sorted Gr1^hiCD11b⁺F4/80⁻ neutrophils. Since secretion varied between experiments but reliably did so in parallel for the different analytes, data were analyzed by determining the fold difference between <i>Irf5−/−</i> and WT in each experiment and applying one-sample T-tests to the fold-differences for the 3 experiments. P values for cells treated with CpG were 0.014 for TNF and <0.01 for the other proteins, and 0.13–0.97 for other TLR ligands.</p

Total food intake and average body weights.

<p><i>A</i>, Food was weighed weekly and total amount ingested was recorded for each group (control, n = 8; MMF, n = 9). <i>B,</i> Evolution of body weight as weighed weekly during the course of the experiment.</p

Management and outcomes of 27 pregnancies in women with myeloproliferative neoplasms

<p><b>Introduction:</b> Philadelphia-negative myeloproliferative neoplasms (MPNs) greatly increase the risk of maternal and fetal complications during pregnancy. Currently, international agreements regarding the management of these women are lacking.</p> <p><b>Patients and methods:</b> Our study aimed to assess the current management and outcomes of MPN pregnancies in a French cohort. We retrospectively analyzed 27 pregnancies in women with MPNs that were associated with a specific mutation. Nineteen pregnancies in nine women with essential thrombocythemia and eight pregnancies in five women with polycythemia vera were identified.</p> <p><b>Results:</b> Our study showed 70% live births, but only 30% uneventful pregnancies. Fetal complications were mainly early spontaneous abortions (22%), fetal growth restriction (15%), and premature delivery (15%). Maternal issues were divided between thrombosis (15%) and hemorrhages (11%). High rates of preeclampsia and hemolysis, elevated liver enzymes, and low platelet count syndrome (15%) were reported. Uterine artery Doppler was performed in 70% pregnancies. Abnormal Doppler results were found in 43% pregnancies. Pregnancies with high platelet counts and packed cell volume remaining static or increasing ended with fetal death and utero-placental dysfunction. According to expert consensus, most of the pregnancies (67%) could be stratified in the high risk group and had a bad obstetrical outcome, with 50% standard-risk pregnancies versus 22% high-risk pregnancies that were uneventful. Higher risk pregnancies were prescribed heparin and/or interferon α in 72%.</p> <p><b>Conclusions:</b> The prognosis of these pregnancies remains very bad and may be improved by a more effective collaboration between specialists as well as a therapeutic intensification including heparin and interferon α.</p

Biological processes showing up-regulation or down-regulation of genes in activated neutrophils.

<p>(<b>A–H</b>). Heat maps show mean expression in neutrophils from blood (BL), synovial fluid (SF), or peritonitis induced by uric acid (UA) or thioglycollate (TG). Mean expression across all four conditions was placed at the center of the gradient (white) for each gene. Red indicates increased expression, and blue indicates decreased expression. The full color gradient for each gene represents an 8-fold difference in expression. Lists of genes of interest were compiled using the KEGG and Ingenuity databases as well as literature reviews; only genes showing at least 2-fold differences in expression comparing conditions and with Q<0.05 by ANOVA are shown. In the pathway diagrams, up-regulated genes are shown in red, and down-regulated genes are shown in green. <b>A.</b> Uptake and metabolism of lipoproteins. <b>B.</b> Nr4a-family transcription factors. <b>C.</b> Glutathione metabolism. <b>D.</b> Synthesis of and response to leukotrienes and prostaglandins. <b>E.</b> Antigen processing and presentation via MHC class II. <b>F.</b> Genes related to apoptosis. <b>G.</b> NFκB subunits and proximal regulators of NFκB. <b>H.</b> Genes related to signaling by innate immune receptors for microbial products. <b>I.</b> Expression of H3 histone genes (<i>Hist1h3a, b, c, d, e, g, h, I,</i> and <i>Hist2h3b</i> and <i>3c1</i>) in neutrophil populations. Mean ± SD of these 10 genes (black) declined after release from bone marrow (BM) to blood (BL) and further after activation (SF, UA, TG). Mean ± SD among 198 non-neutrophil populations is shown for comparison. Although it is not apparent from this plot, the lowest expression among non-neutrophils exceeded the highest expression in UA or TG neutrophils. Expression of genes for the “replacement” H3 histones, shown in red and blue, was maintained after neutrophil maturation and activation, at levels similar to non-neutrophils.</p

Genes with expression most specific to neutrophils in the ImmGen database.

<p>Numbers indicate gene expression levels. BM = bone-marrow neutrophils; BL = blood neutrophils; SF = synovial fluid neutrophils; UA = uric acid-induced peritoneal neutrophils; TG = thioglycollate-induced peritoneal neutrophils; NF = neutrophils; Non-NF = all non-neutrophil leukocyte populations profiled in ImmGen.</p><p>*The number of non-neutrophils populations (of 198 total) in which expression was greater than 120.</p>†<p>Published expression in eosinophils using the same microarray platform <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108553#pone.0108553-Wen1" target="_blank">[40]</a>.</p>‡<p>Included in a published gene-expression signature for neutrophils <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108553#pone.0108553-Hume1" target="_blank">[47]</a>.</p><p>Genes with expression most specific to neutrophils in the ImmGen database.</p

Additional file 1: of Predictive biological markers of systemic lupus erythematosus flares: a systematic literature review

Presenting search strategies. (DOC 201 kb