Serum microRNAs as novel biomarkers for primary sclerosing cholangitis and cholangiocarcinoma.

The diagnosis of primary sclerosing cholangitis (PSC) is difficult due to the lack of sensitive and specific biomarkers, as is the early diagnosis of cholangiocarcinoma (CC), a complication of PSC. The aim of this study was to identify specific serum miRNAs as diagnostic biomarkers for PSC and CC. The levels of 667 miRNAs were evaluated in 90 human serum samples (30 PSC, 30 CC and 30 control subjects) to identify disease-associated candidate miRNAs (discovery phase). The deregulated miRNAs were validated in an independent cohort of 140 samples [40 PSC, 40 CC, 20 primary biliary cirrhosis (PBC) and 40 controls]. Receiver operating characteristic (ROC) curves were established and only miRNAs with an area under the curve (AUC) > 0·70 were considered useful as biomarkers. In the discovery phase we identified the following: 21 miRNAs expressed differentially in PSC, 33 in CC and 26 in both in comparison to control subjects as well as 24 miRNAs expressed differentially between PSC and CC. After the validation phase, miR-200c was found to be expressed differentially in PSC versus controls, whereas miR-483-5p and miR-194 showed deregulated expression in CC compared with controls. We also demonstrate a difference in the expression of miR-222 and miR-483-5p in CC versus PSC. Combination of these specific miRNAs further improved the specificity and accuracy of diagnosis. This study provides a basis for the use of miRNAs as biomarkers for the diagnosis of PSC and CC.

Systemic and cellular consequences of macrophage control of iron metabolism.

Iron is necessary for both mammalian cells and microorganisms, which fiercely compete for this essential nutrient. Accordingly, macrophages exploit the denial of iron from microbial pathogens as an important strategy to accomplish their key role in innate immunity and host defense. Macrophages employ multiple mechanisms to accumulate iron and thus contain microbial infections, but this may come at a price. In particular, at the systemic level iron sequestration in the reticuloendothelial cells can lead to the development of anemia of chronic disease. At the local level, iron sequestration in macrophages, which is targeted to extracellular invaders, can in turn favor intracellular pathogens. Moreover, iron accumulation can per se promote pro-inflammatory activation of macrophages and consequently contribute to maintain the process of inflammation, without resolution. Finally, the peculiar iron trafficking that characterizes alternatively polarized macrophages can influence neighboring cells in the microenvironment and impact on the resolution phase of inflammation. In this review, we describe the role of macrophages in iron metabolism in the context of host defense and iron balance.

Identification of serum and tissue micro-RNA expression profiles in different stages of inflammatory bowel disease.

The altered expression of micro-RNA (miRNA) has been associated with Crohn's disease (CD) and ulcerative colitis (UC). The aim of this study was to establish specific miRNA expression patterns in the serum and mucosa of inflammatory bowel disease (IBD) patients (UC and CD with colonic involvement) at different stages of the disease. Serum and biopsies from nine active CD (aCD), nine inactive CD (iCD), nine active UC (aUC) and nine inactive UC (iUC) and serum from 33 healthy subjects were collected. Up to 700 miRNAs were evaluated by the TaqMan human miRNA array. The ΔCt values were obtained using the mean expression values of all expressed miRNAs in a given sample as a normalization factor for miRNA real-time quantitative polymerase chain reaction data. The levels of serum miRNAs in CD and UC patients were different to healthy subjects. Thirteen serum miRNAs were expressed commonly in CD and UC patients. Two miRNAs were higher and four miRNAs were lower in the serum of aCD than iCD. No serum miRNA was regulated exclusively in aUC compared with iUC patients. Four miRNAs were higher and three miRNAs were lower in the mucosa of aCD than iCD. Two miRNAs were higher and three miRNAs were lower in the mucosa of aUC than iUC. No serum miRNAs coincided with tissue miRNAs in aCD and aUC patients. Our results suggest the existence of specific miRNA expression patterns associated with IBD and their different stages and support the utility of miRNA as possible biomarkers. This pilot study needs to be validated in a large prospective cohort.

[Trend of the asbestos related diseases mortality in workers of a company in the province Cremona which manufactured asbestos products]. / Andamento delle patologie e della mortalità asbesto correlate in lavoratori di un'azienda della provincia di Cremona che produceva manufatti in amianto.

The study analyzes the trend of asbestos-related diseases and mortality in workers of a company in the province of Cremona which manufactured asbestos products. It is confirmed that the exposure to a high concentration of asbestos fibers (estimated to more than 20 fibers/cc) strictly correlates with the onset of pathologies from asbestos. In the studied population were found 19 cases of neoplastic diseases (12 mesotheliomas and 7 bronchopulmonary carcinomas). This figure, compared to the company working population, which over the years has been an average of 80 units, while not enabling to calculate an incidence rate due to the lack of reliable data on population, is indicative of a very significant cause-effect relationship since these are neoplastic diseases that can still arise. So it is necessary to continue the health monitoring of formerly exposed workers and appropriate to try to extend it to all workers of the asbestos compartment.

Identification of CCR8: a human monocyte and thymus receptor for the CC chemokine I-309.

The human CC chemokine I-309 is a potent monocyte chemoattractant and inhibits apoptosis in thymic cell lines. Here, we identify a specific human I-309 receptor, and name it CCR8 according to an accepted nomenclature system. The receptor has seven predicted transmembrane domains, is expressed constitutively in monocytes and thymus, and is encoded by a previously reported gene of previously unknown function named, alternatively, CY6, TER1, and CKR-L1. After transfection with the CY6 open reading frame, a mouse pre-B cell line exhibited calcium flux and chemotaxis in response to I-309 (EC50 = 2 nM for each), whereas 20 other chemokines were inactive. Signaling was sensitive to pertussis toxin, suggesting coupling to a Gi-type G protein. These properties parallel those of endogenous I-309 receptors expressed in an HL-60 clone 15 cell line model. The apparent monogamous relationship between I-309 and CCR8 is unusual among known CC chemokines and known CC chemokine receptors. CCR8 may regulate monocyte chemotaxis and thymic cell line apoptosis.

Interleukin 10 increases CCR5 expression and HIV infection in human monocytes.

The immunosuppressive and antiinflammatory cytokine interleukin (IL) 10 selectively upregulates the expression of the CC chemokine receptors CCR5, 2, and 1 in human monocytes by prolonging their mRNA half-life. IL-10-stimulated monocytes display an increased number of cell surface receptors for, and better chemotactic responsiveness to, relevant agonists than do control cells. In addition, IL-10-stimulated monocytes are more efficiently infected by HIV BaL. This effect was associated to the enhancement of viral entry through CCR5. These data add support to an emerging paradigm in which pro- and antiinflammatory molecules exert reciprocal and opposing influence on chemokine agonist production and receptor expression.

Anti-phospholipid antibody mediated fetal loss: still an open question from a pathogenic point of view.

Antiphospholipid antibodies (aPL) are associated with recurrent miscarriages and pregnancy complications, however their pathogenic mechanisms are still matter of research. Thrombotic events at the placental level cannot explain all of the clinical manifestations. It has been suggested that aPL may be responsible for a local acute inflammatory response mediated by complement activation and neutrophil infiltration eventually leading to fetal loss. However histological and immunohistological studies on human placental samples do support such a mechanism only in part and with no any clear relationship with the pregnancy outcome. A direct effect of aPL on both maternal and fetal placental tissues has been reported through the reactivity of the antibodies with beta2 glycoprotein I (beta2GPI) expressed on the cell membranes. These events do not require an inflammatory response and can be in part related to the inhibition of growth factors favouring a physiological placentation. Understanding the different pathogenic mechanisms of aPL-associated miscarriages may help in improving our therapeutic approach particularly in recurrent cases not responsive to the usual treatment.

Inflammatory reaction and implantation: the new entries PTX3 and D6.

Successful embryonic implantation implies anchoring the conceptus in the maternal uterine wall, establishing a vascular supply to enable optimal growth and development of the conceptus, and promoting tolerance of fetal alloantigens encoded by paternal genes. To achieve these goals, complex molecular dialogues take place among the maternal endometrium, the conceptus, and the placenta. Several factors are involved in the fetal-maternal interaction, including hormones, growth factors, cytokines, chemokines, adhesion molecules, extracellular matrix components, and matrix-degrading enzymes. This complex cross-talk results in the induction of a local inflammatory response and a state of systemic inflammation, as revealed by leukocytosis, endothelium activation, increased activity of innate immune cells, and increased levels of inflammatory cytokines and chemokines. The enriched cytokine milieu associated to implantation is likely to control trophoblast migration and differentiation, leukocyte influx and activation, complement activation, as well as angiogenic and angiostatic processes in the implantation site. Finally, these mediators play a key role in tuning the immune responses to protect the fetus from infections as well as from maternal rejection. Here, the role of pro-inflammatory networks activated in implantation will be discussed. In particular, emphasis will be put on two new players involved in regulating inflammation at the maternal-fetal interface: the long pentraxin PTX3 and the decoy receptor for inflammatory chemokines D6.

Chemokines and bone remodeling.

Bone remodeling is characterized by spatial and temporal coupling of bone resorption and formation and is necessary for skeletal growth and normal bone structure maintenance. Imbalance of this process is related to metabolic bone disorders such as osteoporosis or rheumatoid arthritis. For this reason, bone remodeling is under the control of several local and systemic factors, including molecules of the immune system. The importance of the interplay of both the skeletal and immune systems is reflected by the emerging interdisciplinary research field, called osteoimmunology, focused on common aspects of osteology and immunology. This review focuses on the role of inflammatory mediators, such as cytokines in bone remodeling and, in particular, a subfamily of chemotactic cytokines or chemokines which are involved not only in several aspects of physiological bone remodeling but also in pathological bone disorders, such as rheumatoid arthritis or osteoporosis. Understanding the role of inflammation and chemokines will provide new insights for the treatment of diseases affecting both skeletal and immune systems, by the development of new therapeutic strategies targeting common inflammatory mediators.

Colifagina, a novel preparation of 8 lysed bacteria ameliorates experimental colitis.

Immune reactivity towards the bacterial intestinal flora plays an important part in the pathogenesis of inflammatory bowel disease. Administration of probiotic bacteria has beneficial effects on infectious and inflammatory diseases, principally in bowel disorders. However, little is known about the administration of soluble bacterial antigens in intestinal inflammation. We investigated the therapeutic effects of colifagina in experimental colitis. To assess this effect, C57BL/6 mice with dextran sulphate sodium-induced colitis were treated with colifagina, or with a placebo, for a period of 10 days. The mice were monitored, and inflammation was assessed by disease activity index (DAI). Analysis of fecal IgA concentration and measurement of IgA and inflammatory chemokine production in organ colonic culture was performed by ELISA. Clinically and histologically, bacterial-lysate-treated mice revealed significantly fewer DAI and a reduction of colonic histological inflammation. Treatment of healthy mice with colifagina significantly increased the fecal concentration of IgA and IgA production in organ culture. Colifagina administration in DSS-treated mice significantly increased the fecal concentration of IgA and IgA production in organ culture. MIP-1, MIP-2 and RANTES concentrations in colonic organ culture were significantly lower in colifagina-treated mice than in the placebo group. The use of colifagina is effective in amelioration of murine colitis.

IL-1 beta primes IL-8-activated human neutrophils for elastase release, phospholipase D activity, and calcium flux.

Interleukin-8 (IL-8), the prototype of the alpha (e.i., C-X-C branch) chemokine family, induced elastase release in a concentration-dependent manner (50-1000 ng/mL) in cytochalasin B-treated human polymorphonuclear leukocytes (PMNs). This response was potentiated about twofold if PMNs were preexposed to interleukin-1 beta (IL-1 beta) at concentrations that were by themselves inactive. The effect of IL-1 beta was clearly observed after 5 min and was maximal after a 30-min preincubation of the cells. The effect was present over the whole active concentration range of IL-8 and was completely blocked by the presence of IL-1 receptor antagonist. Priming of elastase release by IL-1 beta was not associated with a change in receptor number or affinity for IL-8. On the contrary, it was correlated with priming of phospholipase D activity and calcium flux activated by IL-8. Preincubation of the cells with ethanol and/or La3+ inhibited IL-8-induced degranulations, suggesting that activation of phospholipase D and increase of [Ca2+]i were important for this response. In contrast, ethanol and La3+ did not decrease the priming effect of IL-1 beta. IL-8 and IL-1 beta have been shown to be released by the same cell types and may be concomitantly present at sites of inflammation, giving rise to an amplification of the inflammatory response.

Receptors, signal transduction, and spectrum of action of monocyte chemotactic protein-1 and related chemokines.

Chemokines are a bipartite family of chemotactic proteins that bear the structural hallmark of four cysteine residues, the first two of which are in tandem. The spectrum of action of C-C chemokines, monocyte chemotactic protein-1 (MCP-1), MCP-2, and MCP-3, in particular, encompasses, in addition to monocytes, other leukocyte populations. Evidence is presented that MCP-1, MCP-2, and MCP-3 are active on natural killer cells. Available information on receptor usage by MCP-1 and related chemokines and signal transduction pathways is reviewed. A better understanding of signaling mechanisms will provide a new basis for therapeutic strategies.

Regulation of the chemokine system at the level of chemokine receptor expression and signaling activity.

The chemokine system is highly influenced by the microenvironmental context. Regulation of the chemokine system occurs not only at the level of agonist production, but also at the level of chemokine receptor expression. This review provides examples of regulation of the system at the receptor level by modulation of receptor expression in canonical cellular targets (tuning of the system), and induction of novel receptors (shaping of the system), with particular attention to dendritic cells as a cellular model. Receptor signaling activity represents a further potential level of regulation of the system. Finally, chemokines can also influence the microenvironment by modulating gene expression in target cells.

Interleukin-1 beta primes interleukin-8-stimulated chemotaxis and elastase release in human neutrophils via its type I receptor.

Interleukin-1 (IL-1) is a pleiotropic proinflammatory cytokine which binds to human neutrophils (PMN) and can directly or indirectly activate their functions. In this study we show that a brief exposure to IL-1 beta induces a potentiation of both PMN elastase release and chemotactic response to interleukin-8 (IL-8), the prototype of C-X-C chemokines. Priming by IL-1 beta was maximal at 100 ng/ml, was completely blocked in the presence of IL-1 receptor antagonist (IL-1ra) and, in the chemotaxis assay, was best observed at suboptimal (3-6 ng/ml) or inactive (0.75 ng/ml) concentrations of IL-8. Priming of PMN by IL-1 beta was completely blocked by M1, a specific antibody against the type I IL-1 receptor (IL-1RI). On the other hand M22, an antibody directed against the IL-1 decoy type II IL-1 receptor did not affect IL-1 beta action and slightly increased the priming effect. Thus, exclusively via its type I receptor, IL-1 beta can act on PMN at multiple levels, by promoting their accumulation in tissues through the induction of chemotactic factors (e.g. IL-8) and the upregulation of adhesion molecules, and by priming their response to chemotactic agonists.

Double-blind study of dothiepin versus placebo in the treatment of primary fibromyalgia syndrome.

A double-blind study comparing the efficacy and tolerability of dothiepin with that of placebo in the treatment of primary fibromyalgia syndrome was carried out. Dothiepin was shown to improve significantly the condition of patients with primary fibromyalgia syndrome and there was a significant difference between dothiepin and placebo in all the clinical variables measured. Only mild and transient side-effects were reported. Further controlled studies are required to define the effects of dothiepin on fibromyalgia.

[Role of inflammation mediators in the pathogenesis of heart failure]. / Ruolo dei mediatori dell'infiammazione nella patogenesi dello scompenso cardiaco.

A number of factors are involved in congestive heart failure pathogenesis. Among these, inflammatory mediators could have a crucial role. Patients with congestive heart failure show increased plasma levels of "proinflammatory cytokines", in particular tumor necrosis factor-alpha and interleukin-6. Clinical and experimental models have demonstrated that these cytokines induce left ventricular dysfunction, pulmonary edema, ventricular remodeling, skeletal muscle abnormalities, myocyte apoptosis and endothelial dysfunction, suggesting the possibility that increased plasma concentration of cytokines could not be just an epiphenomenon, but an effective pathogenetic mechanism of disease progression. Additional inflammatory proteins involved in the acute phase response could play a part in the pathogenesis of heart failure. Pentraxin 3 is a prototypical long pentraxin, structurally related, although with different functions, to C-reactive protein, is produced by immune system cells, fibroblasts and particularly by cardiac endothelial cells and myocytes, as demonstrated in murine and human models. Its synthesis is rapidly induced after exposition to bacterial lipopolysaccharide and proinflammatory cytokines, as interleukin-1beta and tumor necrosis factor-alpha. In heart diseases, pentraxin 3 could be involved in the acute local inflammatory response to myocardial injury (e.g. necrosis) and in heart failure pathogenetic mechanisms, but its exact role is not yet settled. Defining the specific part played by these molecules in the pathogenesis of heart failure could lead to new therapeutic approaches in the treatment of cardiac insufficiency.

The biochemistry and biology of the atypical chemokine receptors.

A subset of chemokine receptors, initially called "silent" on the basis of their apparent failure to activate conventional signalling events, has recently attracted growing interest due to their ability to internalize, degrade, or transport ligands and thus modify gradients and create functional chemokine patterns in tissues. These receptors recognize distinct and complementary sets of ligands with high affinity, are strategically expressed in different cellular contexts, and lack structural determinants supporting Gα(i) activation, a key signalling event in cell migration. This is in keeping with the hypothesis that they have evolved to fulfil fundamentally different functions to the classical signalling chemokine receptors. Based on these considerations, these receptors (D6, Duffy antigen receptor for chemokines (DARC), CCX-CKR1 and CXCR7) are now collectively considered as an emerging class of 'atypical' chemokine receptors. In this article, we review the biochemistry and biology of this emerging chemokine receptor subfamily.

Receptor binding mode and pharmacological characterization of a potent and selective dual CXCR1/CXCR2 non-competitive allosteric inhibitor.

BACKGROUND AND PURPOSE: DF 2156A is a new dual inhibitor of IL-8 receptors CXCR1 and CXCR2 with an optimal pharmacokinetic profile. We characterized its binding mode, molecular mechanism of action and selectivity, and evaluated its therapeutic potential. EXPERIMENTAL APPROACH: The binding mode, molecular mechanism of action and selectivity were investigated using chemotaxis of L1.2 transfectants and human leucocytes, in addition to radioligand and [(35) S]-GTPγS binding approaches. The therapeutic potential of DF 2156A was evaluated in acute (liver ischaemia and reperfusion) and chronic (sponge-induced angiogenesis) experimental models of inflammation. KEY RESULTS: A network of polar interactions stabilized by a direct ionic bond between DF 2156A and Lys(99) on CXCR1 and the non-conserved residue Asp(293) on CXCR2 are the key determinants of DF 2156A binding. DF 2156A acted as a non-competitive allosteric inhibitor blocking the signal transduction leading to chemotaxis without altering the binding affinity of natural ligands. DF 2156A effectively and selectively inhibited CXCR1/CXCR2-mediated chemotaxis of L1.2 transfectants and leucocytes. In a murine model of sponge-induced angiogenesis, DF 2156A reduced leucocyte influx, TNF-α production and neovessel formation. In vitro, DF 2156A prevented proliferation, migration and capillary-like organization of HUVECs in response to human IL-8. In a rat model of liver ischaemia and reperfusion (I/R) injury, DF 2156A decreased PMN and monocyte-macrophage infiltration and associated hepatocellular injury. CONCLUSION AND IMPLICATIONS: DF 2156A is a non-competitive allosteric inhibitor of both IL-8 receptors CXCR1 and CXCR2. It prevented experimental angiogenesis and hepatic I/R injury in vivo and, therefore, has therapeutic potential for acute and chronic inflammatory diseases.