Interactions of cancer cells with the primary tumor microenvironment are important determinants of cancer progression toward metastasis but it is unknown whether additional prometastatic signals are provided during the intravascular transit to the site of metastasis. Here, we show that platelet-tumor cell interactions are sufficient to prime tumor cells for subsequent metastasis. Platelet-derived TGFβ and direct platelet-tumor cell contacts synergistically activate the TGFβ/Smad and NF-κB pathways in cancer cells, resulting in their transition to an invasive mesenchymal-like phenotype and enhanced metastasis in vivo. Inhibition of NF-κB signaling in cancer cells or ablation of TGFβ1 expression solely in platelets protects against lung metastasis in vivo. Thus, cancer cells rely on platelet-derived signals outside of the primary tumor for efficient metastasis.

Bone remodeling depends on the precise coordination of bone resorption and subsequent bone formation. Disturbances of this process are associated with skeletal diseases, such as Camurati-Engelmann disease (CED). We show using in vitro and in vivo models that active TGF-beta1 released during bone resorption coordinates bone formation by inducing migration of bone marrow stromal cells, also known as bone mesenchymal stem cells, to the bone resorptive sites and that this process is mediated through a SMAD signaling pathway. Analyzing mice carrying a CED-derived mutant TGFB1 (encoding TGF-beta1), which show the typical progressive diaphyseal dysplasia seen in the human disease, we found high levels of active TGF-beta1 in the bone marrow. Treatment with a TGF-beta type I receptor inhibitor partially rescued the uncoupled bone remodeling and prevented the fractures. Thus, as TGF-beta1 functions to couple bone resorption and formation, modulation of TGF-beta1 activity could be an effective treatment for bone remodeling diseases.

Interleukin (IL)-13 is a major inducer of fibrosis in many chronic infectious and autoimmune diseases. In studies of the mechanisms underlying such induction, we found that IL-13 induces transforming growth factor (TGF)-beta(1) in macrophages through a two-stage process involving, first, the induction of a receptor formerly considered to function only as a decoy receptor, IL-13Ralpha(2). Such induction requires IL-13 (or IL-4) and tumor necrosis factor (TNF)-alpha. Second, it involves IL-13 signaling through IL-13Ralpha(2) to activate an AP-1 variant containing c-jun and Fra-2, which then activates the TGFB1 promoter. In vivo, we found that prevention of IL-13Ralpha(2) expression reduced production of TGF-beta(1) in oxazolone-induced colitis and that prevention of IL-13Ralpha(2) expression, Il13ra2 gene silencing or blockade of IL-13Ralpha(2) signaling led to marked downregulation of TGF-beta(1) production and collagen deposition in bleomycin-induced lung fibrosis. These data suggest that IL-13Ralpha(2) signaling during prolonged inflammation is an important therapeutic target for the prevention of TGF-beta(1)-mediated fibrosis.

TGF-beta1 is a regulatory cytokine with a pleiotropic role in immune responses. TGF-beta1 is widely expressed in leukocytes and stromal cells. However, the functions of TGF-beta1 expressed by specific lineages of cells remain unknown in vivo. Here, we show that mice with a T cell-specific deletion of the Tgfb1 gene developed lethal immunopathology in multiple organs, and this development was associated with enhanced T cell proliferation, activation, and CD4+ T cell differentiation into T helper 1 (Th1) and Th2 cells. TGF-beta1 produced by Foxp3-expressing regulatory T cells was required to inhibit Th1-cell differentiation and inflammatory-bowel disease in a transfer model. In addition, T cell-produced TGF-beta1 promoted Th17-cell differentiation and was indispensable for the induction of experimental autoimmune encephalomyelitis. These findings reveal essential roles for T cell-produced TGF-beta1 in controlling differentiation of T helper cells and controlling inflammatory diseases.

The Breast Cancer Association Consortium (BCAC) has been established to conduct combined case-control analyses with augmented statistical power to try to confirm putative genetic associations with breast cancer. We genotyped nine SNPs for which there was some prior evidence of an association with breast cancer: CASP8 D302H (rs1045485), IGFBP3 -202 C ->> A (rs2854744), SOD2 V16A (rs1799725), TGFB1 L10P (rs1982073), ATM S49C (rs1800054), ADH1B 3' UTR A ->> G (rs1042026), CDKN1A S31R (rs1801270), ICAM5 V301I (rs1056538) and NUMA1 A794G (rs3750913). We included data from 9-15 studies, comprising 11,391-18,290 cases and 14,753-22,670 controls. We found evidence of an association with breast cancer for CASP8 D302H (with odds ratios (OR) of 0.89 (95% confidence interval (c.i.): 0.85-0.94) and 0.74 (95% c.i.: 0.62-0.87) for heterozygotes and rare homozygotes, respectively, compared with common homozygotes; P(trend) = 1.1 x 10(-7)) and weaker evidence for TGFB1 L10P (OR = 1.07 (95% c.i.: 1.02-1.13) and 1.16 (95% c.i.: 1.08-1.25), respectively; P(trend) = 2.8 x 10(-5)). These results demonstrate that common breast cancer susceptibility alleles with small effects on risk can be identified, given sufficiently powerful studies.

The cytokines controlling the development of human interleukin (IL) 17--producing T helper cells in vitro have been difficult to identify. We addressed the question of the development of human IL-17--producing T helper cells in vivo by quantifying the production and secretion of IL-17 by fresh T cells ex vivo, and by T cell blasts expanded in vitro from patients with particular genetic traits affecting transforming growth factor (TGF) beta, IL-1, IL-6, or IL-23 responses. Activating mutations in TGFB1, TGFBR1, and TGFBR2 (Camurati-Engelmann disease and Marfan-like syndromes) and loss-of-function mutations in IRAK4 and MYD88 (Mendelian predisposition to pyogenic bacterial infections) had no detectable impact. In contrast, dominant-negative mutations in STAT3 (autosomal-dominant hyperimmunoglobulin E syndrome) and, to a lesser extent, null mutations in IL12B and IL12RB1 (Mendelian susceptibility to mycobacterial diseases) impaired the development of IL-17--producing T cells. These data suggest that IL-12Rbeta1- and STAT-3--dependent signals play a key role in the differentiation and/or expansion of human IL-17-producing T cell populations in vivo.

The concentration of transforming growth factor beta (TGF-beta) in plasma has been correlated with the development of several diseases, including atherosclerosis and certain forms of cancer. However, the mechanisms that control the concentration of TGF-beta in plasma are poorly understood. In a study of 170 pairs of female twins (average age 57.7 years) we show that the concentration of active plus acid-activatable latent TGF-beta1 [(a+l) TGF-beta therefore is predominantly under genetic control (heritability estimate 0.54). Single strand conformation polymorphism (SSCP) mapping of the TGF-beta1 gene promoter has identified two single base substitution polymorphisms. The two polymorphisms (G->>A at position -800 bp and C->>T at position -509 bp) are in linkage disequilibrium (correlation coefficient Delta = 0.215, P < 0.01). The C-509T polymorphism is significantly associated with the plasma concentration of (a+l) TGF-beta1, explaining 8.2% of the additive genetic variance of (a+l) TGF-beta1 concentration. It is therefore possible that predisposition to atherosclerosis, bone diseases or various forms of cancer may be correlated with the presence of particular alleles at the TGFB1 locus.

TGF-beta signaling is mediated through two types of serine/threonin kinase-containing receptors, type I (TGF-betaRI) and type II (TGF-betaRII), which form a heteromeric complex. In this signaling complex, ligand binding TGF-betaRII phosphorylates and thereby activates the TGF-betaRI to signal downstream pathways. To determine the role of TGF-betaRII in embryogenesis, we have generated a TGF-betaRII gene (Tgfbr2) knockout mouse line. The heterozygous Tgfbr2 knockout mice are developmentally normal. The homozygous Tgfbr2 mutation causes defects in the yolk sac hematopoiesis and vasculogenesis, resulting in an embryonic lethality around 10.5 days of gestation. This phenotype is indistinguishable from the previously reported embryonic lethality by the homozygous TGF-beta1 gene (Tgfb1) null mutation. In addition, we have generated chimeric mice using a Tgfbr2 (-/-) embryonic stem cell line. Some chimeric mice showed several types of congenital anomalies, suggesting that TGF-beta II is important for normal development in a variety of organs.

Mesenchymal stem cells (MSCs) have been shown to secrete exosomes that are cardioprotective. Here, we demonstrated that MSC exosome, a secreted membrane vesicle, is immunologically active. MSC exosomes induced polymyxin-resistant, MYD88-dependent secreted embryonic alkaline phosphatase (SEAP) expression in a THP1-Xblue, a THP-1 reporter cell line with an NFκB-SEAP reporter gene. In contrast to lipopolysaccharide, they induced high levels of anti-inflammatory IL10 and TGFβ1 transcript at 3 and 72 h, and much attenuated levels of pro-inflammatory IL1B, IL6, TNFA and IL12P40 transcript at 3-h. The 3-h but not 72-h induction of cytokine transcript was abrogated by MyD88 deficiency. Primary human and mouse monocytes exhibited a similar exosome-induced cytokine transcript profile. Exosome-treated THP-1 but not MyD88-deficient THP-1 cells polarized activated CD4(+) T cells to CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) at a ratio of one exosome-treated THP-1 cell to 1,000 CD4(+) T cells. Infusion of MSC exosomes enhanced the survival of allogenic skin graft in mice and increased Tregs.

Liver fibrosis is a reversible wound-healing response involving TGFβ1/SMAD activation of hepatic stellate cells (HSCs). It results from excessive deposition of extracellular matrix components and can lead to impairment of liver function. Here, we show that vitamin D receptor (VDR) ligands inhibit HSC activation by TGFβ1 and abrogate liver fibrosis, whereas Vdr knockout mice spontaneously develop hepatic fibrosis. Mechanistically, we show that TGFβ1 signaling causes a redistribution of genome-wide VDR-binding sites (VDR cistrome) in HSCs and facilitates VDR binding at SMAD3 profibrotic target genes via TGFβ1-dependent chromatin remodeling. In the presence of VDR ligands, VDR binding to the coregulated genes reduces SMAD3 occupancy at these sites, inhibiting fibrosis. These results reveal an intersecting VDR/SMAD genomic circuit that regulates hepatic fibrogenesis and define a role for VDR as an endocrine checkpoint to modulate the wound-healing response in liver. Furthermore, the findings suggest VDR ligands as a potential therapy for liver fibrosis.

Monocytes are a heterogeneous cell population with subset-specific functions and phenotypes. The differential expression of CD14 and CD16 distinguishes classical CD14(++)CD16(-), intermediate CD14(++)CD16(+), and nonclassical CD14(+)CD16(++) monocytes. Current knowledge on human monocyte heterogeneity is still incomplete: while it is increasingly acknowledged that CD14(++)CD16(+) monocytes are of outstanding significance in 2 global health issues, namely HIV-1 infection and atherosclerosis, CD14(++)CD16(+) monocytes remain the most poorly characterized subset so far. We therefore developed a method to purify the 3 monocyte subsets from human blood and analyzed their transcriptomes using SuperSAGE in combination with high-throughput sequencing. Analysis of 5 487 603 tags revealed unique identifiers of CD14(++)CD16(+) monocytes, delineating these cells from the 2 other monocyte subsets. Gene Ontology (GO) enrichment analysis suggests diverse immunologic functions, linking CD14(++)CD16(+) monocytes to Ag processing and presentation (eg, CD74, HLA-DR, IFI30, CTSB), to inflammation and monocyte activation (eg, TGFB1, AIF1, PTPN6), and to angiogenesis (eg, TIE2, CD105). In conclusion, we provide genetic evidence for a distinct role of CD14(++)CD16(+) monocytes in human immunity. After CD14(++)CD16(+) monocytes have earlier been discussed as a potential therapeutic target in inflammatory diseases, we are hopeful that our data will spur further research in the field of monocyte heterogeneity.

Single-nucleotide polymorphisms (SNP) associated with polygenetic disorders, such as breast cancer (BC), can create, destroy, or modify microRNA (miRNA) binding sites; however, the extent to which SNPs interfere with miRNA gene regulation and affect cancer susceptibility remains largely unknown. We hypothesize that disruption of miRNA target binding by SNPs is a widespread mechanism relevant to cancer susceptibility. To test this, we analyzed SNPs known to be associated with BC risk, in silico and in vitro, for their ability to modify miRNA binding sites and miRNA gene regulation and referred to these as target SNPs. We identified rs1982073-TGFB1 and rs1799782-XRCC1 as target SNPs, whose alleles could modulate gene expression by differential interaction with miR-187 and miR-138, respectively. Genome-wide bioinformatics analysis predicted approximately 64% of transcribed SNPs as target SNPs that can modify (increase/decrease) the binding energy of putative miRNA::mRNA duplexes by >90%. To assess whether target SNPs are implicated in BC susceptibility, we conducted a case-control population study and observed that germline occurrence of rs799917-BRCA1 and rs334348-TGFR1 significantly varies among populations with different risks of developing BC. Luciferase activity of target SNPs, allelic variants, and protein levels in cancer cell lines with different genotypes showed differential regulation of target genes following overexpression of the two interacting miRNAs (miR-638 and miR-628-5p). Therefore, we propose that transcribed target SNPs alter miRNA gene regulation and, consequently, protein expression, contributing to the likelihood of cancer susceptibility, by a novel mechanism of subtle gene regulation.

Deregulation of normal regenerative responses to physical, chemical and biological toxins in susceptible individuals leads to abnormal remodelling of extracellular matrix with pathological fibrosis. Processes deregulated after radiotherapy have much in common with processes associated with fibrotic diseases affecting the heart, skin, lungs, kidneys, gastro-intestinal tract and liver. Among the secreted factors driving fibrosis, transforming growth factor beta 1 (TGFβ1) produced by a wide range of inflammatory, mesenchymal and epithelial cells converts fibroblasts and other cell types into matrix-producing myofibroblasts. Even if required for the initiation of fibrosis, inflammation and the continued stimulus of TGFβ1 may not be needed to maintain it. After myofibroblast activation, collagen production can be perpetuated independently of TGFβ1 by autocrine induction of a cytokine called connective tissue growth factor. The role of inflammation, the origins and activation of myofibroblasts as biosynthetic cells and the downstream pathways of extracellular matrix synthesis in common fibrotic states are reviewed. Oxidative stress, hypoxia and microvascular damage are also considered, before examining the same processes in the context of radiotherapy. One of the main uncertainties is the relevance of very early events, including inflammatory responses in blood vessels, to fibrosis. Despite the power of animal models, including genetic systems, the potential contribution of research based on human tissue samples has never been greater. A closer interaction between scientists researching fibrosis and radiation oncologists holds enormous promise for therapeutic advances.

The multifunctional cytokine transforming growth factor (TGF) beta1 is secreted in a latent complex with its processed propeptide (latency-associated peptide [LAP]). TGFbeta1 must be functionally released from this complex before it can engage TGFbeta receptors. One mechanism of latent TGFbeta1 activation involves interaction of the integrins alpha v beta6 and alpha v beta8 with an RGD sequence in LAP; other putative latent TGFbeta1 activators include thrombospondin-1, oxidants, and various proteases. To assess the contribution of RGD-binding integrins to TGFbeta1 activation in vivo, we created a mutation in Tgfb1 encoding a nonfunctional variant of the RGD sequence (RGE). Mice with this mutation (Tgfb1(RGE/RGE)) display the major features of Tgfb1(-/-) mice (vasculogenesis defects, multiorgan inflammation, and lack of Langerhans cells) despite production of normal levels of latent TGFbeta1. These findings indicate that RGD-binding integrins are requisite latent TGFbeta1 activators during development and in the immune system.

Angiotensin II (Ang II), a potent hypertrophic stimulus, causes significant increases in TGFb1 gene expression. However, it is not known whether there is a causal relationship between increased levels of TGF-beta1 and cardiac hypertrophy. Echocardiographic analysis revealed that TGF-beta1-deficient mice subjected to chronic subpressor doses of Ang II had no significant change in left ventricular (LV) mass and percent fractional shortening during Ang II treatment. In contrast, Ang II-treated wild-type mice showed a >20% increase in LV mass and impaired cardiac function. Cardiomyocyte cross-sectional area was also markedly increased in Ang II-treated wild-type mice but unchanged in Ang II-treated TGF-beta1-deficient mice. No significant levels of fibrosis, mitotic growth, or cytokine infiltration were detected in Ang II-treated mice. Atrial natriuretic factor expression was approximately 6-fold elevated in Ang II-treated wild-type, but not TGF-beta1-deficient mice. However, the alpha- to beta-myosin heavy chain switch did not occur in Ang II-treated mice, indicating that isoform switching is not obligatorily coupled with hypertrophy or TGF-beta1. The Ang II effect on hypertrophy was shown not to result from stimulation of the endogenous renin-angiotensis system. These results indicate that TGF-beta1 is an important mediator of the hypertrophic growth response of the heart to Ang II.

In this review, we describe the recent advances in the understanding of the role of microRNAs in idiopathic pulmonary fibrosis (IPF), a chronic progressive and lethal fibrotic lung disease. Approximately 10% of the microRNAs are significantly changed in IPF lungs. Among the significantly downregulated microRNAs are members of let-7, mir-29, and mir-30 families as well as miR-17∼92 cluster among the upregulated mir-155 and mir-21. Downregulation of let-7 family members leads to changes consistent with epithelial mesenchymal transition in lung epithelial cells both in vitro and in vivo, whereas inhibition of mir-21 modulates fibrosis in the bleomycin model of lung fibrosis. Perturbations of mir-155 and mir-29 have profibrotic effects in vitro but have not yet been assessed in vivo in the context of lung fibrosis. A recurrent global theme is that many microRNAs studied in IPF are both regulated by transforming growth factor β1 (TGFβ1) and regulate TGFβ1 signaling pathway by their target genes. As a result, their aberrant expression leads to a release of inhibitions on the TGFβ1 pathway and to the creation of feed-forward loops. Coanalysis of published microRNA and gene expression microarray data in IPF reveals enrichment of the TGFβ1, Wnt, sonic hedgehog, p53, and vascular endothelial growth factor pathways and complex regulatory networks. The changes in microRNA expression in the IPF lung and the evidence for their role in the fibrosis suggest that microRNAs should be evaluated as therapeutic targets in IPF.

To better understand the molecular basis of chronic obstructive pulmonary disease (COPD), we used serial analysis of gene expression (SAGE) and microarray analysis to compare the gene expression patterns of lung tissues from COPD and control smokers. A total of 59,343 tags corresponding to 26,502 transcripts were sequenced in SAGE analyses. A total of 327 genes were differentially expressed (1.5-fold up- or down-regulated). Microarray analysis using the same RNA source detected 261 transcripts that were differentially expressed to a significant degree between GOLD-2 and GOLD-0 smokers. We confirmed the altered expression of a select number of genes by using real-time quantitative RT-PCR. These genes encode for transcription factors (EGR1 and FOS), growth factors or related proteins (CTGF, CYR61, CX3CL1, TGFB1, and PDGFRA), and extracellular matrix protein (COL1A1). Immunofluorescence studies on the same lung specimens localized the expression of Egr-1, CTGF, and Cyr61 to alveolar epithelial cells, airway epithelial cells, and stromal and inflammatory cells of GOLD-2 smokers. Cigarette smoke extract induced Egr-1 protein expression and increased Egr-1 DNA-binding activity in human lung fibroblast cells. Cytomix (tumor necrosis factor alpha, IL-1beta, and IFN-gamma) treatment showed that the activity of matrix metalloproteinase-2 (MMP-2) was increased in lung fibroblasts from EGR1 control (+/+) mice but not detected in that of EGR1 null (-/-) mice, whereas MMP-9 was regulated by EGR1 in a reverse manner. Our study represents the first comprehensive analysis of gene expression on GOLD-2 versus GOLD-0 smokers and reveals previously unreported candidate genes that may serve as potential molecular targets in COPD.

Lung epithelial cells have emerged as a frequent target of injury, a driver of normal repair, and a key element in the pathobiology of fibrotic lung diseases. An important aspect of epithelial cells is their capacity to respond to microenvironmental cues by undergoing epithelial-mesenchymal transition (EMT). EMT is not simply widespread conversion of epithelial cells to fibroblasts but a graded response whereby epithelial cells reversibly acquire mesenchymal features and enhanced capacity for mesenchymal cross-talk. Recent studies elucidate distinct integrin-sensing systems that coordinate activity of TGFβ1, a critical signaling element regulating EMT, with the presence of proinflammatory signals and cell injury. Repeated injury superimposes persistent inflammation and hypoxia onto these highly regulated repair pathways, potentially overwhelming orderly repair and creating sustained fibrogenesis. Understanding specific signaling mechanisms driving the mesenchymal response to TGFβ1 may reveal therapeutics to attenuate fibrogenesis yet preserve the important homeostatic functions of TGFβ1.

TGF-beta1 is a key regulator of diverse biological processes in many tissues and cell types, but its exact function in the developing and adult mammalian CNS is still unknown. We report that lack of TGF-beta1 expression in neonatal Tgfb1(-/-) mice results in a widespread increase in degenerating neurons accompanied by reduced expression of synaptophysin and laminin and a prominent microgliosis. Lack of TGF-beta1 also strongly reduces survival of primary neurons cultured from Tgfb1(-/-) mice. TGF-beta1 deficiency in adult Tgfb1(-/+) mice results in increased neuronal susceptibility to excitotoxic injury, whereas astroglial overexpression of TGF-beta1 protects adult mice against neurodegeneration in acute, excitotoxic and chronic injury paradigms. This study reveals a nonredundant function for TGF-beta1 in maintaining neuronal integrity and survival of CNS neurons and in regulating microglial activation. Because individual TGF-beta1 expression levels in the brain vary considerably between humans, this finding could have important implications for susceptibility to neurodegeneration.

Multiple bone marrow stromal cell types have been identified as hematopoietic stem cell (HSC)-regulating niche cells. However, whether HSC progeny can serve directly as HSC niche cells has not previously been shown. Here we report a dichotomous role of megakaryocytes (MKs) in both maintaining HSC quiescence during homeostasis and promoting HSC regeneration after chemotherapeutic stress. We show that MKs are physically associated with HSCs in the bone marrow of mice and that MK ablation led to activation of quiescent HSCs and increased HSC proliferation. RNA sequencing (RNA-seq) analysis revealed that transforming growth factor β1 (encoded by Tgfb1) is expressed at higher levels in MKs as compared to other stromal niche cells. MK ablation led to reduced levels of biologically active TGF-β1 protein in the bone marrow and nuclear-localized phosphorylated SMAD2/3 (pSMAD2/3) in HSCs, suggesting that MKs maintain HSC quiescence through TGF-β-SMAD signaling. Indeed, TGF-β1 injection into mice in which MKs had been ablated restored HSC quiescence, and conditional deletion of Tgfb1 in MKs increased HSC activation and proliferation. These data demonstrate that TGF-β1 is a dominant signal emanating from MKs that maintains HSC quiescence. However, under conditions of chemotherapeutic challenge, MK ablation resulted in a severe defect in HSC expansion. In response to stress, fibroblast growth factor 1 (FGF1) signaling from MKs transiently dominates over TGF-β inhibitory signaling to stimulate HSC expansion. Overall, these observations demonstrate that MKs serve as HSC-derived niche cells to dynamically regulate HSC function.

Activation of myofibroblast rich stroma is a rate-limiting step essential for cancer progression. The responsible factors are not fully understood, but TGFβ1 is probably critical. A proportion of TGFβ1 is associated with extracellular nano-vesicles termed exosomes, secreted by carcinoma cells, and the relative importance of soluble and vesicular TGFβ in stromal activation is presented. Prostate cancer exosomes triggered TGFβ1-dependent fibroblast differentiation, to a distinctive myofibroblast phenotype resembling stromal cells isolated from cancerous prostate tissue; supporting angiogenesis in vitro and accelerating tumour growth in vivo. Myofibroblasts generated using soluble TGFβ1 were not pro-angiogenic or tumour-promoting. Cleaving heparan sulphate side chains from the exosome surface had no impact on TGFβ levels yet attenuated SMAD-dependent signalling and myofibroblastic differentiation. Eliminating exosomes from the cancer cell secretome, targeting Rab27a, abolished differentiation and lead to failure in stroma-assisted tumour growth in vivo. Exosomal TGFβ1 is therefore required for the formation of tumour-promoting stroma.

This study analyzed gene expression patterns and global genomic alterations in hepatocellular carcinomas (HCC), hepatoblastomas (HPBL), tissue adjacent to HCC and normal liver tissue derived from normal livers and hepatic resections. We found that HCC and adjacent non-neoplastic cirrhotic tissue have considerable overlap in gene expression patterns compared to normal liver. Several genes including Glypican 3, spondin-2, PEG10, EDIL3 and Osteopontin are over-expressed in HCC vs. adjacent tissue whereas Ficolin 3 is the most consistently under-expressed gene. HCC can be subdivided into three clusters based on gene expression patterns. HCC and HPBL have clearly different patterns of gene expression, with genes IGF2, Fibronectin, DLK1, TGFb1, MALAT1 and MIG6 being over-expressed in HPBL versus HCC. In addition, specific areas of the genome appear unstable in HCC, with the same regions undergoing either deletion or increased gene dosage in all HCC. In conclusion, a set of specific genes and areas of genomic instability are found across the board in liver neoplasia.

BACKGROUND

T regulatory (T reg ) cells are characterized by expression of suppressive cytokines and the transcription factor FOXP3. They play a key role in balancing immune responses and maintain peripheral tolerance against antigens and allergens. The loss of peripheral tolerance against allergens causes diseases that can be therapeutically controlled with glucocorticoids.

OBJECTIVE

The present study investigates whether glucocorticoids affect the activity of T reg cells on the basis of FOXP3 and cytokine expression.

METHODS

CD4 + T cells from healthy donors and glucocorticoid-treated asthmatic patients were isolated, and expression of FOXP3, along with IL-10 and TGF-beta1, was determined. The effect of glucocorticoids on T reg cells was measured in vivo before and after GC treatment and in in vitro cultures.

RESULTS

FOXP3 mRNA expression was significantly increased in asthmatic patients receiving inhaled glucocorticoid treatment, systemic glucocorticoid treatment, or both. FOXP3 tightly correlated with IL10 mRNA expression. No correlation of FOXP3 mRNA expression was observed in relation to a (GT)n microsatellite promoter polymorphism on chromosome Xp11.23 or total IgE level. The frequency of CD25 + memory CD4 + T cells and transient FOXP3 mRNA expression by CD4 + T cells significantly increased after systemic glucocorticoid treatment, whereas TGFB1 expression did not change. Furthermore, glucocorticoids induced IL10 and FOXP3 expression in short-term and long-term cultures in vitro.

CONCLUSIONS

These findings demonstrate that glucocorticoid treatment is not only immunosuppressive and anti-inflammatory but also promotes or initiates differentiation toward T R 1 cells by a FOXP3-dependent mechanism. Strategies that convert transient glucocorticoid-induced T reg activity into a stable phenotype might improve allergy and asthma therapy.

Body fluids of cancer patients contain TEXs (tumour-derived exosomes). Tumours release large quantities of TEXs, and the protein content of exosome or MV (microvesicle) fractions isolated from patients' sera is high. TEXs down-regulate functions of immune cells, thus promoting tumour progression. We isolated TEXs from tumour cell supernatants and sera of patients with solid tumours or AML (acute myelogenous leukaemia). The molecular profile of TEXs was distinct from that of circulating exosomes derived from normal cells. TEXs were co-incubated with activated T-cells, conventional CD4(+) CD25(neg) T-cells or CD56(+) CD16(+) NK (natural killer) cells respectively. TEXs down-regulated CD3ζ and JAK3 (Janus kinase 3) expression in primary activated T-cells and mediated Fas/FasL (Fas ligand)-driven apoptosis of CD8(+) T-cells. TEXs promoted CD4(+) CD25(neg) T-cell proliferation and their conversion into CD4(+) CD25(hi)FOXP3+ (FOXP3 is forkhead box P3) Treg cells (regulatory T-cells), which also expressed IL-10 (interleukin 10), TGFβ1 (transforming growth factor β1), CTLA-4 (cytotoxic T-lymphocyte antigen 4), GrB (granzyme B)/perforin and effectively mediated suppression. Neutralizing antibodies specific for TGFβ1 and/or IL-10 inhibited the ability of TEXs to expand Treg cells. TEXs obtained at diagnosis from AML patients' sera were positive for blast-associated markers CD33, CD34, CD117 and TGFβ1, and they decreased cytotoxic activity of NK cells isolated from NC (normal control) donors, induced Smad phosphorylation and down-regulated NKG2D receptor expression. Correlations between the TEX molecular profile or TEX protein levels and clinical data in cancer patients suggest that TEX-mediated effects on immune cells are prognostically important. In contrast with exosomes released by normal cells, TEXs have immunosuppressive properties and are involved in regulating peripheral tolerance in patients with cancer.