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Background. Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intra- and interoperator variabilities in a multicentre project. Methods. The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration. Results. Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of cross-centre variability for naïve/memory subset frequencies particularly in the whole blood setting. Conclusion. Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options.

Study question Have decidual natural killer (dNK) cells a different microRNA (miRNA or miR) expression pattern compared to NK cells circulating in the peripheral blood (pb) of healthy pregnant women in the first trimester of gestation? Summary answer dNK cells have a unique miRNA profile, showing exclusive expression of a set of miRNAs and significant up- or down-regulation of most of the miRNAs shared with pbNK cells. What is known already dNK cells differ from pbNK cells both phenotypically and functionally, and their origin is still debated. Many studies have indicated that miRNAs regulate several important aspects of NK cell biology, such as development, activation and effector functions. Study design, size, duration Decidua basalis and peripheral blood specimens were collected from women (n = 7) undergoing voluntary termination of gestation in the first trimester of pregnancy. dNK and pbNK cells were then highly purified by cell sorting. Participants/materials, setting, methods miRNAs expression was analysed by quantitative RT-PCR (qRT-PCR)-based arrays using RNA purified from freshly isolated and highly purified pbNK and dNK cells. Results from arrays were validated by qRT-PCR assays. The bioinformatics tool ingenuity pathway analysis (IPA) was applied to determine the cellular network targeted by validated miRNAs and the correlated biological functions. Main results and the role of chance Herein, we identified the most differentially expressed miRNAs in NK cells isolated from peripheral blood and uterine decidua of pregnant women. We found that 36 miRNAs were expressed only in dNK cells and two miRNAs only in pbNK cells. Moreover, 48 miRNAs were commonly expressed by both NK cell preparations although at different levels: 28 were upregulated in dNK cells, while 15 were downregulated compared to pbNK cells. Validation of a selected set (n = 11) of these miRNAs confirmed the differential expression of nine miRNAs: miR-10b and miR-214 expressed only in dNK cells and miR-200a-3p expressed only in pbNK cells; miR-130b-3p, miR-125a-5p, miR-212-3p and miR-454 were upregulated while miR-210-3p and miR-132 were downregulated in dNK cells compared to pbNK cells. IPA network analysis identified a single network connecting all the miRNAs as well as their significant involvement in several classes of functions: 'Organismal injury, Reproductive system disease, Inflammatory disease' and 'Cellular development'. These miRNAs target molecules such as argonaute 2, tumour protein p53, insulin and other genes that belong to the same network and significantly influence cell differentiation and pregnancy. Limitations, reasons for caution In the present study, the cellular network and biological functions modulated by miRNAs differentially expressed in dNK and pbNK cells were identified by IPA considering only molecules and relationships that were with confidence 'experimentally observed' in leucocytes. The decidual and pbNK cells that were analysed here are a heterogeneous population and further study will help to disentangle whether there are differences in miRNA production by the different subsets of NK cells. Wider implications of the findings This is the first study describing a different miRNA expression profile in dNK cells compared to matched pbNK cells during the first trimester of pregnancy. Our findings improved the body of knowledge on dNK cell biology and strongly suggest further investigation into the roles of miRNAs that are differentially expressed in human dNK compared to pbNK cells. Our results suggest that specific miRNAs can modulate dNK cell origin and functions, highlighting a potential role of this miRNA signature in human development and diseases. Study funding/competing interest(s) This work was supported by grants from the Istituto Pasteur, Fondazione Cenci Bolognetti, the European NoE EMBIC within FP6 (Contract number LSHN-CT-2004-512040), Istituto Italiano di Tecnologia, and Ministero dell'Istruzione, dell'Universita e della Ricerca (Ricerche Universitarie), and from Universita Politecnica delle Marche. There are no conflicts of interest to declare.

HMGA1 is a member of a small family of architectural transcription factors involved in the coordinate assembly of multiprotein complexes referred to as enhanceosomes. In addition to their role in cell proliferation, differentiation, and development, high-mobility group proteins of the A type (HMGA) family members behave as transforming protoncogenes either in vitro or in animal models. Recent reports indicated that HMGA1 might counteract p53 pathway and provided an interesting hint on the mechanisms determining HMGA's transforming potential. HMGA1 expression is deregulated in a very large array of human tumors, including cervical cancer, but very limited information is available on the molecular mechanisms leading to HMGA1 deregulation in cancer cells. Here, we report that HMGA1 expression is sustained by human papilloma virus (HPV) E6/E7 proteins in cervical cancer, as demonstrated by either E6/E7 overexpression or by repression through RNA interference. Knocking down HMGA1 expression by means of RNA interference, we also showed that it is involved in cell proliferation and contributes to p53 inactivation in this type of neoplasia. Finally, we show that HMGA1 is necessary for the full expression of HPV18 E6 and E7 oncoproteins thus establishing a positive autoregulatory loop between HPV E6/E7 and HMGA1 expression. Copyright © 2008 Neoplasia Press, Inc. All rights reserved.

We present a series of experimental results, and their interpretation, connected to various aspects of the hydrodynamics of laser produced plasmas. Experiments were performed using the Prague PALS iodine laser working at 0.44 μm wavelength and irradiances up to a few 1014W/cm2. By adopting large focal spots and smoothed laser beams, the lateral energy transport and lateral expansion have been avoided. Therefore we could reach a quasi one-dimensional regime for which experimental results can be more easily and properly compared to available analytical models.

Context: Although decidual natural killer (NK) cell accumulation and vascular remodeling are critical steps to ensure successful pregnancy, the molecular mechanisms controlling these events are poorly defined. Objective: Herein we analyzed whether chemerin, a recently identified chemoattractant involved in many pathophysiological processes, could be expressed in the uterine compartment and could regulate events relevant for the good outcome of pregnancy. Design: Chemerin expression in human primary culture of stromal (ST) cells, extravillous trophoblast cells, and decidual endothelial cells (DEC) was analyzed by RT-PCR, ELISA, and Western blot. Migration through ST or DEC of peripheral blood and decidual (d) NK cells from pregnant women was performed using a transwell assay. A DEC capillary-like tube formation assay was used to evaluate endothelial morphogenesis. Results: Chemerin is differentially expressed by decidual cells during early pregnancy being present at high levels in ST and extravillous trophoblast cells but not in DEC. Notably, ST cells from pregnant women exhibit and release higher levels of chemerin as compared with ST cells from menopausal or fertile nonpregnant women. Chemerin can support peripheral blood NK cell migration through both DEC and ST cells. Although dNK cells exhibit lower chemerin receptor (CMKLR1) expression than their blood counterpart, CMKLR1 engagement on dNK cells resulted in both ERK activation and migration through decidual ST cells. Interestingly, DEC also express CMKLR1 and undergo ERK activation and capillary-like tube structure formation upon exposure to chemerin. Conclusions: Our data indicate that chemerin is up-regulated during decidualization and might contribute to NK cell accumulation and vascular remodeling during early pregnancy.

Fetal trophoblast cells invading the decidua in the early phase of pregnancy establish complex interaction with the maternal extracellular matrix. We discovered that C1q was widely distributed in human decidual stroma in the absence of C4 and C3 and was actively synthesized by migrating extravillous trophoblasts. The cells expressed the messages for the three chains of C1q and secreted this complement component that interacted with the proteins of the decidual extracellular matrix. Solid phase-bound C1q promoted trophoblast adhesion and migration, and cell binding to C1q resulted in activation of ERK1/2 MAPKs. Ab inhibition experiments showed that the receptors for the globular head of C1q/p33 and α4β1 integrin were both involved in this process and were colocalized on the cell surface following binding of C1q to trophoblasts. We also found that C1q−/− mice manifested increased frequency of fetal resorption, reduced fetal weight, and smaller litter sizes compared with wild-type mice. C1q deficiency was associated with impaired labyrinth development and decidual vessel remodeling. Collectively, these data suggest that C1q plays an important role in promoting trophoblast invasion of decidua and that defective local production of C1q may be involved in pregnancy disorders, such as pre-eclampsia, characterized by poor trophoblast invasion.

International audience; Smoothing of laser beam non-uniformities using gas jets has been studied. The experiment has been performed with the PALS laser working at 0.44 ?m with an intensity of about 1015 W/cm2. The laser beam has been split in two by a prism thus creating an artificial large-scale non-uniformity (?90 ?m). We recorded time resolved and static images of laser-gas jet interaction with and without an Al target. Multi 1D and 2D simulations show that such interaction acts redistributing the over-intensities over larger surface. This effect has to be attributed to ionization processes with consequent laser beam refraction. Results show that Argon gas jet produces a strong refraction of the laser beam thus strongly reducing the initial two spots separation. © EDP Sciences and Springer 2009.

International audience; The process of laser beam homogenization in a gas medium placed in front of a thin metallic foil has been studied. Experiments were performed using the Prague Asterix Laser System iodine laser [ Jungwirth et al., Phys. Plasmas 8, 2495 (2001) ] working at 0.44 μm wavelength and irradiance of about 10^15 W/cm^2. Homogenization was detected both by directly analyzing the transmitted laser beam and by studying the shock breakout on the foil rear side. Results show that the gas ionization by the laser pulse induces a strong refraction and produces an effective smoothing of large-scale intensity nonuniformities.

International audience; We studied the process of laser beam homogenization in a gas medium placed in front of a thin metallic foil. Experiments were performed using the Prague PALS iodine laser working at 0.44 µm wavelength and irradiances of about 10^15 W cm^−2. Homogenization was detected both by directly analysing the transmitted laser beam and by studying the shock breakout on the foil rear side. Results show that the gas ionization by the laser pulse induces a strong refraction and produces an effective smoothing of large-scale intensity non-uniformities.

International audience; We present the experimental results, and their analysis, connected to the possibility to control laser inhomogeneities exploiting the non-uniform electron density distribution created by the laser while propagating in a gas jet. The induced self-refraction in the plasma created in the gas medium results in re-distributing local over-intensities over larger surfaces. The experiment at the PALS laser facility has been performed creating a large non-uniformity by a wedge arrangement and recording the static and dynamic images with and without the coupling to an Al target.

During early pregnancy, uterine mucosa decidualization is accompanied by a drastic enrichment of CD56highCD16− natural killer (NK) cells. Decidual NK (dNK) cells differ from peripheral blood NK (pbNK) cells in several ways, but their origin is still unclear. Our results demonstrate that chemokines present in the uterus can support pbNK cell migration through human endothelial and stromal decidual cells. Notably, we observed that pregnant women's pbNK cells are endowed with higher migratory ability compared with nonpregnant women's or male donors' pbNK cells. Moreover, NK cell migration through decidual stromal cells was increased when progesterone-cultured stromal cells were used as substrate, and this correlated with the ability of progesterone to up-regulate stromal cell chemokine expression. Furthermore, we demonstrate that dNK cells migrate through stromal cells using a distinct pattern of chemokines. Finally, we found that pbNK cells acquire a chemokine receptor pattern similar to that of dNK cells when they contact decidual stromal cells. Collectively these results strongly suggest that pbNK cell recruitment to the uterus contributes to the accumulation of NK cells during early pregnancy; that progesterone plays a crucial role in this event; and that pbNK cells undergo reprogramming of their chemokine receptor profile once exposed to uterine microenvironment.

An experimental investigation about laser beam homogenization using gas jets is presented in this paper. The results, obtained at PALS iodine laser facility using the 3 omega wavelength and irradiances of about 10(15) W/cm(2), showed that the use of high pressure gas jets (up to 10 bar of Argon) can be effective in reducing strong laser beam non-uniformities artificially introduced by inserting a wedge arrangement on half of the beam.