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SocietàItaliana diCitometria

PROCEEDINGS OF THEXXXIII NATIONAL CONFERENCE

OF THE ITALIAN SOCIETYOF CYTOMETRY

GIC

Lucca, ItalyPalazzo Ducale

September 22-25, 2015

EditorsR. De Vita - G. Mazzini

Lucca atti 2015:Layout 1 15-12-2015 15:53 Pagina 1

con il Patrocinio di

Presidenza del Consiglio dei Ministri

ENEA Agenzia nazionale per le nuove tecnologie,

l’energia e lo sviluppo economico sostenibile

CNR Consiglio Nazionale delle Ricerche

IRCCS Istituto di Ricerche Farmacologiche “Mario Negri”

Istituto Nazionale Tumori “Fondazione G. Pascale”

Stazione Zoologica “Anton Dohrn”

Comune di Lucca

SOCIETÀ ITALIANA DI CITOMETRIA

c/o Unità Biologia delle Radiazioni e Salute dell'UomoENEA Centro Ricerche Casaccia s.p. 016Via Anguillarese, 301 - 00123 Romatel. 06 30484671 - fax 06 30484891

e-mail: [email protected]://gic.casaccia.enea.it

La Conferenza è organizzata dalla Società Italiana di Citometria GIC

Con il Patrocinio ed il supporto della Provincia di Lucca

COMITATO ORGANIZZATORE

R. DE VITA (Roma)E. ERBA (Milano)G. MAZZINI (Pavia)G. PIROZZI (Napoli)

COMITATO SCIENTIFICO

A. AIELLO (Milano)R. CHIANESE (Ivrea)I. D’AGNANO (Roma)M. DANOVA (Pavia)R. DE PALMA (Napoli)L. DEL VECCHIO (Napoli)M.G. DELLA PORTA (Pavia)A. FATTOROSSI (Roma)D. FENOGLIO (Genova)G. GAIPA (Monza)A. KUNKL (Genova)F. LANZA (Cremona)S. LUCRETTI (Roma)A. MANTI (Urbino)O. NAPPI (Napoli)M. ROCCHI (Bari)M. SPANÒ (Roma)V. TIRINO (Napoli)C. USAI (Genova)A. VENDITTI (Roma)L. ZAMAI (Urbino)


SOCIETÀ ITALIANA DI CITOMETRIA

XXXIII CONFERENZA NAZIONALEAGGIORNAMENTI E INNOVAZIONI DELLA CITOMETRIA

IN APPLICAZIONI CLINICHE E DI RICERCA

PALAZZO DUCALELUCCA

22-25 SETTEMBRE 2015

PROCEEDINGS

THE ITALIAN SOCIETY OF CYTOMETRYGIC

EDITED BYR. DE VITA and G. MAZZINI

SUPPORTED BY ENEA - AGENZIA NAZIONALE PER LE NUOVE TECNOLOGIE,L’ENERGIA E LO SVILUPPO ECONOMICO SOSTENIBILE


XXXIII Conferenza Nazionale di Citometria - Palazzo Ducale 22-25 settembre 2015 - Lucca

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The XXXIII Italian Society of Cytometry (GIC) bi-annual Meeting had been again held inLucca (Tuscany, Italy) from September 22th to 25th 2015. The great success of the previous(2013) event allowed by a proper mix of high level of science and the geographical/culturaladvantages offered by Lucca did stimulate the Society to come back to the same place.All abstracts were carefully reviewed by the Scientific program Committee and publishedhere in full and categorized by scientific tracks such as Environmental sciences and toxico-logy, Hematology, Immunology, Methodology-technology, and Oncology.To date there are over 600 members actively involved in educational actions, promotion ofquality control programs, drafting/validation of guidelines and accreditation, providing infor-mation for people involved that actively work in the field of basic and applied cytometry.This years the Meeting had been preceded by a half a day satellite event dedicated to theprogress of the “Professional Certification GIC Register” which will be committed to test,train, certify and monitor the professional levels of the “Italian Cytometrists” under theresponsibility of GIC. The first phase of this action started in 2014 had been very muchappreciated by the members. A Basic Course (split in the three areas of application of flowcytometry) had been proposed to the young attendees with the aim to upgrade their theorybut also to cover some basic practical aspects they may encounter in the lab. After theCourses those members registered to join the “GIC Register” did had the opportunity toapply the examinations (written and oral) organized by the GIC Committee inside the samebuilding. At present more than 60 members have applied to be enrolled in the GIC Register.The Meeting started in the afternoon with the Opening Ceremony, chaired by the GICPresident and Secretary followed by the series of “companies seminars” included in theSession “Analytical methodology”. The first day session closed with the Keynote Lecture“The evolution of cytometry”, dedicated to the memory of Francesco Mauro who died onmarch 25; Francesco had been the real leaving-soul of our Society GIC and spent many ofhis energies in the first organization of what he called “Gruppo Italiano di Cytometria” laterevolved in National Society of Cytometry GIC. He had been in fact one of the foundingmembers as well as the first President of the Society. He had been more “international” thanlinked to the local national organization and thanks to his “american mind” (and also lifestyle) become soon in 1994 ISAC President at XVII ISAC Meeting of Lake Placid (NY-USA)till 1996 when organized the XVIII ISAC Meeting in Rimini (Italy).Regarding the scientific program this year the Scientific Committee selected 20 abstractsfor oral presentation among those submitted (93). Plenary sessions included 21 invitedlectures focused on the contribute of cytometry in different application fields, highlighting theinterdisciplinary profile of GIC. These outstanding talks offered a rational and fascinatingoverview on the role of flow cytometry in the diagnosis and clinical management of acuteleukemias including the future perspectives of monitoring the patent’s individual responseto therapy in modern clinical trials.Each Parallel session, offered along three days, was tailored on the traditional topics such



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as immunology, oncology, hematology, methodology, and environmental sciences withcytometry as the common denominator. Several invited and selected lectures were givenin order to bring the discussion on the more emerging and promising aspects of flowcytometry (FC).Again in the “core” of the Society activities a special session had been programmed to thereports of the ongoing GIC Projects:a) “The role of FC in the immunophenotyping of Acute Leukemia.” G. Gaipa made anexhaustive details of what the board of this Prog did in the recent past and what the enrol-led “Expert Committee” is doing in the evaluation of the selected literature in order to beable to define in the future step of the activity witch is the real impact of FC in this veryimportant and promising area of application of this technology.b) “Standardization of a FC approach for the evaluation of Endothelial Circulating cells andtheir progenitors”. M. Della Porta reported the activities of the group of labs (spread aroundthe north Italy) practically involved in the analyses of reference samples prepared in Paviaand rapidly sent to the participants. The very ambitious Original Design involving a verylarge number of markers did results in interlaboratories CVs of the preliminary phase extre-mely wide. In a successive step and thanks to the “analytical-instrumental-setting” pre-defi-ned by the Committee the actual output of the trial is going to be greatly improved. The sup-port of the companies (Beckman Coulter and Miltenyi Biotec) had been extensively ackno-wledged while the cost of the Project was out of the financial possibility of the GIC Society.c) “Cell therapy and regulatory guidance”. F.Lanza gave an overview on the current EUregulatory framework in the context of practical challenges regarding quality, preclinicaland clinical issues, which should be faced prospectively during the development phase ofcell-based medicinal products, in order to apply for manufacturing authorizations, clinicaltrials, licensing under the hospital exemption clause or for centralized European markingauthorization.A special “joint Session” between the GIC Society and AICC (Italian Society of CellCulture) had been Organized by G.Pirozzi, M.Caraglia and V. Tirino. The Session didfocus on the always actual theme of apoptosis and related cell machinery and their impli-cations in pathology.At the end of the Meeting people attending the Session “Cytometry and Microscopy” hadthe opportunity to watch the moves presented by L. Visai concerning the biomedical expe-riments carried out on the last Mission (2014/15) on board of the International Space Station(as cooperation between NASA and ASI) and dealing with osteoporosis occurring to crewmembers during the spaceflights. The Italian Captain Samanta Cristoforetti (member of thecrew) had the double role to be at the same time “Investigator” and “patient” of the Projectdesigned by L. Visai.Poster session included 55 contributions and a special committee have selected 2 Posteraward winner in each category.A substantial contribution has been provided by the principal companies in the field whichhad been located in a large exhibition area inside the conference center.The President conclusive remarks included the great satisfaction for the very good scienti-fic level of all the sessions with particular reference to the “Posters” definitely establishedas the most common way to introduce novel data and discoveries in Science.

President Elect Past President PresidentGiuseppe Pirozzi Francesco Lanza Giuseppe Gaipa

Edited byR. De Vita and G. Mazzini


table of contents

Invited Speakers 5

Session IEnvironmental Sciences and Toxicology 11

Session IIHematology 17

Session IIIImmunology 23

Session IVMethodology and Technology 29

Session V 33Oncology


EPIGENETIC DEREGULATION IN CANCER:TRANSLATIONAL APPROACHES FROM BENCH TOBEDSIDEAltucci L.Dipartimento di Biochimica, Biofisica e Patologia generale,Seconda Università di Napoli, Napoli, [email protected]

Deregulation of miRNAs expression and/or function con-tributes to initiation and maintenance of cancer, support-ing tumor-suppressor (TS) or oncogene (OG) functions,respectively. Epigenetic modulators (the so-calledepidrugs), such as histone deacetylase inhibitors(HDACis), are currently used in several anticancer thera-pies for their antiproliferative, proapoptotic and differenti-ation action. We identified a specific miRNA (and itsnewly identified target gene) to be modulated by HDACiin acute myeloid leukemias (AMLs). Our results identifythis couple as highly relevant target(s) during leukemoge-nesis. More in details, our data support a causal linkresponsible in keeping the undifferentiated status andslow-drug response of AML cells. As a consequence,forced re-equilibrium of miRNA/Target expression inacute myeloid leukemia cells resets an effective epige-netic program allowing maturation commitment and abetter drug-response. The output of this study may resultin therapeutic implications for a better patient’s stratifica-tion. Taken together our data point at the development ofmiRNA-based interventions in combination with chemo-immunotherapies of AMLs.

QUANTIFICATION AND CHARACTERIZATION OFMICROORGANISMS BY FLOW CYTOMETRY: CASESTUDIES AND APPLICATIONAmalfitano S.Water Research Institute (CNR-IRSA), Via Salaria km 29,300, Monterotondo, Rome, [email protected]

Besides the well-developed medical-clinical applications,flow cytometry allows the quantification and characteriza-tion of several structural and functional properties ofmicrobial cells in liquid samples. The most popular ap-proach combines the scatter and fluorescence signals(i.e., by using of nucleic acid targeted dyes), thusallowing the identification of microorganisms through awide dimensional range that goes from viruses, to bacte-ria, to protists. Moreover, the autofluorescence signals,owing to intracellular photosynthetic pigments (eg.,chlorophylls, phycocyanins, ficoeritrine) may lead to theidentification of specific microbial populations and algae.Nowadays, the common practices for microbiologicalcontamination assessment are based on cultivationmethods on selective media, by following a well-estab-lished practice but developed more than 100 years agofor detecting human pathogens. Given the possibility togenerate a multiparametric dataset much faster than thetraditional microbiological approaches, I will be reportingon some recent experiments carried out in natural andengineered systems, with the aim to highlight the advan-tages and new applications offered by the use of flowcytometry as an efficient method for microbiologicalmonitoring practices.

HUMAN REGULATORY T CELL IDENTIFICATION BYFLOW CYTOMETRYBattaglia A.Gynecology Oncology Dept, Pol. Universitario A. GemelliUniversità Cattolica Sacro Cuore, Roma, [email protected]

Regulatory T cell (Treg)-mediated immunosuppression isconsidered both a major obstacle for successful cancerimmunotherapy and a valuable support in autoimmunedisorders, and association between clinical outcome andTreg in these settings is actively being studied.Unfortunately, no definite consensus has been reachedabout a) the markers and b) the gating strategy requiredfor identification of human Treg by flow cytometry, makingit difficult to compare results among studies. A rationallybased ranking list of Treg markers has been agreed uponduring a workshop on the detection and functional testingof Treg organized by the Cancer Immunotherapy (CIMT)Immunoguiding Program (CIP) in 2013. This workshopresulted in the definition of a minimally required markerset for identification of human Treg as CD3+, CD4+,CD25+/++, CD127- and Foxp3+ cells. Staining for Ki67and CD45RA provides additional information on Tregactivation status. A corresponding robust gating strategyfor the context-dependent analysis of Treg by flowcytometry has also been defined. Additional markers canbe used for refining Treg profile in the different clinicalsettings and get more insights on Treg biology, namely,Helios, CTLA4, CD39 and CD73.Flow cytometry allows Treg functionality to be tested. TheCFSE-based T-cell suppression assay is the most com-mon assay used. However, comparable results can beobtained by testing IL-2 producing capability. This assayis easy-to-perform and less time- and cell-consumingthan the CFSE-based T-cell suppression assay.

REGULATION OF THE AUTOPHAGIC PROCESSCaraglia M., Lombardi A., Grimaldi A., Misso G.,Zappavigna S.Department of Biochemistry, Biophysics and GeneralPathology; Second University of Naples, Naples, [email protected]

Autophagy is an intracellular degradation system thatdelivers cytoplasmic constituents to the lysosomes.Autophagy consists of several sequential steps—seque-stration, transport to lysosomes, degradation, and utiliza-tion of degradation products—and each step may exertdifferent functions. The central machinery of autophagyincludes a series of complexes comprised of autophagy-related (Atg) proteins, executing the formation ofautophagosomes: membrane nucleation, expansion, clo-sure and maturation by fusing with lysosomes. It is gene-rally activated by conditions of nutrient deprivation buthas also been associated with physiological as well aspathological processes such as development, differentia-tion, neurodegenerative diseases, stress, infection, andcancer. In fact, anticancer drugs can induce autophagybut it remains controversial whether this process leads tocancer cell death or protects cancer cells from cellularstress and other forms of cell death such as apoptosis orsenescence. The role of autophagy in cancer is complexand is likely dependent on tumor type, stage, andgenetic context. However, recent evidences demonstratea tight interconnection of autophagy with several celldeath pathways and reveal an active contribution ofautophagy to cell death. When autophagy is directlyinvolved in the death process, the cell death process is

Invited Speakers

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designated “autophagic cell death” (ACD). We havedemonstrated in some studies by FACS, TEM, andwestern blot that autophagy activation and cell death wasinduced by overexpression of some miRNAs (i.e.: miR-423-5p in hepatocellular carcinoma) or by some drugcombinations based upon mTOR inhibitors (i.e.:everolimus) and autophagolysosome inhibitors (i.e.:choloroquine) in renal cell cancer. In conclusion, theunderstanding of the fine tuning of the signal transductionpathways regulating cell death and controlling the switchfrom autophagy to apoptosis and viceversa is pivotal inthe design of new anti-cancer/anti-angiogenic strategies.

IN VITRO EFFECTS OF NANOPARTICLES ONOSTEOBLASTS DIFFERENTIATION IN MICROGRAVITYCristofaro F.,1 Pani G.,2 Pascucci B.,3 Rizzo A.M.,2 ReaG.,3 Vukich M.,4 Visai L.1,51Department of Molecular Medicine, University of Pavia,Biochemistry Unit, Via Taramelli 3/b, 27100 Pavia, Italy2Department of Pharmacological and BiomolecularSciences, University of Milan, Via D. Trentacoste 2,20134 Milan, Italy 3Institute of Crystallography, CNR,00015 Monterotondo Scalo, Rome, Italy 4Kayser ItaliaS.r.l. Via di Popogna, 501, 57124 Livorno, Italy5Salvatore Maugeri Foundation, IRCCS, Lab ofNanothecnology, Via S.Boezio 28, 27100 Pavia, [email protected]

Exposure to microgravity has been associated withseveral physiological changes in astronauts, including anosteoporosis-like loss in bone mass. Osteoporosis is adisease of bones which leads to a high risk of fractureand other problems, mainly due to the reduction of thebone mineral density. The lack of weight-bearing forcesmakes microgravity an ideal physical stimulus to assessbone cell responses. In this study, we aimed to determinethe effect of nanoparticles as a countermesasure tomicrogravity-induced osteoporosis on osteoblasts cells.Nanoparticles of hydroxyapatite (nHaps) and strontium-enriched hydroxyapatite (Sr-nHaps) were obtained bypowder dispersion in bovine serum albumin. Physical-chemical characterizations of nHap and Sr-nHap wereperformed measuring hydrodynamic radius, z-potentialand taking images at transmission electron microscopeobservations (TEM). Biological studies were performed invitro by evaluating osteoblast viability and calcium depo-sition in the presence/absence of simulated microgravitywith/without both type of nanoparticles. The resultsshowed that osteoblast viability like as calcium depositionwere increased by treatment with Sr-nHaps in simulatedmicrogravity if compared to the untreated or treated cellswith nHaps.All these preliminary data seem to suggest that thesenanoparticles could be a useful system for the delivery ofstrontium for the treatment of osteoporosis in simulatedmicrogravity.

CIRCULATING TUMOR CELLS AND CIRCULATINGTUMOR DNA: THE LIQUID BIOPSY FOR CANCERDanova M. e Torchio M.Dipartimento di Area Medica, Azienda Ospedaliera diPavia, e Università di Pavia - [email protected]

Targeted therapies have profoundly changed theapproach to cancer treatment over the past 10 years.However, almost all tumors acquire resistance to sys-temic treatment as a result of tumor heterogeneity, clonalevolution and selection. Although genotyping is the most

currently utilized method for categorizing tumors for clini-cal decisions, tumor tissue provide only a snapshot or isofter difficult to obtain. To overcome these issues meth-ods are needed for a rapid, cost effective and non-inva-sive identification of cellular and molecular biomarkers atvarious time points during the course of the disease. Inorder to assess these issues the use of surrogatesources of DNA such as blood samples which often con-tains circulating tumor cells (CTCs) and circulating tumorcell-free DNA (ctDNA) are emerging as new strategies fortumor genotyping and may provide prognostic and pre-dictive biomarkers for clinicians. This so called liquidbiopsy has the potential to enable non-invasive tests forpersonalized medicine in providing similar information asthat derived from invasive tumor biopsies. Although theanalysis of CTCs and of ctDNA represent a very promis-ing area and despite the several efforts to develop andvalidate suitable and reproducible tools for a comprehen-sive analysis of tumor genomes, is not yet routinely usedas a clinical application. A deeper knowledge of preana-lytical and analytical problems related to both the proce-dures is needed to provide clinical standards for both theapproaches. The dedicated session of the Conferencewill consider how the liquid biopsy has contributed tosignificant insight on the knowledge of the metastaticprocess biology and how it may be utilized in clinicalpractice. The session will also include an overview of therecent studies exploring the diagnostic, prognostic andpredictive potential of CTCs and ctDNA. Biological andtechnical aspects, including recent advances in the ana-lytical sensitivity and accuracy of the various methods forboth CTC isolation and ctDNA analysis will be covered aswell as hurdles that have to be overcome before imple-mentation into the clinical oncology routine.

VALIDATIONOFASTANDARDIZEDMETHODFORENU-MERATING CIRCULATING ENDOTHELIAL CELLS ANDPROGENITORS BY FLOW CYTOMETRY: A STUDYFORM THE ITALIAN SOCIETY OF CYTOMETRY (GIC)Matteo G. Della Porta, MDDepartment of Hematology & Oncology - Fondazione IRCCSPoliclinico San Matteo University of Pavia Medical School P.leGolgi 19, 27100 Pavia - [email protected]

An increase in the number of circulating endothelial cells(CEC) and progenitors (CEP) has been reported in manydiseases, including cancer. Antiangiogenic drugs havebeen recently approved for the therapy of solid tumors, butthere is a need for new biomarkers to define the optimalbiological dose and dosing schedule of these molecules.Several studies indicate that CEC and CEP kinetics corre-late with survival in cancer patients treated with antiangio-genic therapies and might help to stratify patients who arelikely to receive clinical benefit from antiangiogenic or anti-vascular treatments. In these clinical studies, CECs havebeen measured in most cases by multiparametric flowcytometry, an approach that requires accurate sequentialgating and is prone to operator-induced variability.Moreover, antigenic overlap between CECs and plateletshas generated the concern that some of the cells countedas CECs by flow cytometry might not be endothelial cells.Thus, rigorous standardization of the flow cytometric enu-meration procedure is crucial before CECs and CEPs canbe reliably measured in clinical trials.Here, we report the experimental validation of a standar-dized flow cytometry method for enumerating CECs andCEPs in blood samples. CECs and CEPs were evaluatedby six-color flow cytometry using the nuclear staining


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Syto16 and 7-AAD and a panel of monoclonal antibodiesincluding anti-CD45 (to exclude hematopoietic cells),anti-CD133 (a progenitor cell marker), anti-CD31, andanti-CD146 (endothelial cell markers). Appropriate analy-sis gates were used to enumerate viable and apoptoticCECs and CEPs. Cell suspensions were evaluated afterred cell lysis by a FACSCanto (Becton Dickinson) orNavios Flow Cytometer (Beckman Coulter). After acquisi-tion of at least 1 × 106 cells per blood sample, analyseswere considered as informative when adequate numbersof cells (>100, typically 300-400) were collected in theCEC enumeration gates. CECs were defined as DNA(Syto16) positive, negative for the hematopoietic markerCD45, positive for the endothelial markers CD31 andCD146, and negative for the progenitor marker CD133.CEPs were depicted by the expression of CD133. Thecombination of Syto16 and 7-AAD was used to gaininsight into CEC viability: necrotic cells were identified asSyto16low/7-AAD+, apoptotic cells as Syto16low/7-AAD−, and viable cells as Syto16bright/7-AAD−.Toassess the reproducibility of the procedure and validate it,fresh sample were evaluated from different operators.Three ml of peripheral blood from 32 healthy subjects and4 cord blood were collected in EDTA tubes and dispat-ched to nine different laboratories. A full white blood cellcount was performed on an hemoanalyzer and sent to alloperators along with samples.In healthy donors CECs were 81.3 ± 32.8/ml, while CEPswere 144 ± 69/ml. The fraction of apoptotic/necrotic CECswas 69 +/- 13%. Coefficients of variation (CV, interreader)were found to be i) 0.21 (range 0.15-0.39) for Syto16+CD45-CD31+CD146+ CECs, ii) 0.12 (0.07-0.45) for Syto16+/CD34+ progenitors, and CV 0.29 (0.17-0.47) for CD45-/Syto16 +/CD34+ cells. We sorted CD34+/CD45+ andCD34+/CD45- compartments form three cord blood samplesand in all cases and in all cases Endotehlial Colony FormingCells (ECFC, i.e. truly endothelial progenitors) were obtainedexclusively form CD34+/CD45- progenitors. The evaluationof CD45-/ Syto16 +/CD34+/VEGFR + and CD45-/ Syto16+/CD34+/CD133+ endothelial progenitors did not reached anadequate interreader reproducibility (CV 0.60, range 0.39-0.94 and CV 0.97, range 0.67-1.15, respectively).This procedure enumerates a truly endothelial cell popu-lation with limited interreader variability. This methodscan be easily implemented in clinical practice.

T CELLS AND CD161: LINEAGE OR FUNCTION?De Palma R.Dept. of Clinical and Experimental Medicine - SecondUniversity of Naples [email protected]

In the last few years, a growing body of data has changedour knowledge regarding functional specificity of immunecells, unveiling a high degree of plasticity between differentcell subsets and, more intriguingly, between different celllineages. Strikingly, different surface molecules can beexpressed by different cells lineages or by cells with diffe-rent function. This talk will try to address recent findingsabout the “connections” between the expression of CD161and T cells. CD161, KLRB1, is the human homolog ofmurine NKRP1, first identified on NK cells. More recently,several groups have shown that the expression of CD161correlates with IL-17 production in CD4+and CD8+ T cells,bearing α/β or γ/δ receptor. Even more intriguingly,CD161 expression seems to correlate with IL-17 produc-tion by Innate Lymphoyd Cells.These findings led to further investigate the role of thismolecule as phenotype marker, as possible effector mole-

cule or as chemotactic player. Finally, the expression ofCD161 on cells in inflamed tissue seems to pair with theability to induce damage more than with a specific functio-nal subset. A better understanding of role and function ofCD161 expression could be instrumental to set new thera-peutic approach in immune mediated inflammation.

HOW TO GET THE MOST OUT OF YOUR CYTOME-TER: AMPLIFICATION, COMPENSATION AND HOWTO DEFINE POSITIVE-NEGATIVE THRESHOLDFattorossi A.Gynecologic Oncology Unit, Catholic University,Roma, [email protected]

Most proteins in the cell can be measured simultaneous-ly by flow cytometry using appropriate antibodies (abs).Because the emission spectra of fluorescent dyes arebroad, fluorescence compensation is necessary. Propercompensation requires proper PMT voltages. Althoughunstained beads can be used, cells with the same fluo-rescence background as the final sample are the bestchoice. Fluorescence background is peculiar to each cellin a mixed cells population, e.g. lymphocyte vs mono-cytes, and also depends on cell activation status andwhether fresh or fixed cells are to be analyzed. For com-pensation, cells (or abs capture beads) are labelled singlywith each of the fluorochromes. It is essential that singlylabelled cells and beads be brighter than the experimen-tal samples. Several software packages are available tocompensate automatically, once the data from each fluo-rochrome has been recorded. Do not alter PMT voltagesafterwards. Select fluorochrome-conjugated abs combi-nations with minimum spectral overlap to avoid or mini-mize a decrease in sensitivity during analysis of multicol-or cell samples because distribution of compensatedbackground values for a given subset becomes broaderas the extent of spectral overlap that must be correctedby compensation increases. Optimize combinations offluorochrome-conjugated abs such that the low-antigen-density targets are labeled with abs conjugated to brightfluorochromes and targets expressed in abundance arelabeled with ‘‘less bright’’ conjugates. Define positive-negative threshold using isotype controls (not the bestchoice and expensive), isoclonic controls (best choiceand very expensive), unstained controls (acceptabletrade-off and very cheap), each of these using fluores-cence-minus-one sample (not so cheap).

EXTRACELLULAR MICRORNAS IN FLOW CYTOME-TRY: FROM THE BENCH TO CLINICAL APPLICATIONSFelsani A.Institute of Cell Biology and Neurobiology-CNR,Rome, [email protected]

MicroRNAs (miRNAs) are small (18-25 nucletoidi) single-stranded non-coding RNAs, highly conserved from anevolutionary point of view, that recognize by a mecha-nism involving partial base-pairing specific target mes-senger RNAs, resulting either in their degradation or inhi-bition of their translation. MiRNAs are key post-transcrip-tional regulators of gene expression involved in many cel-lular processes such as proliferation, growth, differentia-tion, apoptosis and immune response. Currently morethan 5000 miRNAs, but not their functions, are known.Their expression is frequently altered in many diseasesand cancers and correlates with the prognosis and theprogression of the neoplastic disease.


miRNAs are molecules stable and easily measurable in tis-sues and body fluids, where they are present because ofphysiological and pathological processes. ExtracellularmiRNAs are protected from degradation because are con-tained mainly in two types of vesicles, exosomes andmacrovesicles, produced by different cellular mechanisms.The recent increase in sensitivity of technologies for theanalysis of genetic material has allowed sampling andanalyzing the minute amounts of cell-free DNA/RNAreleased in the blood from the tumors, giving origin to thepractice of liquid biopsy as an approach alternative to thestandard biopsy to monitor the genetic profile of tumors.Liquid biopsies offer many advantages besides being aless painful, easier and less expensive sampling proce-dure. First, liquid biopsies may capture the entire hetero-geneity of the disease, since nucleic acids from differenttumor parts and cell types could be represented simulta-neously in the blood stream. Second, liquid biopsies givethe possibility to take serial samples in order to monitortumor genomic changes under selection pressure.More difficult, but extremely important, is the task ofanalyzing the characteristics of the vesicles carrying nucleicacids and in particular miRNAs. In fact, the same miRNAcanbe produced and released in the blood stream from tumor orhealthy body organs, but clearly with different pathological orphysiological meaning. The analysis of the proteins presentat the vesicles’ surface can help to trace their origin, addingto the profile of their miRNA content an information essentialto understand their diagnostic relevance.Flow cytometry is the most promising technique to per-form this task and should be considered as a method ofchoice for detection and analysis of extracellular vesiclesin biological fluids. Because conventional flow cytome-ters cannot distinguish between vesicles that are <300nm, techniques allowing indirect visualization of extracel-lular vesicles have been developed. Approaches, such asadsorption of exosomes to antitetraspanin coated latexbeads, may overcome the limits of currently availableinstrumentation.

FLOW CYTOMETRY FOR THE DIAGNOSIS OFACUTE LYMPHOID LEUKEMIA (ALL).IS IT POSSIBLE TO IDENTIFY EARLY T AND BCR-ABL1-LIKE ALL?Guarini A., De Propris M.S., Foà R.Hematology, Department of Cellular Biotechnologiesand Hematology, “Sapienza” University, [email protected]

In hematologic diseases, flow cytometry represents themost important technique to identify and characterizenormal and neoplastic cells. At the time of the diagnosisof an acute leukemia, the immunophenotypic work-uphas several objectives: a) lineage assignment, b) evalua-tion of cell maturation, and c) assessment of phenotypicaberrations. Lineage assignment of blast cells byimmunophenotype may still represent a major challengein acute leukemias. The first step of the diagnostic work-up consists in the evaluation of intracytoplasmatic anti-gens: myeloperoxidase (MPO) for the myeloid lineage,and CD79a and CD3 for the B- and T-cell lineages,respectively. The second step analyzes antigens associa-ted with differentiation and maturation, such as CD7,CD1a, CD2, sCD3, CD5, CD99, TdT, TCRαβ, TCRγδ forT-cell acute lymphoid leukemia (T-ALL) and CD10, CD19,CD20, CD22, CD38, CD45, CD58, NG2, CD66c, TdT forB-lineage ALL. Furthermore, CD34 is evaluated in allcases. Through such an approach, it is possible to clas-sify T- and B-ALL according to the maturation stage,

make correlation with response to treatment and progno-sis, and monitor minimal residual disease.Two new subgroups of ALL - early T and BCR-ABL1-likeALL - have been identified by gene expression profiling(GEP) and associated to a poor prognosis. It would beextremely important to identify at presentation such caseswith simpler methods, as flow cytometry, in order to opti-mize/personalize treatment. Indeed, the immunopheno-type of early T-ALL has been characterized:cCD3+/CD7+/CD5low/CD1a-/CD8- and the positivivity forone or more stem or myeloid cell antigens: CD117, CD34,HLA-DR, CD13, CD33, CD11b, CD65. On the contrary, forBCR-ABL1-like ALL no immunophenotypic-baseddiagnostic combination - not even using antibodies againstCD66c, CRLF2, CD99, CD97, suggested by GEP or wholegenome sequencing - has produced reliable data.

FISHIS AND FLOW SORTING SIMPLIFY COMPLEXGENOMICS: ACCESSING THE HEXAPLOID OATGENOMELangdon T.*, Giorgi D.**, Hegarty M.*, FarinaA.**, Bisaga M.*,Grosso V.**, Lucretti S.***IBERS, Aberystwyth University, Aberystwyth, UnitedKindom; **ENEA Casaccia - Italian Agency for NewTechnologies, Via Anguillarese 301, Rome (Italy)[email protected]

The traditional chromosome approach can facilitate thedevelopment of genomics in complex and polyploidgenomes, if single chromosomes differ from each other inDNA amount. In plants, this is a severe limitation sincethis is a rare event to occur. To overcome such an incon-venient, we have deleloped and used FISHIS (Giorgi etal. 2013) for oat chromosomes flow sorting, showing thatthe appropriate use of labelled oligonucleotides wouldallow single chromosomes and their groups to be sepa-rated in sufficient quantity and purity, suitable forsequencing. Preliminary work has been carried out inAvena sativa cv Firth to confirm the method can be effi-ciently adapted to oat and conditions have been foundallowing a clear separation of four specific chromosomes,two on the basis of size alone and two by size andhybridisation with a fluorescently labelled simple repeatoligonucleotide. Several flow sorted fractions, containinga mixture of specific chromosome types were also isolat-ed for GBS, using an internal standard to assess analy-sis and sorting stability all along the experiments. DNAfrom flow-sorted chromosomes can be easily purified in10-20ng quantities, sufficient to allow Nextera librarypreparation and comprehensive Illumina sequencing. Foreach sorted fractions ~20 million paired HiSeq reads2x100bp, were generated, and assemblies (CLC Bio)were compared with GBS data from Huang et al. Genicregions of the sorted chromosomes have been assem-bled to N50s larger than 2kb using diploid genome con-tigs as guides. We have used genotyping-by-sorting andsequencing (GBS2) to produce a reduced genome repre-sentation and target polymorphisms in the genomicspace flanking restriction enzyme sites. Combined withmultiplexing samples using DNA barcoded adapters, thisapproach has enabled high-throughput genotyping withrelatively low per sample costs. The identity of thesechromosomes and the scale of isolation required for com-prehensive sequence coverage is currently being investi-gated and probes identified which would allow the isola-tion of all chromosomes, at least in small pools (regions)not containing homeologous. The application of sub-genome specific probes, such as As120a sequence,could facilitate the isolation of homeologus genomes.

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GBS2 can be a useful tool to disentagle chromosomehomeologus sequencing overlapping and facilitategenomics in complex plant polyploids.

CHARACTERIZATION OF CELLULAR PRODUCTSINTENDED FOR THE MEDICAL USE IN HUMANSLanza F, Brambilla P, Lazzari C, Di Martino G, Ravelli A,*Crotti M.Hematology Institute and Flow Cytometry Unit, *BloodBank Unit, Hospital of Cremona- [email protected]

The emerging scientific and medical progresses havegiven rise to the development of highly-innovative medici-nal products based on cells or tissues. Furthermore, thecombination of cell biology, medicine, and engineering forthe purpose of repairing, replacing or regeneratinghuman tissues (also called “regenerative medicine” ) hasled to the new field of Tissue Engineering. These noveltherapeutic strategies for patients, developers and indus-try have shown a great number of potential clinical appli-cations; however, new risks have to be faced andaddressed appropriately in the field. In December 2008,the “Regulation on Advanced Therapy MedicinalProducts”(ATMPs) has entered into force in the EuropeanUnion (EU) , classifying gene therapy-, somatic cell thera-py- and tissue engineered-products as pharmaceuticals.Subsequently, additional regulatory guidances and pro-cedures have been developed recently in order toaddress the specific properties of ATMPs.It is recognized that the evaluation of ATMPs oftenrequires very specific expertise, which goes beyond thetraditional pharmaceutical field and covers borderlineareas related to other sectors such as biotechnology,medical devices, mechanical sciences, transplantationand surgery. Interestingly, the Art. 28 of the Regulation1394/2007/EC introduced the “hospital exemption” fromthe centralized marketing authorization procedure for anATMP product used in a hospital on a non routine basefor single patients. Other limitations to the exemptionwere that the product should not cross the border of themember state and that should not to be the product of anindustrial process, but only of a “non-routine procedure”instead. Further clarification of the legislation was left tothe competent authorities of the individual memberstates. However, only a few member states emanatedlegislation on the art 28 application so far, with only a par-tial harmonization.This paper focuses on the current EU regulatory frame-work in the context of practical challenges regardingquality, preclinical and clinical issues, which should befaced prospectively during the development phase ofcell-based medicinal products, in order to apply for manu-facturing authorizations, clinical trials, lincensing underthe hospital exemption clause or for centralized europeanmarking authorization. The new legislation has establi-shed a dedicated path for the marketing authorisation(licensing) of ATMPs, specific tools and incentives to fos-ter research and development in this field and to facilitatetheir access to the EU market, while guaranteeing thehighest level of health protection for patients.Furthermore, this paper deals with guidelines to assistphysicians and laboratory technologists with the settingup of a cell processing laboratory (CPL) in support to ahematopoietic stem cell transplant (HSCT) program. Thisguide describes the essential features of designing sucha laboratory and provides a list of equipment and supplyneeds and staffing recommendations. It describes thetypical scope of services that a CPL is expected to per-

form, including product testing services, and discussesthe basic principles behind the most frequent procedures.Quality management principles specific to a CPL are alsodiscussed. References to additional guidance documentsthat are available in Europe to assist with quality manage-ment and regulatory compliance are also provided.

GUIDELINES FOR APPROPRIATE IMMUNOPHENO-TYPE: APPLICATION TO HUMAN MEMORY T CELLSLugli E.Laboratory of Translational Immunology, Humanitas FlowCytometry Core Humanitas Clinical and ResearchCenter, Rozzano, [email protected]

Flow cytometry is routinely used for the analysis of thehematopoietic system and to monitor changes in theimmune system during therapies for infections, immunodefi-ciencies and cancer. The recent success of immunotherapy,especially in the treatment of solid tumors through the use ofcheckpoint blockade, has raised new interest in the study ofthe immune system to combat tumors. Specific changes inimmune system's subpopulations may lead to the identifica-tion of the patients that will respond to such therapies.Polychromatic flow cytometry can nowadays identify up to18 antigens in the same cell, evaluate the presence andenumerate rare populations and identify functional mole-cules and dozens of cellular subsets. In this talk, I will illustra-te the procedures to evaluate these subpopulations in a cor-rect manner, and I will discuss how to proceed woth reagenttitration, multicolor compensation and exclusion of unwant-ed/false positive events. I will apply these procedures to theidentification of naive and memory T cell subsets in thehuman blood and tissues during immunotherapy for solidtumor and during immune reconstitution followingallogeneic bone marrow transplantation.

APPLICATIONS OF FLOW CYTOMETRY TO CLINICALMICROBIOLOGYManti A., Canonico B. and Papa S.Department of Biomolecular Science, University ofUrbino “Carlo Bo”, [email protected]

In order to provide an overview of the applications of flowcytometry in microbiology, several protocols will be fur-nished.Flow cytometry is a powerful tool that allows the rapidanalysis of entire cell population on the basis of singlecells characteristics. In the last years of 1990s, the appli-cations of FCM in microbiology have significantlyincreased. In fact, several articles propose flow cytome-try to identify, to enumerate and to characterizefunctional properties of bacterial populations from envi-ronmental samples, qualifying FCM also as a rapid diag-nostic tool in the fight against infections in clinical micro-biology.In particular, FCM has been also used in physiologic andmetabolic studies of microrganisms (cell count, viabilitytest in bacteria and yeasts, discrimination of gram-posi-tive from gram-negative bacteria, antibiotic resistance,membrane potential, etc). Furthermore, the use ofspecific antibodies fluorochrome- conjugated could be ahelpful method for identifying specific bacteria in hetero-geneous samples. FCM can also be used to detect andquantify virus, fungi and parasites present in clinical sam-ples.


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MECHANISMS OF REGULATION OF APOPTOSISMarchini S.IRCCS 'Mario Negri' Institute for PharmacologicalResearch, Milan, [email protected]

Apoptosis is the most common word used to identify theway through which cells decide to suicide. Studies per-formed so far over the last 30 years have demonstratedthat this is an “umbrella” term, and that in a given tissue,at the same time, cells are able to activate different kindsof programmed cell death. From a molecular point ofview, different coding and non coding genes have beenidentified to be key players in controlling and driving thedifferent features of apoptosis. These programs aregenerally conserved along the evolution from C. elegansto Homo sapiens. The knowledge of the way throughwhich programmed cell death is regulated in both normaland pathological tissues is of utmost importance toimprove current therapeutic protocols.The different kinds of programmed cell death, their mecha-nisms of activation and regulation will be discussed indetail with a special attention to the biology of cancer cells,where impairment of programmed cell death has beenrecognized as one of the mechanisms through whichtumor cell survive along with neoplastic transformation.

GENETIC VARIANTS REGULATING IMMUNE CELLLEVELS IN HEALTH AND DISEASEOrrù V.1, Steri M.1, Serra V.1,2, Marongiu M.1, Sole G.1, SidoreC.1,2, Virdis F.1, Dei M.1, Lai S.1, Zoledziewska M.1, MulasA.1,2, Piras M.G.1, Lobina M.1,2, Whalen M.B.1, Sanna S.1,Fiorillo E.1, Cucca F.1,2.1.Istituto di Ricerca Genetica e Biomedica (IRGB), CNR,Monserrato, 09042, Italy; 2.Dipartimento di ScienzeBiomediche, Università di Sassari, 07100, Sassari, [email protected]

The recent combination of polychromatic flow cytometrywith genome-wide association scans (GWAS) identifiedgenetic factors underlying complex immune-related di-seases or quantitative variation of broadly classified cellpopulations. We considered that cell subtypes, whichplay specific roles in the tight balance and regulation ofthe immune system could have much more definitegenetic specification (and hence larger effect size) thancould be assessed in combined cell populations. Hence,a detailed analysis of the different cell subtypes wouldprovide a more comprehensive view of immune cell regu-lation and the underlying genetic factors.Aiming to uncover genetic factors involved in physiologi-cal and pathological immune cell quantitative variations,we measured more than 300 immune-related phenotypesin about 4,000 individuals, and performed genetic analy-ses using up to ~26 Million variants resulting from theintegration of high density genotyping data and low passwhole genome sequencing. Our results show that the lev-els of the majority of the circulating immune cells arehighly heritable (mean 41%), and a number of specificDNA changes account for a significant part of their inher-ited variation. Furthermore, some of these variants areassociated with immune-related pathologies and othermaladies in which the role of the immune system was notpreviously highlighted.

THE TELOMERES IN AGEING AND DISEASERossiello F.IFOM, the FIRC Institute of Molecular Oncology,Milano, [email protected]

All the cells in our body, with the exception of germ cells,are mortal. This is due to the shortening of telomeres,the structures at the ends of linear chromosomes, whichcan not be replicated in full at each cell cycle. Whentelomeres get too short, cells undergo a permanentarrest of proliferation, known as cellular senescence.Indeed, when they reach a critical length, the telomeresinduce cell cycle arrest, by activating an intracellularpathway, the DNA damage response (DDR). Thispathway is activated also when the genomic DNA isdamaged. By treating cells with DDR inhibitors, or byknocking down the genes needed for its activation,senescent cells start proliferating again.However, cellular senescence is not exclusively causedby telomere shortening. Indeed, when the telomeres aredamaged by endogenous or exogenous sources of DNAdamage, the mechanisms of DNA repair are inhibitedspecifically at telomeres, in order to prevent chromoso-mal fusions, and telomeres show a persistent DDR,which induces cellular senescence. This recapitulatewhat happens in non-proliferating tissues of the body,where senescence acitvation can not be explained bytelomere shortening, as a result of cell proliferation.Cancer is caused by the deregulation of cellularpathways that determine the uncontrolled proliferationof cells. When this occurs, replicative stress causesDNA damage at telomeres, which in turn induces sene-scence. Thus, cellular senescence is considered as ananticancer mechanism, which prevents the uncontrol-led proliferation of cells expressing an oncogene.Consistently, when DDR genes are repressed, oncoge-ne-induced senescence is not activated, and cancercells continue to proliferate, resulting in tumor progres-sion. In conclusion, different types of cellular senescen-ce (replicative, induced by DNA damage and oncoge-ne-induced) are caused by activation of the DDR attelomeres.

PROGRAMMED CELL DEATH IN THE CELLULARRESPONSE TO ANTICANCER AGENTSTagliaferri P., Ventura M., Di Martino M.T., Botta C.,Raimondi L., Rota R. and Tassone P.Magna Graecia University of Catanzaro and BambinGesù Hospital Rome, [email protected]

Programmed dell death has a pivotal role in the cellularresponse to a variety of anticancer agents. It is now beco-ming clear that two main factors have a prominent role inthe benefit of treatment: the activation of survival mecha-nisms and the induction of immunogenic cell death. Thesurvival response can be genetically driven by escapemutations but may also involve transient activation of sur-vival signalling. At this aim we have demonstrated thatsensitivity of BRCA1 mutated breast cancer cells tocisplatinum (CDDP) is not only mediated by BRCA1-rela-ted deficiency in DNA repair mechanisms, but also tochanges in the Notch signaling pathway. We providedproof-of-concept that silencing Notch3 Intracellulardomain (ICD) potentiated CDDP activity that was, howe-ver, antagonized by dominant positive Notch3 ICD. Thisfinding opens a new avenue to combinatory approachesinvolving Notch inhibitor as gamma secretase inhibitors in



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improving apoptosis of breast cancer cells by conventio-nal cytotoxic agents.An additional important finding is that immunogenic celldeath induced by anticancer agents can indeed activateanticancer T cell response opening a new avenue forcombinatory approaches involving immunogenic celldeath inducers with immune check point inhibitors.All these experimental approaches can be based on insilico integrative genomic prediction tools to be validatedin translational wet-biology models within the evolvingprecision medicine paradigm.

NANOVECTORS AND CYTOMETRY TO STUDY THENEUROBIOLOGICAL DETAILSVeglianese P.Istituto di Ricerche Farmacologiche Mario Negri,Milano - [email protected]

Many efforts have been performed to understand the roleof recruited macrophages in the progression of the spinalcord injury (SCI). Different studies revealed a pleiotropiceffect of this cellular compartment associated to distinctphenotypes (M1 and M2) that show a predictable spatialand temporal distribution in the injured site after SCI.Differently, the role of activated microglia in injury pro-gression has been poorly investigated so far, mainlybecause of the unfeasibility to target and modulate selec-tively microglia in the injury site. Here by exploiting a deli-very nanovector tool able to treat/target selectively micro-glia we overcome these limitations and clarify the tempo-ral and spatial involvement of the pro-inflammatoryresponse associated to the activated microglia in SCI.We show that the treatment with minocycline, a well-known anti-inflammatory drug, when administered prom-ptly by nanoparticles in the early phase of SCI is able tomodulate efficiently resident microglial cells reducing thepro-inflammatory response, maintaining a pro-regenerati-ve milieu and ameliorating the behavioural outcome up to63 days post injury. Furthermore, we provide a mechani-stic link between early microglia activation and M1macrophage recruitment via CCL2 chemokine, furtherrevealing a detrimental contribution of macrophages ininjury progression after SCI.

ASSESSING PREY-PREDATOR INTERACTIONSAMONG AQUATIC MICROBES BY FLOW CYTOMETRY:AN EXPERIMENTAL STUDYAmalfitano S.,1 Callieri C.,2 Corno G.,2 Bertoni R.21Water Research Institute (CNR-IRSA), Via Salaria km29, 300, Monterotondo, Rome, Italy; 2Institute ofEcosystem Study (CNR-ISE), Largo Tonolli 50, 28922Verbania, [email protected]

Prey-predator interactions are among the major ecologi-cal factors inducing structural changes within aquaticmicrobial communities. In particular, the active formationof microbial aggregates was proposed as an efficientdefence strategy against size-selective predation by pro-tists. To better understand this ecological mechanism, thetraditional microscopic counting is time-consuming andcost-effective, given the variety of the microorganismsinvolved. Instead, the use of flow cytometry could allowthe rapid quantification of several different interactingmicrobes.In this study, we set-up and applied a flow cytometric pro-tocol, based on scatter and fluorescence signals, to fol-low the dynamics of bacterial and protist single cells andmicrocolonies in semi-continuous cultures under differenttreatments (single and co-cultures, with and withoutpredators). During four days of incubation, we assessedthe impact of grazing by the mixotrophic nanoflagellatePoteriochromonas on the aggregation of two freshwatercyanobacterial strains and their associated bacteria. Thephotosynthetic efficiency of the two strains, belonging todifferently pigmented phylogenetic clades ofSynechococcus (phycoerythrin-rich cells, PE, Group A;phycocyanin-rich cells, PC, Group I), was also measured(PhytoPAM).All results indicated that grazing unfavourably affectedthe development of bacterial single cells, while promotingthe formation of microcolonies. In single cultures underpredation, we observed the formation of grazing-inducedmonoclonal PE microcolonies, whereas PC cells did notaggregate. In co-culture, an interaction between PE andPC emerged, with microcolonies containing both PE andPC and an increase of PC photosynthetic fitness (Fv/Fm).Again, in co-culture, the microenvironment, generated bythe formation of PE microcolonies, PC cells,heterotrophic bacteria and predators, can be the site of abeneficial “quorum sensing” interaction between the twophylotypes of Synechococcus.

AN INTEGRATED APPROACH OF FLOW CYTOME-TRY, CARD FISH AND NEXT GENERATIONSEQUENCING TOASSESSABUNDANCE, DIVERSITY,AND ACTIVITY OF PICOPLANKTON IN THE SARNORIVER PLUME (Gulf of Naples, Italy).Balestra C., Casotti R.Stazione Zoologica A. Dohrn, Villa Comunale 80121Naples, [email protected]

Abundance, diversity, and activity of picoplankton in theSarno River plume (Gulf of Naples) were investigated usingmultiple approaches. Samples were collected at the surfacealong an increasing salinity gradient from the station closest

Environmental Sciences andToxicology



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to the Sarno River (St.1) to the farthest station (St.5) in April2013. For autotrophic picoplankton, Synechococcus,Prochlorococcus, and Picoeukaryotes and for heterotrophicbacteria, abundances were estimated by flow cytometry.Bacterial community composition and activity were estimat-ed by CARD-FISH and MAR-CARD-FISH, respectively.Total environmental DNA and RNA were also sequenced inorder to provide a high-resolution estimate of biodiversityand functional diversity in terms of transcripts as a proxy ofpotential activity of heterotrophic bacteria. BothProchlorococcus and Synechococcus increased towardsmarine waters so as picoeukaryotes, while heterotrophicbacteria were more abundant in the river-influenced waters,confirming their role as remineralizers of organic matter andas nutrient recyclers.Total Eubacterial cells were on average 56% ± 8.7%, sug-gesting the presence of Archaea as well as other non-Eubacteria. Within the Eubacteria, Alphaproteobacteriawere the most abundant group (>40% average) from sta-tion 2 to station 5 and also most active while in station 1they were the less abundant suggesting a negative effectof the river. Within the Alphaproteobacteria, SAR 11 andRoseobacter showed an opposite trend with Roseobactermore active and abundant at st.1 and less abundant andactive at all the other stations. Abundance and activity ofGammaproteobacteria and Cytophaga-Flavobacterium-Bacteroides increased from st5 to st.1 confirming theirability to degrade organic matter from land or river inputs.

DIEL VARIATIONS OF A SUMMER MICROBIAL COM-MUNITY IN THE GULF OF NAPLESBalestra C., Trano A.C., Casotti R.Stazione Zoologica A. Dohrn, Villa Comunale 80121,Napoli, [email protected]

Diel variability is an important time scale to consider wheninvestigating the physiology and ecology of microbial com-munities. The picoplankton community (autotrophic andheterotrophic) of the Gulf of Naples was investigated inJuly 2012 during a Lagrangian experiment (Tosca Diel, TD)by sampling surface water every 2 h while following afloating device. The sampling strategy consisted inreleasing a floating buoy and following its path for 28 h,with the aim of analysing the same water body and itsassociated picoplankton community. Picoplankton abun-dances, both autotrophs (Synechococcus andPicoeukaryotes) and heterotrophic bacteria, wereestimated by flow cytometry, and the heterotrophicbacterial composition was estimated by CARD-FISH every6 h. Although the water masses characteristics shouldhave stayed constant, when the floating device encoun-tered a flowing coastal current, then a missing occurredand an unexpected evolution was observed. This wasreflected into a shift from more oligotrophic to rathereutrophic seawater, in terms of nutrients and also chloro-phyll content. Also, higher picoplankton concentrationswere observed and also a lower contribution ofAlphaproteobacteria (56%±3%) suggesting this as a mark-er of river influenced. In fact, the Sarno river plume showsa northbound flow, carrying nutrients and biomass. 66%(±4%) of heterotrophic bacteria were detected using theEubacteria probe by CARD-FISH. Among these, the mostabundant group was Alphaproteobacteria (56%±3%) andwithin them, SAR11 (32%±5%) was the most abundantgroup and showed an opposite trend to Roseobacter(16%±7%). Gammaproteobacteria (26%±4%) andCytophaga/Flavobacterium/Bacteroides clade (28%±4%)showed no variations during the 28 h sampling.

Altogether, these data point to a complex dynamics of thebacterial community which even in the summer, when cir-culation is reduced, depends upon the hydrological fac-tors and water mass characteristics which can vary atvery short time scales.

EVALUATION OF CYTOTOXIC AND GENOTOXICEFFECTS OF ALKALINE EARTH SILICATE WOOLSIN TWO KIND OF HUMAN RESPIRATORY CELLSCavallo D., Ursini C.L., Fresegna A.M., Ciervo A., MaielloR., Tassone P., Iavicoli S.INAIL - Italian Workers’ Compensation Authority –Research Area, Department of of Occupational andEnvironmental Medicine, Epidemiology and Hygiene,Rome, [email protected]

The toxicity of fibers currently used for thermal insulationas substituted of asbestos, is still not well understood.Among these, alkaline earth silicate wool (AESW) repre-sent a new generation of high-temperature insulationmaterial characterized by low biopersistence and highbiosolubility and then considered not hazardous.Cytotoxic, proliferative and genotoxic effects of AESW(isofrax), a fiber designed for high temperature (1260°C)usage and characterized by high magnesium percenta-ge, were investigated in human bronchial (BEAS-2B) andalveolar (A549) cells exposed to 2-200 µg/ml. Cytotoxiceffects were studied evaluating cell viability reduction bytrypan blue and proliferative effects by MTT and WST1assays, after 24h exposure. Membrane damage wasanalysed by LDH release after 30 min, 2h and 24h expo-sure. The genotoxic potential was evaluated after 4hexposure by Fpg comet assay. No cytotoxicity in terms ofviability reduction and cell proliferation was found in bothcell types. While an induction of membrane damage interms of increased LDH release was found at the lowerconcentrations with a peak at 20 µg/ml in BEAS-2B andat the highest concentrations in A549 cells. We found amoderate direct DNA damage at the highest concentra-tions in BEAS-2B and slight genotoxic effect at the lowerconcentrations in A549 cells. Oxidative DNA damagewas found only in BEAS-2B at the lower concentrationssuggesting an higher genotoxic susceptibility for normalbronchial cells. The findings show, in both cell lines, lowcytotoxicity in terms of cell viability and moderate induc-tion of membrane damage by AESW that could berelated to its specific chemical composition and biosolu-bility. Moreover, early genotoxic-oxidative effects werefound only in BEAS-2B cells suggesting further investiga-tions with longer exposure time to study the persistenceof effect found and clarify the potential toxicity of this kindof fibre whose adverse effects are not yet studied.

ACTIVITY OF “EXCRETED BIOSURFACTANTS” OFSEVERAL LACTIC ACID BACTERIA AGAINSTSTREPTOCOCCI ORAL BIOFILMCiandrini E., Campana R., Manti A., Casettari L., FagioliL., Papa S. and Baffone W.Department of Biomolecular Science, University ofUrbino “Carlo Bo”, [email protected]

Lactic acid bacteria (LAB) can interfere with pathogensthrough different mechanisms. One is the production ofbiosurfactants, a group of surface-active molecules whichinhibits the growth of potential pathogens. In the presentstudy the excreted biosurfactants of L. reuteri DSM17938, L. acidophilus DDS-1, L. rhamnosus ATCC 53103



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and L. paracasei B21060, were characterized in term oftheir activities. Excreted biosurfactanst were dialysed(molecular weight cut-off 1 and 6 kDa), freeze-dried andcharacterized in reduction of surface tension (ST) andemulsification index (E24). Then, standard methods andflow cytometry (FCM) were used to evaluate anti-biofilmproperties of biosurfactants against S. mutans ATCC25175 and S. oralis ATCC 9811 during the formation ofbiofilm on titanium surface.All the dialysed biosurfactants showed to reduce ST, witha mean of 38.90 mN/ for those dialyzed at 6 kDa com-pared to 53.0 mN/m of control culture medium, and toemulsify paraffin oil (mean 58.02 and 45.82 % for biosur-factants dialyzed at 6 and 1 kDa respectively). Moreover,dialysed biosurfactants were able to significantly inhibitformation of S. mutans ATCC 25175 and S. oralis ATCC9811 biofilm on titanium surface in dose-dependent way,as demonstrated by the remarkable decrease of cfu/mlvalues and in term of biomass. FCM viability test,assessed by double staining based on SYBR Green I andPropidium Iodide fluorochromes, showed a strong biofilminhibition percentages in term of total and viable cells ineach treated samples.This work highlights the potential application ofLactobacillus spp. and their excreted substances againstmicro-organisms responsible of oral diseases. MoreoverFCM facilitates rapid data acquisition and multiparameteranalysis, leading to increased popularity also in microbio-logy applications.

CAMPYLOBACTER JEJUNI CELL LYSATES INDUCEMODIFICATIONS AT DIFFERENT CELLULAR LEVELSON PERIPHERAL BLOOD MONOCYTESDi Sario G.1, Campana R.1, Cesarini E.1, Luchetti F.1,Zamai L.2, Baffone W.1, Gabrielli S.2, Papa S.1 andCanonico B.21Department of Biomolecular Sciences, University ofUrbino Carlo Bo, Urbino, Italy; 2Department of Earth, Lifeand Environmental Sciences, University of Urbino CarloBo, Urbino, [email protected]

Campylobacter jejuni bacterium is both a common com-ponent of mammal intestinal microbiota and it is associa-ted with more gastroenteritis and myocarditis in humans,probably due to the production of a cytolethal distendingtoxin (CDT). Guillain-Barrè syndrome (GBS) is a neu-ropathy strictly correlated to C. jejuni infection; recentstudies have suggested a presumed role of macrophagesin the pathogenesis of GBS.To investigate this issue, we studied the interaction ofmonocyte with cell lysates of two distinct CDT producerstrains, C. jejuni ATCC 33291, C. jejuni ISS1, and C. jeju-ni 11168H cdtA mutant as a negative control. Monocytesfrom peripheral blood mononuclear cells from donorswere treated with the different C. jejuni cell lysates(CCLys).Cells were analysed to characterize and quantitate celldeath, to analyse DNA content, mitochondrial and lysoso-mal features, and to determinate CD14, CD54 and CD59modulation. After CCLys treatment, cytometric and confo-cal imaging results well depicted mitochondrial and lyso-somal alterations, mitochondrial superoxide increment,apoptotic behaviour (particularly induced by ATCC33291) and increment of their endolysosomal/ER com-partments with concomitant modulation of CD14, CD54and CD59.In conclusion, we demonstrated that CCLys induce modi-fication at different cellular level on monocytes,

depending on the specific strain, regarding CDT internali-zation and subsequent sublethal, lethal or reservoir-transforming cellular effects.

FLUORESCENT IN SITU HYBRIDIZATION ON NUCLEIIN SUSPENSION: A NEW FLOW CYTOMETRY-BASEDMETHOD FOR GENOME REARRANGEMENT EVALU-ATIONFarina A*, Grosso V.*, Aracri B.**, Pashkoulov D.** GiorgiD.*, Lucretti S.**ENEA, CASACCIA Research Centre, BiotechnologyLab. , Via Anguillarese 301, 00123 Rome, Italy; **FLO-RAMIATA, Località Casa del Corto, 53025 Pian-castagnaio (SI), [email protected]

We have developed a novel flow molecular cytogeneticmethod which makes use of the FISHIS technique toevaluate the fluorescence of nuclei labeled with differentmolecular probes (e.g. pTa71-Cy3, (GA)10-Cy3,(TTAGGG)5-Cy3 and (GAA)7-Cy3) by flow cytometryanalysis. FISHIS labeling of nuclei with microsatellites,and other synthetic repetitive probes, allowed a qualita-tive and quantitative discrimination of a genomic re-ma-stering due to, e.g. specific chemical treatments orgenetic instability during in vitro cell culture. We haveused this technique in Orchid genera to evaluate theeffects of induced polyploidization with metaphaseblocking agents; following this approach we were able tocharacterize the presence of repetitive sequences andtheir redundancy in genomes. Driven by the fluorescenceemitted from the hybridized labeled DNA probes, we haveobserved repetitive sequence dubling in polyploids, whilein partial polyploids we monitored a corresponding partialincrease of repetitive sequences, demonstrating thepresence of a phenomenon known as "genomedownsizing". These variations can’t be assessed easilyby standard cytological observation; in Orchids this is avery limiting factor because of the very tiny size of theirchromosomes, and then this technique could find a fullexploitation in these species. Several other economicrelevant plants, such as spinach, tomato, lettuce andpepper have all a small "chromosome size" and theircomplement can’t be easily investigated. In the mean-time, these species are also hybrids and their breeding ishampered by the lack of genetic markers. FISHIS onnuclei, and single chromosomes, will be a valuablesource of genetic alteration scoring and specific chromo-some markers from every species where specific molecu-lar probes are available for labeling.This work is supported by MIPAAF, ProjectNOVAORCHID (D.M.11074/7643/09)

ANTIMICROBIAL ACTIVITY OF LACTIC ACID BACTE-RIA (LAB) AGAINST CRONOBACTER SAKAZAKIIFederici S.1, Ciandrini E.2, Campana R.2, Manti A.2,Patrone V.3, Papa S.2 and Baffone W.21Centro Ricerche Biotecnologiche, Universita’ Cattolicadel Sacro Cuore, Cremona, Italy; 2Department ofBiomolecular Science, University of Urbino “Carlo Bo”,Italy 3Faculty of Agrarian, Alimentary, and EnvironmentalSciences, Università Cattolica del Sacro Cuore,Piacenza, [email protected]

Cronobacter sakazakii is a pathogen isolated in pow-dered infant formula (PIF) and occasionally associatedwith meningitis and necrotizing enterocolitis in neonates.Lactic acid bacteria (LAB) are commonly used in food


production for their antimicrobials properties against food-borne pathogens. In this work, a reference strain, C. sakaza-kii ATCC 51329, and one human isolate, C. sakazakii 304,were first characterized for their growth ability at differenttemperatures, survival to heat and cold shocks, resistance tosimulated gastric conditions, and invasiveness on Caco-2cells. Then, the antimicrobial effect of cell-free supernatants(CFCSs) obtained from several LAB strains, owning previ-ously assessed probiotic abilities, was evaluated by agarwell assay, time-kill experiments and flow cytometry (FCM).C. sakazakii strains were able to grow at different temper-ature (up to 48°C), their survival was affected neither byheat and cold shock nor by simulated gastric conditionsand both the strains showed the ability to invade intestin-al cells. The strongest antimicrobial effect over time wasdisplayed by CFCS of L. casei subsp. rhamnosus thatwas able to reduce C. sakazakii vitality of 100% within 2h.Moreover, FCM revealed a compromised plasmaticmembrane of C. sakazakii cells exposed to CFCSs andallowed to distinguish quickly, accurately and repro-ducibly the coexistence of different subpopulations afterantimicrobial treatment. The use of FCM, capturingviable, dead, and injured cells can explain more accurate-ly efficacy of treatment, in respect with culture-dependenttechniques that only detect vital cells. Moreover, theresults obtained encourage the use of safe probiotics asfood additives in PIF development and production, forimproving formula safety and promoting infant health.

CHROMOSOMALLY ENGINEERED RECOMBINANTCHROMOSOMES CAN BE FLOW SORTED USINGFLOW MOLECULAR CYTOGENETICS: USING FISHISTO SORT T.DURUM X THINOPYRUM PONTICUMHYBRID CHROMOSOMESGiorgi D.*, Kuzmanovic L.**, Farina A.*, Grosso V.*,Ceoloni C.**, Lucretti S.**ENEA, C. R. CASACCIA, Unità Sviluppo Sostenibile edInnovazione del Sistema Agroindustriale - UTAGRI INN, ViaAnguillarese 301, 00123 Roma **Dep. of Agriculture, Forestry,Nature and Energy (DAFNE), Univ. of Tuscia, Viterbo, [email protected]

Chromosome engineering is a strategy which enables thetransfer of alien chromosome segments from an interestingand useful donor to a valuable plant species, and specifical-ly from various wild Triticeae into cultivated wheats.Chromosome transfer it is based on the use of wheat ph1mutants, carrying a mutated gene which permits chromo-some pairing and recombination between homoeologouschromosomes, normally not occurring in wild type wheat,and in its hybrids with related alien species. This approachwas successfully applied to engineer the durum “pasta”wheat genome with small alien segments from Thinopyrumponticum, a wild species containing several traits of interest(e.g. disease and drought resistance genes) for wheatbreeding, thus obtaining promising introgression lines forpractical achievements. The recent development of the FlowMolecular Cytogenetic (FMC) approach makes possible tofocus on a specific chromosome from potentially anyspecies or variety of interest. FMC combines FluorescenceIn Situ Hybridization In Suspension (FISHIS), at first usingsimple sequence repeats as probes with Flow Cytometry, toanalyse and flow sort specific chromosome types, not onlyon the basis of total DNA content, but also on their FISHlabelling pattern. Here we have successfully applied theFMC approach to durum wheat/Th. ponticum chromosomal-ly engineered lines, containing different amount of alien DNAon the same type recipient wheat chromosome, with the aimto specifically isolate the different recombinant chromo-

somes of each of them. The gene content of the “hybrid” flowsorted chromosomes will be characterized by genetic andgenomic analyses.

DIRECT DETERMINATION OF CARBON CONVER-SION FACTORS FOR ECOLOGICALLY-RELEVANTPHOTOSYNTHETIC PICOEUKARYOTESLefort T.1, Balestra C.1, Not F.2, Marie D.2, Probert I.2,Zingone A.1, Casotti R.1 and Gasol J.M.31Stazione Zoologica Anton Dohrn di Napoli, Napoli, Italy;2UPMC (Paris 6) et CNRS, UMR7144, Station Biologiquede Roscoff, Place G. Teissier, 29680 Roscoff, France;3Department of Marine Biology and Oceanography,Institut de Ciències del Mar (CSIC), Barcelona,Catalonia, [email protected]

Picophytoplankton are a key component of photosyntheticaquatic microbial communities. They are represented bycyanobacteria and small eukaryotes (in the size range of <1to 5 microns). Discrepancies in conversion factor (CF)values used to translate abundance to biomass limit deter-mination of the ecological importance of photosyntheticpicoeukaryotes (Peuk, < 3 µm). In order to constrain theseCFs, we used two direct methods based on the determina-tion of the cell size and the C and N content of 1) 16different monospecific Peuk cultures representative in termof contribution to natural marine peuk populations and 2)flow cytometric sorting and microscopical identification ofnatural peuk populations from a coastal station in the Gulf ofNaples. Cellular C and N contents varied from 230 fgC cell-1 (±1.21%) and 38.8 fgN cell-1 (±2.73%) for Ostreococcus to21800 fgC cell-1 (±23.61%) and 4920 fgN cell-1 (±14.11%)for Pycnococcus. Since the cultures used in the firstapproach were not axenic, two different protocols were usedto correct for the presence of bacteria: 1) estimation of bac-terial C and N content in each culture by flow cytometry,image analysis and standard bacterial conversion factors; 2)flow cytometric sorting of cells to remove bacteria prior toanalysis. The first correction method lowered by 7 to 33%cellular C content and was more accurate in correcting thebacterial carbon bias than the second, for which the efficien-cy of bacterial removal was limited to only 11 of the 16 cul-tures due to the sorting efficiency. We describe new linearrelationships between C and N content and cell volume forsmall eukaryotes (1.38-5.06 µm) and a constant averagecellular carbon per unit volume (C/V) ratio for global unspe-cific Peuk communities of 467 fgC µm-3 (±4%). An averageCF of 1540 fgC cell-1 (±12.01%) for a cell volume of 2.14µm3 was estimated from a mixture of Peuk cultures. We alsosuggest that more specific CFs can be chosen for certainecosystem types based on the known composition of thePeuk communities.

THE FLOW CYTOMETRIC SPERM DNA GLOBALMETHYLATION LEVEL IN MALE REPRODUCTIVE EPI-DEMIOLOGYLeter G.1, Eleuteri P.1, Pacchierotti F.1, Bonde J.P.2, ToftG.3, Jönsson B.A.G.4, Giwercman A.5, Pedersen H.P.6,Ludwicki J.K.7, Zviezdai V.8, and Spanò M.11Lab. of Biosafety and Risk Assessment, ENEA Casaccia,Rome, Italy; 2Bispebjerg Univ. Hospital, Copenhagen,Denmark; 3Dpt. of Clinical Epidemiology, Århus Univ.Hospital, Århus, Denmark; 4Lund Univ., Lund, Sweden;5Molecular Reproductive Medicine, Lund Univ., Malmö,Sweden; 6Greenland Institute of Natural Resources, Nuuk,Greenland; 7National Institute of Public Health, Warsaw,Poland; 8Kharkiv State Medical Univ., Kharkiv, [email protected]


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We have recently described an optimized method to meas-ure, by flow cytometry, human sperm DNA global methyla-tion level (DGML). The assessment relies on the immuno-detection of 5-methyl Cytosine (5-mC) in situ after properrelaxation of the highly compact sperm chromatin struc-ture. DNA methylation is pivotal for a wide array of biologi-cal processes and is the epigenetic change most studied inrelation to environmental exposures. Aberrant DNA methy-lation has been associated, mainly in somatic cells, withexposure to a variety of environmental contaminants,POPs (persistent organic pollutants) included. To investi-gate whether POPs may operate at an epigenetic levelalso in the male germ cell line, sperm DGML was evaluat-ed in frozen archived samples from some 270 healthy fer-tile men from Greenland, Ukraine, and Poland. Generallinear models were used to analyse associations betweenPOP body burden, measured in serum, and DGML. Ourapproach was able to detect a weak hypomethylationeffect associated with exposure to some of the POPs, thuswarranting further investigation.

USING FLOW CYTOMETRY FOR THE MULTI-PARA-METRIC CHARACTERIZATION OF BIOAEROSOL INATMOSPHERIC PARTICULATE MATTER: INSIGHTSFROM A FIELD STUDYMarcovecchio F.,1,2 Perrino C.,2 Amalfitano S.31Department of Chemistry, Sapienza University of Rome,P.le Aldo Moro, 5, Rome, 00185, Italy; 2C.N.R. Institute ofAtmospheric Pollution Research, Monterotondo St.,Rome, 00015, Italy; 3C.N.R. Water Research Institute,Monterotondo St., Rome, 00015, [email protected]

The crucial contribution of biogenic particles to organicambient aerosol has become increasingly apparent inrecent years, since bioparticles have been found to beallergenic, harmful themselves or carriers of harmful sub-stances. Moreover, because of their size, they might con-tribute significantly to the mass concentration of atmos-pheric particulate matter (PM). This study was aimed atbioaerosol characterization and bioparticle size assess-ment by using flow cytometry as detection technique.A multi-stage impactor, running for two consecutiveweeks, collected an air volume of about 350 m3 andsegregated particles in ten size stages between 0.18 and18 µm, according to their aerodynamical diameter (AD).The particle extraction from collection filters was per-formed by shaking separately each filter within adetaching solution. The liquid suspension was stainedwith Sybr Green I and Propidium Iodide. A further sub-aliquot was air-dried on a well glass slide to double-checkthe integrity of biogenic particles by epifluorescencemicroscopy after extraction.The use of flow cytometry allowed the detection andquantification of single particles passing through the laserinterrogation point. The total abundance of bioparticleswas 3.5x104 particles/m3, mostly retrieved within the sizeclasses 1.8 – 3.2 µm and 3.2 – 5.6 µm. Only 6.3% of thetotal particles were found in the size range below 1 µm.After three consecutive extractions, the particle recoveryefficiency was, on average, 92% of the total particles,with lower efficiency (78%) for particles with AD<5.6 µm.Considering that methodological advances for the deter-mination of PM different components are of growingimportance to complement air quality issues, our out-comes highlighted that flow cytometry could represent anappropriate analytical tool to provide rapid and multi-paramet-ric data for a better understanding of the bioaerosol size dis-tribution and concentration in the atmospheric environment.

CLOFAZIMINE INDUCED SUICIDAL DEATH OFHUMAN ERYTHROCYTESOfficioso A.,1,2 Alzoubi K.,1 Manna C.,2 Lang F.11Department of Physiology, University of Tübingen,Germany 2Department of Biochemistry, Biophysics andGeneral Pathology, School of Medicine, SecondUniversity of Naples, Naples, [email protected]

Background/Aims: The antimycobacterial riminophenazineclofazimine has previously been shown to up-regulate cel-lular phospholipase A2 and to induce apoptosis. In erythro-cytes phospholipase A2 and cyclooxygenase dependentformation of prostaglandin E2 triggers Ca2+ entry with sub-sequent stimulation of eryptosis, the suicidal death charac-terized by cell shrinkage and cell membrane scramblingwith phosphatidylserine translocation to the erythrocytesurface. Stimulators of Ca2+ entry and eryptosis furtherinclude oxidative stress. The present study tested, whetherand how clofazimine induces eryptosis. Methods: To this end,phosphatidylserine exposure at the cell surface was estimat-ed from annexin V binding, cell volume from forward scatter,hemolysis from hemoglobin release, cytosolic Ca2+ activity([Ca2+]i) from Fluo3-fluorescence, reactive oxidant species(ROS) from 2′,7′-dichlorodihydrofluorescein diacetate(DCFDA) fluorescence, and cytosolic ATP level utilizing aluciferin–luciferase assay kit. Results:A24-48 hours exposureof human erythrocytes to clofazimine (≥1.5 µg/ml) significant-ly increased the percentage of annexin-V-binding cells withoutappreciably modifying forward scatter. Clofazimine significant-ly icreased [Ca2+]i, significantly decreased cytosolic ATP, butdid not significantly modify ROS. The effect of clofazimine onannexin-V-binding was significantly blunted, but not fully aboli-shed by removal of extracellular Ca2+, and by phospholipaseA2 inhibitor quinacrine (25 µM). Conclusions: Clofaziminestimulates phospholipid scrambling of the erythrocyte cellmembrane, an effect in part dependent on entry of extracellu-lar Ca2+ and cellular energy depletion. and sensitive to phos-pholipase A2 inhibitor quinacrine.

THE EFFECT OF SILVER NANOPARTICLES ON SEAURCHIN SKELETAL DEVELOPMENTUsai C.1, Gambardella C.2, Ramoino P.3, Gatti A.M.4,Bianchini P.5, Diaspro A.5, Faimali M.2, Falugi C.61Istituto di Biofisica, CNR, Genova; 2ISMAR, CNR, Genova;3DISTAV, Università di Genova; 4Nanodiagnostica Srl,Modena; 5Dipartimento di Nanofisica, IIT, Genova; 6DISVA,Università Politecnica delle Marche, [email protected]

Silver nanoparticles (AgNPs) have been applied inproducts like household appliances, cleaners, clothing,cutlery, toys and electronics. Due to their widespread use,it is likely that substantial amounts of AgNPs will ultimatelyend up in aquatic ecosystems, getting negative effects dueto their anti-bacterial activity. This study is aimed to evalu-ate the effects of exposure to different concentrations ofAgNPs on the morphology and biochemistry of the seaurchin Paracentrotus lividus at the first developmentalstages.Sperm was exposed to sea water containing suspensions ofAgNPs ranging from 0.1 µg/L to 1 mg/L. Fertilization abilitywas not affected, but morphological alterations of the skele-tal rods were identified in the gastrula and pluteus stages.The skeleton of sea urchin embryos is shaped by migrationof primary mesenchymal cells (PMCs), then larvae startfeeding and develop additional arms. We observed by con-focal laser scanning microscopy that AgNPs interfere with


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the correct deposition of the organic matrix because adifferent distribution pattern of molecules related to theskeletogenic cell identification occurred: ID5 (a marker of themsp130 protein, responsible for Ca2+ and Mg2+ mineraliza-tion), Wheat Germ Agglutinin (WGA, a bi-univocal marker ofPMCs) and Fibronectin (FN, along which cells migrate viainteractions with integrins of the glycocalix).Our results identified a spatial correspondence amongPMCs, ID5 and WGA affinity sites. Moreover the FN pat-tern, the features of Golgi apparatus skeletogenic cells andthe ID5 distribution were altered according to the degree ofanomalies caused by AgNP concentrations and to thedegree of skeleton mineralization.In conclusion, this study suggests that AgNPs suspendedin seawater interfere with the bio-mineralization processesin marine organisms.

PHYTOPLANKTON COMMUNITY STRUCTURE IN THEGULF OF NAPLES FROM HIGH-FREQUENCYPORTABLE FLOW CYTOMETRY (CYTOSENSE)van Dijk M.A. and Casotti R.Stazione Zoologica A. Dohrn, Villa Comunale, Napoli, [email protected]

Conventional flow cytometers can only handle a limitedrange of particle sizes and are optimized for single, usual-ly spherical, eukaryotic cells. New instruments, developedspecifically for ecological research mainly in aquatic micro-bial ecology, are capable of analyzing a wider range of par-ticles and allow for the analysis of the entire phytoplanktoncommunity, including rarer large phytoplankton species.In the present study, a portable scanning flow cytometercalled Cytosense was used to analyze the phytoplanktoncommunity in the Gulf of Naples. The instrument records fullsignal profiles of each particle, providing several comple-mentary parameters besides the integrated peak values andcan analyze particles with a diameter up to several 100 µmand a few millimeters in length. Therefore, in addition to ana-lyzing the abundance of the dominant photosyntheticpicoplankton clusters of picoeukaryotes, Synechococcus,and Prochlorococcus, the recorded pulse shapes allow for amore detailed discrimination of larger species that possessstructural variability. The instrument’s efficacy was testedonboard a research vessel, measuring at high frequencyfrom a sample-loop while sailing, in order to obtain detailedcommunity structure data at the spatial scale. Cytosenseresults for picoplankton concentrations are compared tostandard counts by conventional lab-based flow cytometers,and the accuracy of capturing spatial variability is evaluatedby comparing total particulate red fluorescence recorded bythe Cytosense to onboard high-frequency fluorometry data.

ASSESSING THE PERFORMANCES OF ADVANCEDTREATMENT PROCESSES FOR WATER REUSE: AFLOW CYTOMETRIC APPROACHVergine P.1, Amalfitano S.2, Salerno C.1, Pollice A.11C.N.R. Water Research Institute, Bari, 70132, Italy;2C.N.R. Water Research Institute, Monterotondo St.,Rome, 00015, [email protected]

Wastewater reclamation and reuse is becoming a wide-spread practice for irrigation purposes, owing to newdevices and techniques that have been decreasing theoverall cost of water recycling in the agro-industry.Recently, the introduction of a membrane filtration stageimproved significantly the effectiveness of wastewatertreatment schemes, thus contributing to reach higher sus-tainable productivities.

In this study, we aimed at monitoring the performances ofadvanced treatment processes, recently introduced withina traditional activated-sludge wastewater treatment plantof a vegetable processing industry. The added tertiarytreatments included sand filtration, membrane ultrafiltration(nominal pore size 0.05 µm) and UV disinfection (6x 300Wmercury-vapor lamps). The company produced on aver-age 5*104 m3/y of wastewater, characterized by high elec-trical conductivity (2.5±0.9 mS/cm), high organic content(1.0±0.3 gCOD/L), low pH (5.6±0.4), and a variable fecalcontamination (106-107 CFU/100mL of E. coli). Flowcytometry (FCM) was applied as a diagnostic tool to eval-uate the removal of potential microbial contaminants. Byquantifying the total and living microorganisms before andafter each treatment, we found a moderate effectiveness ofdisinfection treatments, with a four-time increase of deadcells after UV exposure. Instead, the ultrafiltration stagewas highly efficient, with the removal of >99% of the inletmicrobial biomass. Whereas microbiological water qualityassessment is traditionally based on cultivation methodswith substantial manual handling and results only availableafter 2-5 days, FCM was suited uniquely for this applica-tion, due to its speed and potential for on-site measure-ments. Our cytometric outcomes provided a solid informa-tion basis for operational decisions concerning the treat-ment regime and operation, hence contributing to the safe-ty of water reuse.



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Hematology

LEUKOCYTE QUALITY CONTROLON BLOOD COMPO-NENTS BY FLOW CYTOMETRY: OUR EXPERIENCEBasile F., Graziano D., Piscitelli S., Valentino M.,Lo Pardo C.UOSS Immunologia Cellulare in Emato-Oncologia, SIMTAORN A. Cardarelli, Napoli, [email protected]

Leucodepletion is critical to prevent transfusion reactionscaused by leukocytes. From January to April 2015, we car-ried out WBC contamination quality checks in 16 leucocyte-depleted red cells pre-storage units, in 11 leucocyte-deple-ted red cells post-storage units and in 20 leucocyte-depletedplatelet pools, each prepared with 5 buffy-coats.Materials and Methods: We used the BD LeucoCOUNTassay in single platform and the cytometer FACSCantoII BDto estimate residual leukocyte count (rWBC). 100 µL of sam-ples were incubated with 400 µL of LeucoCOUNT reagent inTruCOUNT tube for 5’at room temperature. We acquired10000 events in the TruCOUNT region.Results: White blood cell average count in pre-storage unitswas 1.43 rWBC/µl (range 0-6.65), corresponding to a valueof 0.33x106 rWBC/Unit (range 0-1.45); in post-storage unitswe found 0.07 rWBC/µl (range 0-0.28), corresponding to avalue of 0.13x106 rWBC/Unit (range 0-0.74). WBC averagecount in platelet pools was 0.09 rWBC/µl (range 0-0.30),corresponding to 0.03x106 rWBC/Unit (range 0-0.15).Conclusions: Our quality controls showed the value of leu-codepleted blood components within the SIMTI standard:<0.5x106 rWBC/Unit in pre-storage red blood cell units;≤0.5x106 rWBC/Unit in post-storage red blood cell units;<1x106/Unit in platelet units. Thus we avoided transfusionreactions caused by white blood cells, such as febrile non-hemolytic reaction, TRALI and platelet alloimmune refracto-riness. The leucoCOUNT assay provides excellent preci-sion, linearity and accuracy moreover it is a fast and easytest, giving the highest level of confidence in the processcontrol.

SINGLE CELL ANALYSIS OF PI3K/Akt/PTEN/mTORSIGNALLING PATHWAY IN PEDIATRIC T-CELL ACUTELYMPHOBLASTIC LEUKEMIA AND COMPARISONWITH WESTERN BLOTTINGBonaccorso P.1,2, Bugarin C.1, Buracchi C.1, Fazio G.1,Biondi A.1,3, Lo Nigro L.2 and Gaipa G.11M.Tettamanti Research Center, University of MilanoBicocca, San Gerardo Hospital, Monza, Italy; 2Center ofPediatric Hematology Oncology, Azienda Policlinico-OVE, University of Catania, Catania, Italy; 3PediatricClinic, University of Milano Bicocca, FondazioneMBBM/Ospedale San Gerardo, Monza, [email protected]

Background: T-lineage acute lymphoblastic leukemia (T-ALL) accounts about 15% of pediatric ALL cases. Most ofpediatric T-ALLs are characterized by constitutive activa-tion of PI3K/Ak/tmTOR signaling pathway. Western blotting(WB) represents the method of reference to study the pro-teomic profile in hematological malignancies, however flowcytometry (FC) can provide several advantages comparedto WB. Aim: In order to validate the use of Phosphoflowapproach for T-ALL signaling profiling, we studied thesignaling pathway of PI3K/Akt/mTOR in pediatric T-ALL by

the contemporary use of FC and WB techniques. Patientsand Methods: Sequencing of PTEN Exon7 was performedby PCR amplification. PTEN and mTOR protein expres-sion were analyzed in BM samples from 9 children with T-ALL, enrolled in AIEOP ALL2000/R2006 protocols. Basalsignaling state of AKT-S473, AKT-T308, mTOR, PTEN,pS6, p4EBP1 was studied by Phosphoflow. PTEN andmTOR protein levels were also assessed by Western Blotanalysis. Results: Both leukemia blasts and normal resi-dual T-cells were analyzed in each sample. All sampleshad a very low-undetectable p-AKT-S473 basal level inboth cell populations. Basal state expression of totalmTOR was higher in normal residual T-cells than in leuke-mia blasts, by contrast PTEN expression was significantlyhigher in T-ALL blasts than in normal T-cells. Completeconcordance was obtained by parallel use of WB. Threeout of 9 patients analysed by phosphoflow were PTENExon7 mutated and PTEN protein expression was comple-tely absent in these patients (confirmed by both techni-ques), moreover we observed that PTEN Exon7 mutatedblasts showed significantly lower pS6 levels compared toPTEN Exon7 wild type blasts. Discussion: Dysregulation ofPI3K/Akt/PTEN/mTOR pathway represents one of themost common events in aggressiveness in pediatric T-ALLs: it needs a more sensitive method to evaluate T-ALLproteomic profile. We showed a different PTEN and mTORbasal state expression in blasts and normal residual T-cellsand PTEN was completely absent in PTEN Exon7 mutatedpatients. Finally, our data demonstrate FC provides seve-ral advantages such as the ability to measure events in sin-gle cells, to assess several parameters simultaneously, toanalyze a rare subset of cells within heterogeneous sam-ples and to provide results in shorter time.

FLOW CYTOMETRY ANALYSIS OF EVs IN WHOLEBLOOD OF PATIENTS WITH HEMATOLOGICALDISORDERSBoselli D.1, Canonico E.1, Di Terlizzi S.1, Romanò M.1,Villa C.1.1Flow Cytometry Resource, Advanced CytometryTechnical Applications Laboratory (FRACTAL) IstitutoScientifico San Raffaele, Milano, [email protected]

Extracellular vesicles (EVs) are a population of membrane-enclosed vesicles released from a variety of cells into theextracellular space. They have been detected in the blood ofhealthy individuals, but undergo phenotypic and quantitativechanges hiring a pathophysiological role in several diseases.In hematological malignancies the involvement of EVs hasbeen poorly investigated, even if some recent papers recogni-ze them a crucial role in thrombosis, inflammation, angiogene-sis, in affecting metastatic processes and multidrug resistan-ce. Characterization of EVs could be of interest as it couldrepresent a biomarker that might significantly add informationto the risk assessment of critical individuals. High-sensitivityflow cytometry allows the simultaneous identification of diffe-rent EVs subpopulations, permits to count them and to cha-racterize their phenotype. The aim of this study is to characte-rize circulating EVs in blood from healthy individuals and tocompare them with EVs in blood from patients carrying hae-matological disorders.Sample preparation will be performed by progressive cen-trifugations of whole blood. This strategy allows the elimi-nation of cells, platelets and debris, without inducing plate-let activation.Moreover EVs will be stained with FLAER, a fluorescentGPI binding protein that permits a further discriminationbetween EVs, remaining debris (FLAER negative) and


cells (FLAER high). EVs will be also counted by usingFlowcount beads and percentage of EVs derived from pla-telets will be assessed by using CD41 mAb.Preliminary experiments showed that in blood circulation ofhealthy individuals (N=20)EVs are 5022±2231/µl and from them 7.2±3.2% are CD41positive. Moreover markers involved in activation, adhe-sion, migration and metastasis formation will be investiga-ted (CD9, CD63, CD81, MHC-I and MHC-II).

EXPRESSION OF THE IMMUNOGLOBULIN SUPERFA-MILY CELL MEMBRANE ADHESION MOLECULECD146 IN ACUTE LEUKEMIACampioni D.1, Cavazzini F.1, Ferrari L.1, Buldini B.2, BardiM.A.1, Moretti S.1, Michielotto B.2, Lazzari M.C.3, DabustiM.1, Basso G.2, Cuneo A.1, Lanza F.31Section of Hematology, University-S.Anna Hospital, Ferrara,Italy; 2Onco-Hematology Division, SDB Department,University of Padova, Italy; 3 Section of Hematology, Hospital ofCremona, Cremona, [email protected]

Key Words: acute lymphoblastic leukemia, Philadelphiachromosome, immunophenotype, CD146Background: The expression of the immunoglobulin super-family cell membrane adhesion molecule CD146 has beenreported on several normal and pathological cell types inhuman. Aim of this study was to investigate CD146 expres-sion in acute leukemia using a multiparametric cytofluori-metric approach.Methods: cytofluorimetric and cytogenetic studies wereperformed on peripheral blood and bone marrow samplesfrom 162 patients with acute myeloid leukemia (AML,n=121) and acute lymphoblastic leukemia (ALL, n=41).ALL patients were subdivided in B-ALL (n=38) and T-ALL(n=3). Adult (n=18) and pediatric (n=20) B-ALL were consi-dered as a whole group.Results: 4/121 (3.3%) AML cases, 14/38 (36.8%) B-ALLand 2/3 (66.6%) T-ALL expressed CD146 on 12-98% ofblasts (p<0.001). CD146 expression was not observed inhealthy subjects. Among B-ALL CD146-positive cases78.6% were associated to a “common”/BII-ALL and 21.4%to a pre-B/BIII-ALL immunophenotype while pro-B/BI-ALLand mature-B/BIV-ALL cases were negative. Statisticalanalysis showed the CD146 expression as strongly asso-ciated to Ph+ positivity in B-ALL since we observed thehighest percentage of CD146-positive blasts in the totalityof Ph-positive B-ALL cases (84 +/-22% Ph-positive B-ALLSD vs. 40 +/-24% SD in Ph-negative B-ALL; p<0,001).Conclusion: in our series CD146 was expressed in allcases of Ph-positive B-ALL and in the vast majority T-ALL,whereas it was rarely expressed by AML blasts. We sug-gest that the CD146 may be considered as an additionalmarker for acute lymphoblastic leukemia diagnosis andmonitoring of minimal residual disease in those casesresulting CD146-positive at diagnosis.

CD34+ FLOW CYTOMETRIC COUNT IN THE COLLEC-TION OF HEMATOPOIETIC STEM CELLS: COMPARI-SON OF DIFFERENT TECHNIQUESCoppola G., Piemonte I., Iorio M., Palumbo D., Vaccaro E.,Reggiani S., Paura G., Massari A.Flow Cytometry Unit – Dept. Clinical Pathology – AOUSalerno, [email protected]

Flow cytometry laboratory plays a key role in the mobiliza-tion and collection of hematopoietic stem cells for tran-splant use. For this purpose, there are two main protocols

of cell count: Milan/Mulhouse and Ishage. Milan/Mulhouseprotocol, the first used for CD34+ cells determination byflow cytometry, doesn’t effect a direct calculation of CD34+,but determines them indirectly by relating the CD34+ cyto-metric percentage to the total leukocytes count obtained bya blood cell counter. The Ishage protocol performs a directdetermination of CD34+ number through the use of fluoro-spheres and a sequential gating strategy. In this work wecompared the two cell count techniques testing them on 5patients undergoing mobilization protocols for the collec-tion of peripheral hematopoietic stem cells. The countswere performed with the two techniques on both the peri-pheral blood of patients either on their respective collectionbags. The results show a overestimated CD34+ count,especially on the collection bags, by Milan/Mulhouse thanIshage protocol. Analyzing in detail the two techniques, weidentified the cause of this overstatement ofMilan/Mulhouse protocol in double platform used (theCD34+ number derived from the comparison betweencytometric percentage of these elements and the numberof leukocytes obtained by CBC). In particular, the blood cellcounter would count within total leukocytes precursors ofred cells and cell debris disrupting this determination andconsequently the count of stem cells. Instead, Ishage pro-tocol, eliminating this error by stem cells direct count (usingfluorescent beads) and by using the 7-AAD that restrictsthe count to only vital elements, is a substantial innovationin hematopoietic stem cells count. Ultimately, the Ishageprotocol allows a more precise and reliable stem cellsabsolute count than the Milan/Mulhouse protocol and ena-bles a better reproducibility of data by various cytometrylaboratories dealing with stem cells.

CIRCULATING REGULATORY T-CELLS IN MULTIPLEMYELOMAD’Arena G.1, Statuto T.2, D’Auria F.2, Simeon V.3,Laurenti L.4, Innocenti I.4, Mansueto G.1,Pietrantuono G.1, Villani O.1, Musto P.51Hematology and Stem Cell Transplantation Unit,IRCCSCentro di Riferimento Oncologico della Basilicata, Rionero inVulture; 2Laboratory of Advanced Diagnostics, IRCCSCentro di Riferimento Oncologico della Basilicata, Rionero inVulture; 3Preclinical and Translational Research, IRCCSCentro di Riferimento Oncologico della Basilicata, Rionero inVulture; 4Hematology Chair, Catholic University of SacredHeart, Rome; 5Scientific Direction, IRCCS Centro diRiferimento Oncologico della Basilicata, Rionero in [email protected]

A general agreement on the pathogenetic and prognosticrole of regulatory T-cells (Tregs) in human tumors exists.However, their frequency and function in multiple myeloma(MM) is still matter of debate. Aiming to evaluate their pro-gnostic significance in monoclonal gammopathies we stu-died the percentage and absolute number of circulatingTregs in a cohoort of patients with MM and monoclonalgammopathies of indetermined significance (MGUS) andin a group of healthy subjects, as control.Peripheral blood and bone marrow samples from 39patients with untreated MM (mean age 69 years; range 37-90 years; 18 M; 21 F) and 44 patients with MGUS (meanage 65 years; range 39 – 87 years; 25 M) were collectedafter informed consent. Peripheral blood samples from 12healthy subjects (mean age 54 years; range 32-75 years; 5M; 7 F) were used as controls. Briefly, 100 µL of whole bloodwere incubated with CD4-PerCP/CD25-PE-Cy7/CD127-PE/CD45-APC-Cy7 monoclonal antibodies. Tregs weredefined by the expression of CD4, CD25 at high density andCD127 low density or undetectable levels.


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Overall, the number of circulating Tregs, detected as per-centage of all lymphocytes, were found: 2.1 %±1 (range0.7-5.1%) in MM patients; 2.1%±0.9 (range 0.3-4.4%) inMGUS; 1.5%±0.4 (range 0.9-2.1%) in controls. Absolutenumber of Tregs was: 36.3/µL ± 23.7 (range 6.7-149/µL) inMM; 38.8/µL ±19.1 (range 4.3-87/µL) in MGUS; 39.4/µL ±12.5 (range 18-63/µL). No statistical differences weredetected. Moreover, the percentage of Tregs showed acorrelation with beta2microglobulin in MGUS patients(r=0.38). Only in MM patients, the percentage number ofTregs correlates with erythrocyte sedimentation velocity(ESV) (r=0.39) and platelet number (r=0.39). The absolutevalue of Tregs correlates with white blood cell number(r=0.66). Finally, the percentage number of bone marrowTregs was found to correlate with plasma cell numberdespite not reaching a statistical significance.Our results indicate that circulating number of Tregs do notdiffer in monoclonal gammopathies and normal subjectsand do not correlate with clinical feature of MM and MGUS,as well. As reported by others (Foglietta et al,Haematologica 2014), Tregs seem to be not influenced bythe disease status of MM. In addition, no prognostic signi-ficance has been found.

FLOW CYTOMETRIC DETECTION OF MICROVESICLES(MV) IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)D'Arena G.1, De Luca L.2, Laurenti L.3, Molica S.4, TrinoS.2, Laurenzana I.2, Caivano A.2, La Rocca F.2, SimeonV.2, Pietrantuono G.1, Musto P.5, Del Vecchio L.61Hematology and Stem Cell Transplantation Unit,IRCCSCentro di Riferimento Oncologico della Basilicata, Rioneroin Vulture; 2Laboratory of Preclinical and TranslationalResearch, IRCCS Centro di Riferimento Oncologico dellaBasilicata, Rionero in Vulture; 3Hematology Chair, CatholicUniversity of Sacred Heart, Rome; 4Hematology Unit,"Pugliese Ciaccio" Hospital, Catanzaro; 5ScientificDirection, IRCCS Centro di Riferimento Oncologico dellaBasilicata, Rionero in Vulture; 6CEINGE, AdvancedBiothecnology, University "Federico II", Naples, [email protected]

Normal and malignant cells secrete extracellular MV,usually defined as shedding membranous vesicles that areproduced by budding from the plasma membrane.Elevated levels of circulating MV in CLL are able to activa-te bone marrow stromal cells and enhance production ofVEGF, a pro-survival factor for CLL B-cells. Sera fromuntreated CLL pts searching for the number of circulatingMV in order to test their possible prognostic role when eva-luated at diagnosis. Sera from 101 CLL pts (mean age 70yrs, range 41–89; 60 M, 41 F; 52 patients with A, 36 withB, and 13 with C Binet clinical stage) were analyzed at dia-gnosis. Briefly, 1 ml of serum was processed with serialcentrifugations: 2,000xg for 15 min; 10,000xg for 30 min;100,000xg for 70 min. Resulting pellets were washed withpre-filtered (0.22 µm) PBS and centrifuged at 100,000xgfor 70 min. MV enriched pellets were analyzed by flowcytometry (FACSCalibur, Becton Dickinson). Standardmicro beads (0.3–0.9–3 µm) were used to define the sizelimit for MV and their size assessment. Pre-filtered PBSwas also used to set the lower limit of MV gate. TruCOUNTbeads (BD) were added immediately prior to analyze sam-ples and to determine the number of MV/µl. To identify andcharacterize MV, CD19-APC was used. A higher meannumber of MV was found in CLL pts with respect to 28healthy subjects (991.8±768.4/µl vs 270±325/µl; p<0.001). Of interest, in CLL pts about 15% of MV werefound to be CD19+. A higher number of MV was found inpts with advanced clinical stage (Binet A+B vs C, p 0.02),

while a trend was observed with surface expression ofCD38 (p 0.06). No correlation was found according toCD49d and ZAP-70 expression, IgVH mutational statusand cytogenetics abnormalities. A correlation was alsofound with the number of circulating B-lymphocytes andtotal and CD19+ MV (r=0.23 and r=0.25, respectively,p<0.05). Overall, pts who required therapy showed a moreelevated number of MV at diagnosis (1,333/µl±941) withrespect to those who did not (764/µl±565) (p 0.003). Thetime to treatment was longer in pts with lower number ofboth total (p 0.05) and CD19+ MV (p<0.05). Finally, overallsurvival was shorter in pts with higher levels of total MV(p0.001), while the number of CD19+ MV was not signifi-cant. Our study suggests a possible prognostic relevance ofthe circulating MV number detected at diagnosis in CLL pts.

CASE REPORT: PATIENT AFFECTED BY ANGIOIMMU-NOBLASTIC T-CELL LYMPHOMA AND SEVERE ANE-MIAWITHOUT HEMOLYSIS INDICES IN PRESENCEOFPANAGGLUTINATIONGraziano D., Basile F., Alberti M., Criscuoli M., ValentinoM., Lo Pardo C.UOSS Immunologia Cellulare in Emato-Oncologia, SIMTAORN A. Cardarelli, Napoli - [email protected]

B.A., a 66 year old man, was hospitalized for anemia (Hb=4.5 g/dl) and severe fatigue, with Toxoplasma lymphade-nopathy diagnosis, performed elsewhere. In our UOSS thepatient received diagnosis of angioimmunoblastic T- cellnon-Hodgkin Lymphoma.Pretransfusion tests, performed in emergency, showedpositive indirect antiglobulin test (IAT) with serological evi-dence of panagglutination and positive direct antiglobulintest (DAT), for complement and IgG. For 9 days the patientreceived 19 incompatible red blood cells units, but the Hbvalue remained <5,5 g/dl. There were not hemolysis signs,splenomegaly or bleeding.Materials and Methods: Flow cytometric tests were perfor-med by six color multiparametric analysis (MoAbs BD)using FACSCanto II (BD). Immunohematological investi-gations were conducted by column agglutination technique(Biocard System - Innova Ortho), solid phase (Neo GalileoSystem - Immucor) and monospecific AHG column System(DC screening BioRad).Results: T pathological population showed CD2+ CD5+CD4+ CD10+ CD3- CD7- CD1a- CD8- CD19- CD20-CD34- phenotype. DAT was positive (score 3) by both solidmethods. IAT was positive (score 3/3/3) by both methods.Biochemical parameters remained within normal range; onthe contrary the Hb, WBC and PLT value stayed very low(Hb =4.4 g/dl, WBC=2200/µl, PLT=8000/µl, reticulocy-tes=1.3%)Conclusions: The synergy between Immunohematologyand Cytometry produced early diagnosis. Unfortunatelythe patient died in a few days. Probably various noxae ledto a cascade of pathological events: lymphoma caused anautoimmune disease and the presence of cold IgM autoan-tibodies complicated the immunohematological picture.Perhaps CID occurred in the final stage.

FLOW CYTOMETRIC IDENTIFICATION OF TWO DIFFE-RENT PATHOLOGICAL B-LYMPHOCYTE POPULA-TIONS IN THREE PATIENTS AFFECTED BY B-NHLGraziano D., Basile F., Piscitelli S., Altomare L., Gravetti A.,Valentino M., Lo Pardo C.UOSS Immunologia Cellulare in Emato-Oncologia, SIMTAORN A. Cardarelli, Napoli - [email protected]


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During last year we studied 282 patients affected by denovo Non Hodgkin Lymphoma (NHL). 3 of them showedtwo different pathological B lymphocyte populations and inone case we found a myeloid pathological population too.Materials and Methods: Peripheral blood and bone marrowsamples were studied by six color multiparametric analysis(MoAbs BD) in flow cytometry by FACSCantoII (BD).Results: 282 patients had de novo Non HodgkinLymphoma diagnosis: 5 were affected by T-NHL and 277by B-NHL; 3 of these showed the presence of two differentpathological populations of B lymphocytes in each patientand, in one case, we found a myeloid pathological popula-tion too.The pathological B lymphocyte phenotypes of the threepatients respectively were:- patient P.L., lymphocytes 40%: CD19+ CD20+ CD5-

CD22+ CD23± CD10± SmIgLambda+ and CD19+CD20+ CD5- CD22+ CD23- CD10± SmIg-;

- patient D.M., lymphocytes 18%: CD19+ CD20+dim CD5+CD22+ CD23± CD200+ SmIgLambda+dim and CD19+CD20+ CD5- CD22+ CD23- SmIgKappa+;

- patient R.V., lymphocytes 29%: CD19+ CD20+dim CD5+CD22- CD23± CD200+ SmIgKappa+dim and CD19+CD20+ CD5- CD22+ CD23- SmIgkappa+bright. In thispatient we found a population of pathological myeloidcells (8%) CD34+ CD117+ CD13+ CD33± CD7±CD45±.

Conclusions: Multiparametric analysis in flow cytometry isavailable for an accurate diagnosis, especially in detectionof very small cell populations.

PROGNOSTIC VALUE OF CD47 EXPRESSION INACUTE LEUKEMIASGraziano D., Piscitelli S., Basile F., Valentino M., Lo Pardo C.UOSS Immunologia Cellulare in Emato-Oncologia, SIMTAORN A. Cardarelli, Napoli - [email protected]

In the last six months we evaluated CD47 expression in123 patients: 52 affected by acute leukemias and 71 con-trols.Materials and Methods: The flow cytometric evaluationwas performed by six color multiparametric analysis byFACSCantoII (BD). CD47 expression was evaluated by thefluorescence histogram analysis and the MeanFluorescence Intensity calculation.The patients were affected by the following acute hemato-logical diseases: 29 de novo acute myeloid leukemias(AML); 4 follow-up AML; 3 chronic myeloid leukemias(CML) in blast crisis; 8 de novo acute lymphoid B leuke-mias (B-ALL); 4 follow-up B-ALL; 1 de novo T-ALL; 1 fol-low-up T-ALL; 1 de novo Mixed M/B leukemia; 1 de novoMixed M/T leukemia.Results: In peripheral blood control samples CD47expression was homogeneous, between 103 and 104 onall hematological cells, except for granulocytes (≤103). Inhealthy subjects bone marrow samples, all hematologicalcells expressed CD47 at the same fluorescence intensity(between 103 and 104). Among patients affected by acuteleukemia, CD47 was overexpressed in only 7 cases, 1 stu-died on peripheral blood, 6 on bone marrow, including the3 patients affected by CML in blast crisis.Conclusions: In spite of literature we did not find any cor-relation between CD47 expression and prognosis in mye-loid and lymphoid acute leukemias, both in de novo and infollow-up diseases. As a matter of fact, we found CD47overexpression in one case of promyelocitic AML in whichthe patient obtained complete remission, as well as in allCML in blast crisis with very poor prognosis.

AN INTEGRATED ASSESSMENT OF GAMMA HEAVYCHAIN DISEASE (GHCD) IN THE BONE MARROW: ACASE REPORTKunkl A., Contini P., Mangerini R., Grasso L., Megna M.,Barbaresi M., Ravetti J.L., Fraternali-Orcioni G.,SS Diagnostica Citofluorimetrica, SC Anatomia PatologicaOsp-Univ, IRCCS -AOU- San Martino- IST, Genova, [email protected]

GHCD is a rare mature B cell neoplasm that produce atruncated monoclonal gamma immunoglobulin heavychain that loses the ability to form disulfide bonds with thelight chains.A 72-year-old male patient, diagnosed in 2013 with ocularadnexal marginal zone B cell lymphoma showing IgG4+plasma cells (clonality tested by PCR), elevated IgG4serum level and free kappa light chains in urine, presentedrecently with a generalized disease: lymphoadenopathy,lymphocytosis, acute renal failure without bone lesions. Abone marrow aspirate and threphine biopsy were perfor-med and a a lymphoplasmacytic mature B cell neoplasmwas described by using multicolor flow cytometry (MFC)and immunohistochemistry (IHC). Histology and IHC sho-wed a polymorphous proliferations of CD138+, CD31+ pla-smacells with atypical cytology and CD20+ B lymphocyteswith mitotic figures. MFC showed a predominant medium-sized monoclonal B plasmacell population (34% of nuclea-ted cells) (CD19+, CD138+, CD38+, CD56neg, CD20neg,CD22neg) containing cytoplasmic gamma chain withoutlight chains and a small-sized monoclonal B lymphocyticcomponent (6% nucleated cells) (CD19+, CD20+, CD22+,CD79b+) negative for membrane and cytoplasmic gammachain, light chains, CD5 and CD10. Polyclonal CD138+plasmacells and polyclonal CD20+ B cells were concomi-tant populations. IHC showed rare IgG4+ plasmacells. Inconclusion, the integrated approach of MFC and histologyplus IHC allowed the diagnosis of GHCD in the bone mar-row, while immunofixation of free monoclonal heavy chainfragments of IgG in serum and urine were not informative.

FLOW CYTOMETRY ENUMERATION OF CIRCULA-TING ENDOTHELIAL CELLS IN HEALTHY PERIPHE-RAL BLOOD: A STANDARDIZED ITALIAN NETWORKSTUDYLanuti P.1,2, Rotta G.3, Almici C.4, Avvisati G.5, BudillonA.6, Doretto P.7, Malara N.8,9, Di Martino M.L.10,Simeone P.1,2, Marini M.4, Neva A.4, Gregorj C.5, DiCerbo M.5, Di Gennaro E.6, Leone A.6, Falda A.7, TozzoliR.7, Trunzo V.8, Mollace V.8, Muggianu E.10, Pinna S.10,Marchisio M.1,2 and Miscia S. 1,2.1Dept. of Medicine and Aging Science, School of Medicineand Health Science, Univ. “G. d’Annunzio” of Chieti-Pescara, 66013 Chieti, Italy; 2Center for Ageing Sciences(Ce.S.I.), “Univ. G. d’Annunzio” Foundation, 66013 Chieti,Italy. 3BD Biosciences Italia, 20090 Milano, Italy; 4Dept. ofTransfusion Medicine, Lab. for Stem Cells Manipulationand Cryopreservation, AO Spedali Civili di Brescia, 25123Brescia, Italy; 5Dept. of Hematology, Stem CellTransplantation, Transfusion Medicine and CellularTherapy, Campus Bio-Medico Univ. Hospital, 00128Rome, Italy; 6Experimental Pharmacology Unit, IstitutoNazionale Tumori Fondazione G. Pascale - IRCCS, 80131Naples, Italy; 7Clinical Pathology Lab., Dept. of Lab.Medicine, “S. Maria degli Angeli” Hospital, 33170Pordenone, Italy; 8Interregional Research Center for FoodSafety & Health (IRC-FSH), Dep. of Health Science,Univ. "Magna Graecia" of Catanzaro, 88100 Catanzaro,Italy; 9BioNEM Lab, Dept. of Experimental and ClinicalMedicine, Univ. “Magna Graecia” of Catanzaro 88100



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Catanzaro, Italy; 10Unit of Internal Medicine, Allergy andClinical Immunology, Dept. of Medical Sciences “M.Aresu”, Univ. of Cagliari, Monserrato, [email protected]

Circulating endothelial cells (CEC) are very rare events inhealthy peripheral blood (PB, < 50 cells/ml) and have highpotential diagnostic value in many endothelium-involvingdiseases. Polychromatic flow cytometry (PFC) appears thegold standard technique for their identification, and theapplication of standardized PFC approaches in the deter-mination of CEC numbers results crucial. However, where-as the interest in CEC studies has grown exponentially inrecent years, their level of standardization has not.In order to standardize CEC identification and count in PBsamples, a network of 6 laboratories carried out the stan-dardization of reagents, protocols, instruments, data acqui-sition and analysis.CEC were identified as CD45-/CD34bright/CD146+ nuclea-ted live events and counted by a double platform method.Results obtained from the intra-assay test demonstrated con-sistent CEC enumerations. The application of the protocol bythe sites involved in this study, allowed the achievement ofcomparable results among centers, with not inter-site variabi-lity in terms of CEC counts (p > 0.05). Standardization wasdemonstrated by testing PB samples from healthy donors (n= 4) harvested, delivered to all six sites and processed simul-taneously: considering the rareness of CEC, results demon-strated a very low variability among sites (% CV range = 28-56). Furthermore, by aggregating data obtained from all sites,CEC numbers in the healthy population (n = 154) were captu-red: medianfemale = 8.8 CEC/ml; medianmale = 11.4 CEC/ml.Biological variability was also assessed by analysing healthyPB samples (n = 112) on two different time points (0 and 90days), and no differences (p > 0.05) were found.Our data demonstrated that the here established procedu-res might be adopted as standardized method for CECmonitoring in PB; we also obtained CEC physiologicalbaselines in healthy subjects, useful as starting point fortheir monitoring in several endothelial dysfunctions.

SINGLE CENTRE VALIDATION OF CD34+ VIABILITYAND RECOVERY ACCORDING JACIE STANDARDSLongoni P.D.1, Vendramin A.2, Di Nuzzi P.A.1, Magni M.1,Carniti C.2, Corradini P.1,2,1Stem Cell Processing Laboratory; 2Department ofHematology, IRCCS Istituto Nazionale Tumori Milano, [email protected]

Jacie Standards regarding Processing Facility indicate thatprocessing procedures should be validated in order to resultin acceptable target cell viability and recovery. A limited num-ber of assays are required according to these Standards(Total Nucleated Cell count, cell viability and CD34 testing),but methods that should be used are not specified.The aim of this study was to assess the quality of the cel-lular therapy products by:1. analyzing the cellular products immediately after tha-wing to avoid possible DMSO toxicities2. applying the single platform ISHAGE Protocol to enume-rate CD34 cells.Assessment of CD34+ cells in thawed samples fromaphaeretic bags cryopreserved for autologous transplanta-tion procedures is time consuming and technically difficult.Thus we decided to evaluate cryovial samples that hadbeen cryopreserved at the same time and in the same con-ditions as what happens to bags cryopreserved for autolo-gous transplantation. One hundred and forty five frozenvials from the apheretic products were analyzed. The follo-

wing parameters were considered: viability (7-AAD negati-ve), CD45+ leukocytes and CD34+ stem cells absolutenumber, in order to correlate the results of the cryopreser-ved samples to the initial conditions after apheretic collec-tion. In 95% of the samples, the CD34+ viability was >80%(mean 90%; range 64%-98%) and the recovery >70%(mean 88%; range 59%-99%). Only in one case (0,7%)viability was <65% and in 4 cases (2,7%), CD34+ recove-ry was <65%. Nevertheless, when the engraftment at thetime of transplant was evaluated, no delay in neutrophilsand platelets recovery was observed.Based on our results we demonstrate that evaluation ofviability and CD34+ recovery on cryopreserved vials maybe used as a standard for validation of cellular productsprocessed by cryopreserving facilities.

STUDY OF MINIMAL RESIDUAL DISEASE IN CHIL-DHOOD ACUTE LYMPHOBLASTIC LEUKEMIA BYFLOW CYTOMETRY: IMPACT OF METHODOLOGICALADVANCES ON ANALYTICAL PERFORMANCESMarino N.1, Maglia O.1, Sala S.1, Silvestri D., ValsecchiM.G.2, Songia S.1, Cazzaniga G.1, Biondi A.1, andGaipa G.11Centro Ricerca Tettamanti, Clinica Pediatrica, UniversitàMilano-Bicocca; 2Dipartimento di Scienze della salute,Università Milano-Bicocca. [email protected]

Minimal Residual Disease (MRD) in children with acutelymphoblastic leukemia (ALL) is a strong and independentprognostic factor. The two techniques primarily used for theevaluation of MRD are PCR able to detect rearrangementsof genes encoding Immunoglobulins and/or T cell Receptorand flow cytometry (FCM) able to detect aberrant leuke-mia-associated immunophenotypes.We studied 309 pediatric patients enrolled in the treatmentprotocol AIEOP-BFM 2000. Bone marrow (BM) samplesaspirates were taken at different time points (day +15,day+33 and day+78 after initiation of treatment). We com-pared the results obtained at each time point by the simul-taneous use of the two methods, and we also took intoaccount the changes introduced in the technical procedu-res of FCM.The results obtained showed a high level of overall concor-dance (88%), confirming the results of previous studies.The concordance rate at single time points (day15, day 33,day 78) was 93%, 79%, 94% , respectively, whereas FCMsensitivity, was 95.4%, 58%, 38.4%, respectively.Most of discrepancies occured at day +33 with PCR-posi-tive samples but FCM-negative. We then asseses theimpact of increasing the number of events acquired in FCMprotocol (from up to 300.000 to up to 1.000.000). Indeed,this enhanced cell acquisition contributed to an increasedsensitivity at day +33 (from 54% to 60%). By contrast, thiswas not the case at day 78 due the contemporary increa-se of hematogones.In this study we confirmed that the comparison betweenFCM and PCR techniques for measuring MRD has to beconsidered in the context of the time point of treatment(which affects both the level of disease and the residualnormal hematopoiesis). At day +15 a high degree of con-cordance exists since the level of disease does not requi-re a high sensitivity. Most of discrepancies are at day +33mainly due to the lack of FCM sensitivity. This limitation isdirected associated with the low cellularity of samplestaken at this time point. To overcome this limitations itwould be necessary to refine and improve the technicalprocedures for bone marrow sampling, sample preparationand reducing the number of tubes. .



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In conclusion, although both methods can provide power-ful MRD-based prognostic information an improvement inFCM technical procedures are needed to overcome somesensitivity limitations of FCM.

CD47 EXPRESSION IN B-CELL CHRONIC LYMPHO-PROLIFERATIVE DISEASES: CORRELATION WITHTHE TENDENCY TO SPREADPiscitelli S., Graziano D., Basile F., Valentino M., Lo Pardo C.UOSS Immunologia Cellulare in Emato-Oncologia, SIMTAORN A. Cardarelli, [email protected]

In the last year we evaluated CD47 expression in 135 sam-ples: 64 patients affected by B cell chronic lymphoprolife-rative disorders (B-CLPD) and 71 controls.Materials and Methods The flow cytometric evaluation wasperformed by six color multiparametric analysis byFACSCantoII (BD). CD47 expression was evaluated by thefluorescence histogram analysis and the MeanFluorescence Intensity calculation.Results In control peripheral blood samples CD47 expres-sion was homogeneous between 103 and 104 on all hema-tologic cells, except for granulocytes (≤103). In healthysubjects bone marrow samples all hematologic cellsexpressed CD47 at the same fluorescence intensity (bet-ween 103 and 104). Among patients affected by B cell chro-nic lymphoproliferative disorders CD47 was overexpres-sed in 26 patients: 13 studied on peripheral blood and 13on bone marrow. In 4 patients we evaluated both the peri-pheral and bone marrow blood, without finding differences.CD47bright expression on pathological B lymphocytepopulations in B-CLPD correlated neither with the histolo-gical diagnosis nor with the type of sample (peripheralblood or bone marrow). On the contrary we found CD47overexpression in spread diseases.Conclusions CD47 overexpression has an important role indisease spreading of patients with blood disorders, particu-larly in B-CLPD. Interestingly two patients studied for HairyCell Leukaemia had CD47 over-expression in both patho-logical peripheral and bone marrow cells. This correlateswith the characteristic hairy cell tendency to spread.Therefore CD47 expression may be used as a prognosticfactor and as a possible therapeutic target in B-CLPD. Anti-CD47 antibodies could be used to prevent lymphomaspreading in early stage and to treat the disease in anadvanced stage.

THE VALUE OF COLLABORATIVE SCREENING NET-WORKS FOR OPTIMIZING DIAGNOSIS IN PAROXYSMALNOCTURNAL HEMOGLOBINURIA (PNH): RESULTS OFTHE RAP-CaRe STUDYRaia M.1, Graziano D.2, Palmieri S.2, Borino G.3,Iaccarino S.3, Iovane E.4, Marotta C.4, Volpe S.5,Volpe A.5, Rocco M.6, Serio B.6, Morelli G.7, Barone M.7,Tommasino C.8, Della Cioppa P.8, Risitano A.M.9,Del Vecchio L.1,9.1CEINGE Institute, Naples; 2Cardarelli Hospital, Naples;3S. Anna and S. Sebastiano Hospital, Caserta; 4MoscatiHospital, Aversa; 5Moscati Hospital, Avellino; 6S. Giovannidi Dio e Ruggi D’Aragona Hospital, Salerno; 7TortoraHospital, Pagani; 8S. Gennaro Hospital, Naples;9Federico II University Hospital, [email protected]

Background. PNH is an acquired form of hemolytic anemia,based upon the clonal expansion of a hematopoietic stemcell carrying a somatic mutation in PIG-A gene (involved inGPI anchor synthesis). Hemolysis, increased risk for throm-

bosis and variable grade of bone marrow failure characteri-ze PNH. Incidence and prevalence of PNH are 1/10-6 and10/10-6, respectively. Flow Cytometry (FCM) is the goldstandard for PNH diagnosis.Methods. The project “RAP-CaRe” (Rapid Approach toPNH Identification in Campania Region) aimed to identifynew PNH cases in Campania Region thanks to a rapidassay based on CD15 (for gating neutrophils) and FLAER(a GPI identifier). The study lasted two years (2013-2015)and involved 11 Centers. Patients with aplastic anemia,refractory cytopenia, Coombs negative, non-schistocytichemolytic anemia were included in the study. Positive sam-ples were confirmed by a complete polychromatic typing.Results. The collaborative group carried out 482 analyses.Twenty-one new cases were detected (4.35%).Interestingly, confirmatory testing demonstrated the absen-ce, in 2/21 cases, of red cell clonal involvement (“white”PNH). Analyzing 100 healthy donor samples and a batteryof 50 previously studied PNH samples, “rapid test” provedto be 100% sensitive and 100% specific.Conclusion. “Rapid test” showed the same efficiency indetecting new cases as the extended assay, with theadvantage of strongly facilitating the setting up of a colla-borative screening network.

POLYCHROMATIC FLOW CYTOMETRY ON SURGICALNODE BIOPSY IS DECISIVE IN THE DIAGNOSIS OF 3%OF CASES WITH SUSPECTED NON-HODGKINLYMPHOMAVisconte F.1, Mattioli M.1, Rosamilio R.1, Raia M.1, ScaliaG.1, Del Vecchio L.1, Panico L.21CEINGE Institute, Naples and 2San Giuseppe MoscatiHospital, Avellino, [email protected]

Background. The diagnosis of NHL is essentially basedupon histology and immunohistochemistry (IHC).However, a series of ancillary technologies may improveaccuracy of NHL identification and characterization. Todate, the impact of flow cytometry (FCM) on NHL diagno-stic accuracy of has not been quantitatively assessed.We studied, by FCM on surgical node biopsies, a series of340 cases with suspected NHL. Final diagnoses were:SLL (21 cases), NMZL (20), FL (55), MCL (13), DLBCL(62), PMLBCL (2), BPDCN (2), DCS (1), ALCL CD30+ (2),B-T Rich (2), Myeloid Tumor (2), MM (1), Composite NHL(3), ALK + LBCL (1), TCL (5), Metastasis (16), ReactiveHyperplasia (RH, 132).Methods. We assumed that the aim of FCM in NHL is toassess: 1) asynchronous expression of T cell antigens andTCR-Vβ restriction in T-cell NHL; 2) anomalous assemblyof B cell antigens and κ/λ restriction in B-cell NHL; 3)absence of point 1 and 2 features in T-cell and B-cell RH;4) absence of CD45 expression in epithelial tumors. Themain goal of this study was to assess, on a non-competiti-ve basis, the role of FCM in implementing accuracy of NHLdiagnosis.Results. IHC was accurate in 329/340 cases (96.76%).FCM was accurate in 328/340 cases (96.47%). In the 11cases in which IHC was inaccurate, the contribution ofFCM enabled a combined final diagnosis. These caseswere one SLL, three NMZL, three Composite NHL, oneTCL, and three RH. Thus, a decisive contribution of FCMto an accurate diagnosis occurred in 3.23% of cases.Conclusion. This study, for the first time, demonstrated thatmore than 3% of cases with suspected NHL could bemisdiagnosed by IHC alone, so that an ancillary techniqueas FCM is mandatory.



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PIVOTAL ROLE OF MULTICOLOUR FLOW CYTOME-TRY IN DETECTING AND MONITORING BICLONAL B-CELL LYMPHOPROLIFERATIVE DISEASESVittoria L.1, Testa F.1, Testi M.A.1, Spina F.2, Gobbo M.1,Cabras A.1, Aiello A.1Fondazione IRCCS Istituto Nazionale Tumori,1Dipartimento di Patologia Diagnostica e Laboratorio;2S.C. di Ematologia, Milano, [email protected]

Biclonal lymphoproliferative disorders (LPD) are rare enti-ties characterised by the coexistence of two or moredistinct aberrant clones that might have different biologicand clinical behaviour. Their incidence has been estimatedaround 4-5% by the few comprehensive studies available.In order to both ascertain the role of multicolour flow cyto-metry (MFC) and sort crucial markers in the identificationof cases with more than one neoplastic clone, we retro-spectively evaluated a series of 488 consecutive leukemicB-cell LPD (B-LPD) by 8-colour FC with a 20 Abs standar-dized panel.At least 2 coexisting clonal populations were identified inPB and/or BM of 23 patients (4.7%), two of which contai-ned 3 neoplastic clones. Most cases showed unbalancedk/λ ratio and concurrent analysis of CD5, CD10, CD19,CD20 and CD45 allowed further subclonal dissection. Fourout of 5 apparently polyclonal B-LPD harboured 2 or 3 clo-nes with assorted light chain expression (k-λ, k-k-λ, k-λ-λ).Conversely, subpopulations with the same light chainrestriction often displayed dissimilar k or λ expression pat-tern. Along with Ig light chains, the most helpful markers todissect clones were CD5 (13 cases), followed by CD20,CD22, CD38, CD79b (12) and CD19, CD81, CD200 (10).Ig gene rearrangement was investigated by PCR in 5 LPDand 3 proved to have different clonal origin. In our series,one MZL patient in apparent remission had two relapsingclones identified by MFC, only one of which is now respon-ding to therapy. Furthermore, in a biclonal CD5+ and CD5-SMZL, we have recently demonstrated dissemination toCNS by the single CD5+ component.In summary, by using a broad antibody panel we foundbiclonality in 4.7% of B-LPD, in keeping with previouslyreported values. We thus provide further evidence thatMFC is highly sensitive in spotting different LPD coexistingclones and is decisive to identify and monitor more aggres-sive and therapy-resistant subpopulations.

Immunology

EFFECTSOFHYMENOPTERAVENOM IMMUNOTHERAPYONCTLA-4EXPRESSION INCD4+CD25high LYMPHOCYTESChiappori A.1, Folli C.1, Parodi A.2, Rogkakou A.1,Fenoglio D.2, Riccio A.M.1, Passalacqua G.1, Filaci G.2,Canonica G.W.11Allergy and Respiratory Diseases Clinic, DIMI, University ofGenoa, IRCCS AOU San Martino-IST, Genoa, Italy. 2Centreof Excellence for Biomedical Research; DIMI, University ofGenoa, Genoa, [email protected]

Introduction: Venom immunotherapy (VIT) allows to modu-late the response to Hymenoptera venom allergens and toavoid severe anaphylactic reactions in allergic patients. Theperipheral tolerance can be achieved by different mecha-nisms including regulatory T-cells (Tregs), a subset of CD4+T lymphocytes that regulates the ongoing immune respons-es toward several antigens with direct cell contact andrelease of cytokines. These lymphocytes express on theirsurfaces activatory and inhibitory receptors and smallchanges in their expressions may have strong effects on theoutcome of T-cell activation. The cytotoxic T-cell antigen 4(CTLA-4) is an inhibitory receptor localized in sub-mem-brane vesicles that rapidly are recycled to/for the cell sur-face. Once expressed at cell membrane level, CTLA-4 cansuppress naive T-cell activation using an impressive varietyof different mechanisms still matter of study.Aim: To evaluate the role of CTLA-4 receptor in the inductionof immunologic tolerance following VIT in allergic patients.We compared CTLA-4 expression levels on CD4+CD25high

Treg cells isolated from peripheral blood at baseline (t0) andsix months after VIT (t6).Materials e methods: Peripheral blood mononuclear cells(PBMCs) were obtained from 7 Hymenoptera venom allergicpatients and 9 healthy non-allergic controls. CTLA-4 expres-sions, both at cell surface and intracellular level, were assessedon Treg CD4+CD25high lymphocytes by flow cytometry.Results: Our data did not show differences in CTLA-4expression between healthy controls and allergic subjectsbefore VIT, both at cell surface and intracellular level.The cytometric analysis has evidenced a significantdecrease in intracellular CTLA-4 expression in allergicpatients following immunotherapy.Conclusions: Our data demonstrated the involvement ofCTLA-4 molecule in the peripheral tolerance induction fol-lowing VIT. However, CTLA-4 regulatory mechanismsshould be further studied to completely understand its spe-cific role in the acquisition of immune peripheral tolerance.This work was partially supported by ARMIA.

DIHYDRORHODAMINE 123 (DHR) FLOW CYTOMETRICASSAY IN CHRONIC GRANULOMATOUS DISEASE(CGD)Comini M.1, Beghin A.1, Soresina A.2, Zucchi M.1,Villanova S.1, Baffelli R.1, Bolda F.1, Porta F.3,Lanfranchi A11-U.O. Microbiologia e Virologia, Sezione di Ematologia eCoagulazione, Laboratorio Cellule Staminali, Ospedaledei Bambini, Brescia; 2-Unità di Immunologia Pediatrica,Clinica Pediatrica, Università di Brescia, Brescia; 3-U.O.Oncoematologia Pediatrica e Trapianto Midollo Osseo,Ospedale dei Bambini, Brescia, [email protected]



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CGD patients(pts) show impaired function of cellularNADPH oxidase that catalyzes the reduction of O2 to ROSdetermining a defect in the PMNs respiratory burst activity.Flow cytometric(FC) DHR assay is a rapid and sensitivemethod for investigation of PMNs respiratory burst and con-sequently for diagnosis of CGD pts.Activation of PMNs with PMA results in oxidation of DHR torhodamine 123,which can be measured by FC assay.Healthy controls produce a strong fluorescence whereasthere is no evidence of rhodamine fluorescence in CGD pts.Female carriers of the X-linked CGD gene show the pres-ence of 2 peaks that represent a bimodal pattern of activity:astrong positive peak and a weaker/negative peak.PMNs harvested by dextran sedimentation of peripheralblood,were incubated with DHR and catalase for 5 min at37°C.PMA was added in “stimulated” tube for 30 min at37°C.Samples were analyzed by BD FACSCANTO II.Astaining with CD45 APC-H7 and CD15 HorizonV450,performed before cells stimulation,allowed us togate out other non-reactive cells morphologically similarto PMNs and to obtain more specific analysis.We collected and analyzed by DHR test 2347 pts withrecurrent infections from 1996 to 2015.We identified 38 pts with impairment of respiratory burst activ-ity;22 pts were given a diagnosis of X-CGD since the DHRassay performed was unreactive and their mothers showedthe coexistence of functional and disfunctional PMNs subpop-ulations. Molecular diagnosis analysis was recently intro-duced in our laboratory and up to now 12/38 CGDpts,diagnosed by DHR assay, were confirmed by this test.8/38 CGD pts underwent HSCT in our center. In 7/8 CGDpts DHR assay was performed to monitor the recovery ofPMNs function after HSCT.Quantitative results of theDHR tests correlate well with donor cell chimerism statusby STR-PCR.In conclusion,FC DHR assay is a rapid andhighly sensitive test for assessing the oxidative burst inPMNs and a useful diagnostic tool for chimerism statuspost-HSCT.

EVALUATION OF HLA-G IN SYSTEMIC SCLEROSISContini P.1, Ghio M.1, Negrini S.1, Puppo F.1.1Department of Internal Medicine, University of Genoa,Genoa, [email protected]

Systemic sclerosis (SSc) is a complex diseasecharacterized by immune dysregulation, extensive vascu-lar damage and wide-spread fibrosis, mostly driven bythe over-production of TGF-β. From a clinical point ofview SSc is divided into limited (lSSc) and diffuse (dSSc)forms. Human leukocyte antigen-G (HLA-G) is a non-classic class I HLA molecule which is characterized byimmuno-modulating properties exerted by soluble (sHLA-G) and membrane-bound isoforms. The purpose of thisstudy is to analyze the membrane expression and plasmalevels of sHLA-G in SSc patients and healthy donors andcorrelate them with TGF-beta, IL-10 and IL-4.Materials and Methods. Thirty-five patients with SSc (12dSSc and 23 lSSc) were enrolled. The membraneexpression of HLA-G1 molecules was evaluated by flowcytometry (Beckman Coulter Navios). Plasma concentra-tion of sHLA-G, TGF-beta, IL-10 and IL-4 was determinedby immuno-enzymatic technique (ELISA).Results. Our results show a significant higher percentageof HLA-G+ cells, mainly monocytes and T cellsCD4+CD8+, and a significant increase in sHLA-G in bothdSSc and lSSc compared to control. Significant higherlevels of TGF-β in both lSSc and dSSc are observedcompared to healthy donors, and a strong correlation

between sHLA-G and TGF-β defines the lSSc. ASignificant reduction in IL-10 is observed in both clinicalcourses of the disease compared to controls. On theother hand no differences are identified in IL-4 concentra-tion between patients and healthy subjects.Conclusion. Little attention has been devoted to the roleof HLA-G in autoimmune and chronic inflammatory disor-ders; however increasing evidences show that HLA-Gplays a role in immune surveillance as well as in main-taining tissue integrity. Our results suggest that HLA-G isinvolved in the pathogenesis of SSc. Further studies areneeded to fully understand the mechanisms associatedwith the function and regulation of HLA-G in SSc.

AN OPTIMIZED METHOD TO SELECTIVELY MEASU-RE APOPTOSIS IN HIV-1 INFECTED CELLSCugia G, Poddesu B, Lori F, De Forni D.ViroStatics s.r.l., Drug Development Unit, Porto ConteRicerche, Alghero, [email protected]

Background: Apoptosis, or programmed cell death, is akey event in biological homeostasis but it is also aninnate defense of cells against viral invasion. Althoughmultiple mechanisms contribute to the gradual T celldepletion that occurs in HIV-1 infected patients, it hasbeen proposed that HIV-1 also has the ability to preventapoptosis of infected cells while inducing apoptosis ofuninfected bystander T lymphocytes, including CD4+ andCD8+ T cells, an important determinant in HIV-1 patho-genesis and immunodeficiency.Methods and Results: We have optimized a flow cytome-try method to measure selective apoptosis induction inHIV-1 infected CD4+ T cells by using a three-color panel.CD4+ T cells were isolated by magnetic bead-basedseparation from PBMC derived from healthy donors.PHA-stimulated cells were infected using a wild type HIV-1 strain (NL4-3) and cultured with IL-2 for 4 days. Viablecells were identified by the amine reactive viability dyeexclusion (FVS450) and KC57 antibody was used todetect intracellular HIV-1 Gag p24 protein in infectedcells. Annexin V and FVS450 were employed to distin-guish apoptotic from necrotic cells in fixed samples.CD4+ T cell depletion following HIV-1 infection wasobserved while intracellular p24 and apoptosis werereproducibly detected 4 days post infection. We con-firmed that apoptosis was significantly (p<0.05) more fre-quent in uninfected compared to infected cells.Conclusions: The assay in its present form is useful forstudying selective apoptosis induction in HIV-1 infectedsamples. Such an assay could be useful to better under-stand the multiple mechanisms of T cell depletion in HIVinfection and to identify in vitro compounds that might beable to selectively induce apoptosis in infected cells.

REGULATORY T CELL MODULATION IN CORDBLOOD AFTER ANTENATAL GLUCOCORTICOIDADMINISTRATIONDe Vita M.1., Catzola V.1., Rosati P.2., Mappa I.2.,Battaglia A.1., and Fattorossi A.11Lab Immunol, Dept Gynecol Oncol; 2Dept Obst andGynecol, Catholic University Rome, [email protected]

Studies in laboratory rodents have shown that exogenousglucocorticoid hormones (GC) administration produces aselective depletion of immature cortical thymocytes and arelative enrichment of more mature thymocyte subsets inthe medulla. Analogous studies in humans are scanty


because the study material would be CG-treated thymusat the birth in the absence of concurrent morbidities.Antenatal GC administration (AGC) to otherwise healthypregnant women at risk of preterm delivery is currentlyused to accelerate maturation of the fetal lung. In this clin-ical setting, a healthy fetal thymus is exposed to exoge-nous GC. Because direct analysis of the thymus in healthyhuman newborns is obviously not a feasible option, wetook the T cell pool in cord blood (CB) as surrogate indica-tor of thymus functionality. Flow cytometry analysisshowed that AGC induced modest modifications of major Tcell subset distribution; frequency of recent thymic emi-grants (CD45RA+ CD62L+ CD31+ CD4+ T cells) was alsonot affected by AGC. AGC had no impact on the wholepool of regulatory T cells (Tregs, CD25+ Foxp3+ CD4+ Tcells). Most CB Tregs are of direct thymic origin andexpress CD45RA. We found that CD45RA+ Tregs werecharacterized by a significantly lower expression of CD25and Foxp3 level as compared to their CD45RA− counter-part, in analogy with adult CD45RA+ Tregs. Only minuteproportions of Tregs, both CD45RA+ and CD45RA , pro-duced IL-2, thereby confirming they were bona fide Tregs.AGC selectively reduced CD45RA+ Tregs proportion with-out affecting CD25 and Foxp3 expression and suppressiveactivity. Thus, one may conclude that AGC reduces Tregoutput from thymus but a homeostatic expansion in theperiphery fills the Treg pool.

COMPARISON OF DIFFERENT METHODS TO INVITRO DIFFERENTIATE PORCINE MONOCYTES INTOMACROPHAGESFranzoni G.1,2, Oggiano A. 2, Graham S.P.3, Dei GiudiciS.2, Pilo G.4, Nicolussi P.4, Bonelli P.41Università degli Studi di Sassari, Dipartimento di ScienzeBiomediche, Sassari, Italy; 2Istituto Zooprofilattico dellaSardegna, Laboratorio di Malattie Esotiche, Sassari, Italy;3School of Veterinary Medicine, University of Surrey,Guildford, United Kingdom; 4 Istituto Zooprofilattico dellaSardegna, Laboratorio di Diagnostica Clinica e Stabulari,Sassari, [email protected]

Monocytes are immune cells that circulate in the bloodbefore entering tissues and replenish the macrophagepopulations, which maintain tissue homeostasis and areimportant in defence against intracellular pathogens. Todate there are no standardized protocols available for invitro differentiation of porcine monocytes to macrophage.Differentiation is achieved through incubation of mono-cytes with porcine serum/plasma (10-30%) for 3-4 daysor with the addition of hM-CSF (human macrophage-colony stimulator factor) to culture media for 4-7 days.Our aim was to provide a standard method to in vitro dif-ferentiate porcine monocytes into macrophages and toevaluate their susceptibility to African Swine Fever Virus(ASFV).Monocytes were cultured for 4-5 days in media supple-mented with 30% porcine plasma or with 50 ng/ml of hM-CSF and then phenotype and susceptibility to ASFV(levels of late protein p72) were assessed using flowcytometry. Monocytes cultured with both methodsincreased in dimension (FSC) and granularity (SSC). Nodifference were observed between macrophage popula-tions in terms of FSC, instead SSC was higher inmacrophages differentiated with porcine plasma. Asexpected CD203 and CD163 expression were up-regu-lated during monocytes differentiation into macrophages.CD203 expression in macrophages were similar usingboth protocols, while CD163 expression was higher in

those differentiated with hM-CSF. CD14 and CD16 per-centages of positive cells did not showed any differences.As expected, macrophages were more susceptible toASFV infection than monocytes, but macrophages differ-entiated using hM-CSF showed higher proportions ofASFV+ cells. Results from ongoing experiments beingconducted to determine cytokine responses to LPS stim-ulation will also be displayed.Data generated in this study suggest that use of hM-CSFrepresents a suitable method to differentiate porcinemonocytes into macrophages in vitro.

THE ROLE OF THE IMMUNE SYSTEM IN THE PATHO-GENESIS OF MULTIFACTORIAL DISEASES: THEINFLUENCE OF IGH ENHACER' HS1.2 ON CROHN'SDISEASEFrosali S.,1 Cianci R.,1, Lolli S.,2 Pagliari D.,1 MarmoR.,3 Melioli G.,4 Orlando A.,5 Serone E.,2 Pandolfi F.,1Frezza D.21.Institute of Internal Medicine and Geriatry, CatholicUniversity, Rome, Italy. 2.Department of Genetic, TorVergata University, Rome, Italy. 3.Department of.Hygiene, Catholic University, Rome, Italy. 4.BiomedicDepartment of Internal and Specialistic Medicine,“V.Cervello” Hospital Palermo. 5.Central Laboratory,I.R.C.C.S. G. Gaslini, Genoa, [email protected]

Crohn's disease is a complex multifactorial disease.Although the precise etiology of inflammatory bowel di-sease (IBD) remains obscure, several reports haveindicated that an altered function of the mucosal immunesystem plays an important role in its pathogenesis.Host genetic susceptibility plays a key role in the risk ofdevelopment of IBD. Many IBD susceptibility genes havebeen identified and some of these are associated withhost immune function, including epithelial barrier func-tions, host defense mechanisms in response topathogens and host immune response.Through B cell development, the Ig heavy chain expres-sion is modulated by a regulatory region at the 3' of theconstant alpha gene (3'RR). The human 3'RR1 and3'RR2 are both characterized by three enhancers, thecentral of which, namely hs1.2, is highly polymorphic.Human hs1.2 has four different variants with uniquebinding sites for transcription factors (e.g. NF-kB andSP1) and shows variable allelic frequencies in popula-tions with immune disorders.The aim of our study was to investigate the impact of IgHHS1,2 alleles on the pathogenesis of Crohn’s disease.Results do not show any significant differences withrespect of genome and allele frequencies of the enhancerHS1,2-A in Crohn’s patients (cases) and healthy subjects(controls). No differences between cases and controlswere observed with respect to serum and secretory IgA,IgG1, IgG2 and IgG3. On the contrary, statistically signifi-cant differences were found between cases and controlswith respect to ASCA positivity (p<0.001), serum IgG4(p<0.001) and IgD (p=0.001).The genotypes and alleles frequencies of the enhancerHS1,2-A do not seem association with this disease.However, the data on antibody level, high anti-Saccharomyces Cerevisiae IgA and IgG and high IgD inthe patients, are interesting and further studies will beneeded to better understand their significance in patho-genesis of Crohn’s disease.


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DEVELOPMENTAL RELATIONSHIP BETWEEN TYPE-1AND TYPE-2 INNATE LYMPHOID CELLSGabrielli S., Buccella F., Canonico B., Luchetti F.,Cesarini E., Palma F., Papa S. and Zamai L.Department of Biomolecular Sciences, University ofUrbino “ Carlo Bo”, [email protected]

Innate lymphoid cells (ILCs) are immune effector cellswhich, differently from T and B lymphocytes, do notexpress rearranged receptors. Besides NK cells thatbelong to the ILC1 group, the novel lineages have raiseda question regarding their developmental relationship.The ILC lineages have different effector functions andanatomical localization, suggesting the possibility that acommon progenitor may migrate and differentiate into thedifferent ILCs, depending on the tissue microenviron-ment. Nevertheless, we have recently suggested thatCD56dim and CD56bright NK cells, both of the ILC1group, originate from two distinct precursors which dif-ferentiate into cells with convergent phenotypes andfunctions. CD56bright /CD117+ NK cells, differently fromCD56dim/CD117neg NK cells, early express natural cyto-toxicity receptors (NCRs), while lately upregulategranzyme-B, perforin, LFA-1, and CD94-CD159a hete-rodimer (Zamai et al. Cytometry A 2012). Within the ILCfamily, the type-2 ILCs (ILC2), characterized byCD161+/NKp44neg phenotype, typically produces IL-13and IL-5 type-2 cytokines (Mjosberg et al. Nat. Immunol.2011). Under opportune cytokine stimulation, this subsethad been described to produce IFN-gamma and tobecome cytotoxic (Loza et al. Blood 2002). Similar to theCD56bright lineage, ILC2 early express CD117, CD127and CD161 antigens and supsequently upregulate CD94(a late marker of CD56bright NK cells), resembling theCD56dim lineage. The results show that ILC2 are closelyrelated to CD56bright NK cells, however, after IL-2 stimu-lation, they resemble the CD56dim NK cell lineage in theirdevelopmental pattern of the effector molecule andreceptor expression.

IMMUNOPHENOTYPICAL AND FUNCTIONAL CHA-RACTERIZATION OF PBMC AND FIBROBLASTSFROM SCLEROMYXEDEMA PATIENTSKalli F.1, Parodi A.1, Curto M.1, Ferrera F.1, Bernardi C.1,Marini C.1, Nasi G.1, Cioni M.3, Rongioletti F.3, FenoglioD.1,2, Filaci G.1,21CEBR; 2Dept. of Internal Med, Univ. of Genoa; 3Dept.of Health Sciences, Univ. of [email protected]

Scleromyxedema (SME) is a chronic connective tissuedisease characterized by diffuse skin sclerosis due toabnormal proliferation of fibroblasts and accumulation ofmucopolysaccharides. It is associated with monoclonalgammopathy and severe systemic disorders, leading to aguarded prognosis. The association of the disease withthe presence of a monoclonal gammopathy and thetherapeutic effect shown in some cases by intravenousimmunoglobulin administration suggest that immunologi-cal alterations may be involved in disease pathogenesis.Hence, the project has been to characterize peripheral Tcell subpopulations (Th1, Th2, Th17, Treg cells) andstudy the function of skin fibroblast primary lines(proliferation and mucin secretion) derived from 10 SMEpatients (pts) compared to 6 healthy donors. The resultsshowed: a) any significant difference in the frequencies ofCD4+ and CD8+Treg populations between SME pts andcontrols; b) effector and terminal effector memory per-

centages resulted increased in SME pts both in CD4+and in CD8+ T cells; c) the cytokine analysis of CD4+ Tcells showed an increase of Th2 profile both ex-vivo andafter in vitro expansion with a polyclonal stimulus orCandida bodies (Ca) cells in SME patients. Regardingfibroblast data, SME fibroblast growth was higher inpresence of autologous plasma and only SME fibroblastsproduce mucin at high level. Collectively, the study pro-vides at the first time a comprenhensive picture of periph-eral T cell populations showing the existence of Th2prevalence in inflammatory compartment that is couldcorrelated with altered functions of fibroblas.

IMMUNOPHENOTYPING OF TH1, TH2, TH17 ANDTREG LYMPHOCYTES IN AN OUTPATIENT POPULA-TION OF AN ACCREDITED LABORATORY INNORTHEAST ITALYMarenda B., Lenzo G., Quartesan R., Signorelli A.,Fortinguerra S., Farnesi M., Deoni D., Zorzi G., Buriani A.Laboratorio Analisi, Gruppo Data Medica [email protected]

T helper cells of Th1, Th2, Th17 and Treg phenotypesplay a key role in the orientation of the immune responseand can be usefully tested in immune dysregulations.Selective expression of chemokine receptors and othersurface molecules in Th1, Th2 and Th17 cells can beused to identify these subpopulations. CD195 (CCR5)has been shown to be expressed by Th1 lymphocytes,while CD194 (CCR4) and CD161 are expressed by Th2and Th17 lymphocytes, respectively. At present referencevalues for these subpopulations are still few. The aim ofthis work is to contribute to the development of referencevalues for Th1, Th2, Th17 and Treg. The present retro-spective analysis on data collected over the last fewyears using a CyFlow Cube 8 flow cytometer (SysmexPartec), provides values for each subpopulation from out-patients selected for the study, so as to exclude lym-phoblastic as well as lymphopenic subjects. The pheno-types were identified as follows: CD3/CD4/CD195(CCR5)for Th1; CD3/CD4/CD194(CCR4) for Th2; CD3/CD4/CD161for Th17; CD4/CD25bright/Foxp3 for Treg lymphocytes.Sample description after inclusion/exclusion criteria: age46±13; sex: F=60 M=34; Lymphocytes 2106±539/µl; Tlymphocytes 1588±421/µl (71%±20); T helper lympho-cytes 973±284/µl (47%±10) – motive of the exam: aller-gopathy (6); autoimmune disease (51); tumor (16); infec-tion (16); check-up (5). Results: Th1 Th2 (N=59): Th1108±64/µl (5%±2,7); Th2 92±63/µl (4,2%±2,4); Th1/Th21,4±0,8 – Th17 (N=62) 181±79/µl (8,8%±3,9); Treg(N=89): 19±17/µl (0,9%±0,7). (Values are mean±SD).The results show that despite the high variability of thedata, which suggest a high sensitivity of these type ofcells for disease-related variations, the mean values arerather consistent in all age or sex subgroups, suggestingthat the mean values obtained from this sample couldactually be used as initial laboratory internal reference,for the evaluation of the cytokinergic phenotype with rela-tion to specific pathophysiological pattern.

ANALYSIS OF MEMORY LIKE NK CELLS IN HCMV-INFECTED PEDIATRIC PATIENTS UNDERGOING αα//ββ+T-AND B-CELL DEPLETED HSCT FOR HEMATOLOGICALMALIGNANCIESMuccio L.1, Bertaina A.2, Falco M.3, Pende D.4, MeazzaR. 4, Lopez-Botet M.5, Moretta L.3, Locatelli F.2,6,Moretta A.1* and Della Chiesa M.1*1Dipartimento di Medicina Sperimentale and Centro diEccellenza per la Ricerca Biomedica, Università di Genova,


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Genova, Italy; 2Dipartimento of Ematologia/OncologiaPediatrica, IRCCS Ospedale Pediatrico Bambino Gesù,Roma, Italy; 3IRCCS Istituto Giannina Gaslini, Genova, Italy;4Istituto di Ricovero e Cura a Carattere Scientifico, AziendaOspedaliera Universitaria San Martino-Istituto Nazionale perla Ricerca sul Cancro, Genova, Italy; 5Universitat PompeuFabra and Institut Hospital del Mar d'InvestigacionsMèdiques, Barcelona, Spain; 6Dipartimento di ScienzePediatriche, Università di Pavia, Pavia, [email protected]

We analyzed the impact of human cytomegalovirus(HCMV) infection on the development of NK cells in acohort of 27 pediatric patients, affected by hematologicalmalignancies, who had received a novel type of HLA-haploidentical HSCT, depleted of both α/β+ T cells and Bcells. In line with previous studies in adult recipients ofumbilical cord blood transplantation (UCBT), we foundthat HCMV reactivation promoted a fast generation ofmature NK cells also in pediatric patients. In particular,upon HCMV reactivation, most children showed a pro-gressive expansion of a memory-like NK cell subsetcharac terized by the expression of both NKG2C, a puta-tive receptor for HCMV, and CD57, an NK marker of ter-minal differentiation. These cells were detectable bymonth 3 after HSCT, and kept expanding at least until 12months after HSCT, in higher percentage than HCMVseropositive healthy donors. NKG2C+CD57+ NK cellexpansion was accompanied by high levels of KIRs andLIR-1 and low levels of Siglec-7, NKG2A and IL-18Rα,was active against tumor targets and could also efficient-ly respond to cells expressing HLA-E (a known ligand forNKG2C). This HCMV-induced NK cell subset displayedpoor ability to produce IFN-γ in response to IL12 andIL18. The diminished response to these cytokines,together with their highly differentiated profile, couldreflect their skewing toward an adaptive condition spe-cialized in controlling HCMV. In conclusion, in this cohortof pediatric patients, HCMV induced the generation offunctional memory-like NK cells that could play a role inpreventing infections and might contribute to anti-leukemia effects.

TARGETING CXCR4 REDUCED T REGULATORYCELLS (TREGS) -MEDIATED CELL PROLIFERATIONSUPPRESSION IN RENAL CELL CARCINOMA (RCC)PATIENTSNapolitano M.1, Santagata S. 1, D'Alterio C. 1, CecereS.1, De Domenico R. 1, Cacciapuoti C. 1, Dimaro S. 2,Marinelli L. 2, Longo N. 3, Pignata S. 1, Perdonà S. 1,Scala S. 11National Cancer Inst. Pascale, Naples, Italy; 2Departmentof Pharmaceutical Chemistry, Naples, Italy; 3Federico II,Medical University, Naples, Italy [email protected]

Introduction: T regulatory cells CD4+CD25+Foxp3+(Tregs) accumulate in renal cell carcinoma (RCC) andhave been implicated in tumor immune escape.Chemokine receptor axis CXCR4/CXCL12/CXCR7 playsa crucial role in RCC biology. Aim: To evaluate patients Tregs function from tumor tis-sue compared to periphery and peritumoral unaffectedtissue and to study the effect of CXCR4 antagonism onTregs immuneresponse.Materials and methods: 40 samples from tumor, peritu-moral tissue and relative blood plus blood from 15 healthydonors (HD) were collected. Tregs and circulating T effec-tor (Teff) cells were isolated using a magnetic cell kit.

Tregs were identified as CD4/CD25/Foxp3 and charac-terized for surface markers (CTLA-4; CXCR-4; PD-1;CD39; ICOS) by flow cytometry. Tregs suppressive activ-ity was evaluated through in vitro co-colture of purifiedTregs versus CFSE-labeled autologous Teff cells.CXCR4 antagonism on Tregs suppressive function wasevaluated through a new class of CXCR4 antagonistsrecently developed.Results: Higher number of Tregs was detected in theRCC tumor, peritumoral tissue and periphery comparedto HD (p<0.001). Higher expression of surface markersCTLA-4; CXCR4; PD-1; CD39 and ICOS were detectedon Tregs isolated from tumor samples compared to circu-lating Tregs cells (p<0.01) and on peripheral Tregs cellscompared to HD. Functional analysis displayed that inRCC patients suppression induced by tumoral isolatedTregs was higher than suppression induced to both circu-lating and peritumoral tissue Tregs toward activated Teffector cells (p<0.05). CXCR4 antagonism revertedTreg-mediated proliferation suppression (p<0.05).Conclusions: Higher number and suppressive activitywas detected for Tregs isolated from primary RCC tis-sues compared to peritumoral unaffected tissue andblood isolated Tregs. CXCR4 antagonism prevented Teffcell suppression Tregs induced. TargetingCXCL12/CXCR4 axis in RCC might be a strategy forimmunotherapeutic intervention.

RETICULUM ENDOPLASMIC (RE) STRESS IMPACTSON GENERATION OF T REGULATORY LYMPHOCYTES(TREG) WITHIN THE TUMOR MICROENVIROMENTNasi G.1, Mahadevan N.3, Parodi A.1, Tardito S.1, CurtoM.1, Kalli F.1, Fenoglio D.1,2, Zanetti M.3, Filaci G.1,21CEBR; 2Dept of Internal Medicine- Univ of Genoa;3Dept of Medicine and Cancer Center, Univ of San Diego [email protected]

Unfolded protein response (UPR) produced by RE stresshas been involved in tumor growth and progression. Intumor cells UPR activates a pro-inflammatory cascade,transmissible to tumor-infiltrating myeloid cells, whichfavors tumor development. Moreover, RE stress may beinvolved in intratumoral Treg generation and tumorimmune escape through the induction of IL10 secretionby Treg.Thus, our purpose was to investigate if peripheral CD8+and CD4+ T effector cells could be converted in Treg byexposure to RE stressed tumor conditioned medium. Inparticular, we investigated on the induction of Tregbelonging to the known CD8+28-127low andCD4+25high127low Treg subpopulations analyzing theirsuppressor activity and expression of CD73 and CD39ectoenzymes, molecules involved in their suppressionfunction.Our results showed that both circulating CD4+ and CD8+T cells, conditioned by RE stressed tumor medium,differen tiated in Treg, acquiring a remarkable suppres-sion function. However, this process was not strictlydependent on either CD73 and CD39 molecules (sinceno significant differences in their expression wereobserved between lymphocytes conditioned by REstressed tumor and controls) or IL10 secretion (since aneutralizing anti-IL10 monoclonal antibody did notantago nized it).These data demonstrate that RE stressed tumor cellsproduce a still unidentified factor able to convert T effec-tor cells in Treg.


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B7-H6-MEDIATED DOWNREGULATION OF NKP30 INNK CELLS CONTRIBUTES TO OVARIAN CARCINO-MA IMMUNE ESCAPEPesce S.1, Tabellini G.2, Cantoni C.1,3, Patrizi O.2,Coltrini D.2, Rampinelli F.4, Matta J.5, Vivier E.5, MorettaA.1, Parolini S.2, Marcenaro E.1 1Dipartimento di Medicina Sperimentale and Centro diEccellenza per le Ricerche Biomediche, Università degliStudi di Genova, Via L.B. Alberti 2, 16132 Genova, Italy;2Dipartimento di Medicina Molecolare e Traslazionale, VialeEuropa 11 25123 Brescia Italy; 3Istituto Giannina Gaslini,L.go G. Gaslini 5, 16148 Genova, Italy; 4Dipartimento diOstetricia e Ginecologia, Spedali Civili di Brescia, 25123Brescia, Italy; 5Centre d'Immunologie de Marseille-Luminy,UM2 Aix-Marseille Université, INSERM U1104, CNRSUMR7280, Marseille, France and Service d'Immunologie,Assistance Publique-Hôpitaux de Marseille, Hôpital de laConception, Marseille, France [email protected]

In this study the phenotype and function of tumor-associ-ated NK cells from peritoneal fluids of a selected cohortof patients with seropapillary ovarian carcinoma wereanalyzed. In > 50% of these patients, the expression ofthe activating receptor NKp30 in tumor-associated NKcells was substantially reduced as compared to autolo-gous peripheral blood (PB) NK cells. The impairedexpression of this receptor was associated with thepresen ce of one of its cellular ligands (B7-H6), which wasdetectable as a surface/cytosolic molecule in tumor cellsand as a soluble molecule in the peritoneal fluid. NK cellsfrom patients expressing this NKp30low phenotype dis-played an impaired interferongamma (IFNg) productionand cytolytic function when tested against target cellsexpressing surface B7-H6. Our data also suggest that inthese patients, the defective expression and function ofNKp30 may be induced by the chronic engagement ofthis receptor by soluble B7-H6 or by tumor cells expres -sing this ligand. The impairment of NK cell functionsdescribed herein could represent a novel mechanism bywhich the tumor microenvironment may contribute to theescape from immune surveillance.

CHARACTERIZATION OF SIGNALING PATHWAYSINVOLVED IN THE CROSSTALK BETWEEN MDSCsAND ACTIVATED T CELLSPinton L.1, Solito S.2, Masetto E.3, Berizzi A.4, PozzuoliA.5, Zanovello P.6, Bronte V.7, Mandruzzato S.81Lab. of Tumor Immunology, Dept. of Surgery, Oncology andGastroenterology, Oncology and Immunology Section, Univ. ofPadova, Italy; 2Lab. of Tumor Immunology, Dept. of Surgery,Oncology and Gastroenterology, Oncology and ImmunologySection, Univ. of Padova, Italy; 3Lab. of Tumor Immunology,Dept. of Surgery, Oncology and Gastroenterology, Oncologyand Immunology Section, Univ. of Padova, Italy; 4OrthopedicClinic, Dept. of Surgery, Oncology and Gastroenterology, Univ.of Padova, Italy; 5Lab. of bio-orthopedics, Orthopedic Clinic,Dept. of Surgery, Oncology and Gastroenterology, Univ. ofPadova; 6Dept. of Surgery, Oncology and Gastroenterology,Oncology and Immunology Section, Univ. of Padova, Italy;Istituto Oncologico Veneto IOV-IRCCS, Padova, Italy;7Verona University Hospital and Dept. of Pathology,Immunology Section, Univ. of Verona, Italy; 8Dept. ofSurgery, Oncology and Gastroenterology, Oncology andImmunology Section, Univ. of Padova, Italy; IstitutoOncologico Veneto IOV-IRCCS, Padova, [email protected]

Tumor cells are able to orchestrate several tolerogenic

responses in order to evade the control of the immunesystem. One of these mechanisms involves the expan-sion of Myeloid-Derived Suppressor Cells (MDSCs), apopulation of immature myeloid cells that have beenshown to accumulate in the blood, lymph nodes, bonemarrow and tumor sites in cancer patients and to inhibitboth innate and adaptive immunity. We demonstratedthat MDSCs can be derived in vitro from fresh bone mar-row (BM) cells (BM-MDSCs) and that only one immaturesubset (i-BM-MDSCs), resembling promyelocytes, isresponsible for the whole immunosuppression. Weobserved that activated T cells are able to inducechanges in MDSC phenotype and to sustain theirprolifera tion, thus suggesting the existence of a cross-talkbetween T cells and MDSCs. We thus decided to dissectthe molecular mechanisms involved in MDSC activity andwe showed that the production of the immunosuppres-sive cytokine IL-10 is significantly enhanced whenMDSCs are cultured in the presence of activated lympho-cytes and that T cells are responsible of IL-10 secretion.Since IL-10 can modulate the activation of STAT3, a tran-scription factor involved in immunosuppression, we eval-uated the phosphorylation of STAT3 on iBM-MDSCs andwe observed that it is up-regulated after the co-culturewith activated T cells. Considering that both IL-10 andSTAT3 can increase B7-H1 in myeloid cells, we investi-gated the expression of this molecule on i-BM-MDSCsand found that it is significantly increased only if thesecells are co-cultured with activated T cells. Our resultsthus suggest that a loop between IL-10, STAT3 and B7-H1 could be involved in the immunosuppressive programexerted by MDSCs. These data are supported by the up-regulation of PD-1 on the surface of T cells in contact withiBM-MDSCs. In order to validate these results we arepresently testing the existence of this loop also in MDSCspresent in the blood and biopsies of cancer patients.


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Methodology and Technology

LLLT. The objective of this study was the investigation oflow-level laser therapy potentiality on cell-based bonetherapy. Firstly, the LLLT was applied to a humanosteoblast-like cells (SAOS-2) in the absence/presenceof osteogenic factors. The obtained results showed thatdaily laser stimulation (doses of 1-3 J/cm2) enhances theproliferation potential of Saos-2 cells without changingtheir telomerase pattern or morphological characteristics.Enhanced secretion of proteins specific for differentiationtoward bone as well as calcium deposition and alkalinephosphatase activity were also observed in irradiatedcells cultured in a medium not supplemented withosteogenic factors. All these data represent an argumentsupporting the power of low-level laser treatment on boneregeneration. Therefore, the following step was the appli-cation of laser on hBM-MSCs. Our preliminary resultsshowed that the laser application influences the stemcells proliferation potential by modulation cell-cycle geneexpression and production of IGF-1, which is reported toregulate stem cell proliferation and differentiation.Currently, we are performing a comparative study tounderstand further the effects of laser irradiation at differ-ent energy doses on the human mesenchymal stem cellsdifferentiation towards osteogenic phenotype and as wellas the biochemical mechanism underlying these effects.Based on these findings we suppose a possibility using ofLLLT as a “photoceutical” for in vitro stem cells precondi-tioning prior to transplantation in order to enhance theirhelpful application on bone tissue regeneration.

IMPROVED FLOW CYTOMETRY ANALYSIS OF ANTI-BODY BIOCONJUGATED NANOPARTICLES (NPs) BYPRIMARY AMINES QUANTIFICATION ON NPs SURFACECalabrese V.1, Barattini C.1, Menna M.1, Juris R.1, VolpeA.1, Ventola A.1 1AcZon Srl, Via Lavino 265-D, 40050 Monte San Pietro,Bologna, Italia [email protected]

Antibody conjugated fluorescent silica nanoparticles areemerging as a promising tool in immunofluorescenceassays, thanks to the improved features compared tomolecular fluorophore-based probes, such as better pho-tostability and enhanced emission.As might be expected, a crucial point is the site-specificbioconjugation between the nanoparticle (NP) and theantibody (Ab). In order to optimize this process, a com-plete control and characterization of the NP surface hasproven to be essential. Since our silica NPs are synthe-sized with an outer layer of PEG to improve stability inwater, by adding precise amounts of an amino-PEG mod-ified reactant, it is possible to fine-tune the degree func-tionalization of the NPs shell. The quantification of theamino groups is performed by means of coupling withFITC and then analyzing the resulting increase inabsorbance at 495 nm: collected data shows a linearbehavior within the tested range.Thanks to the fine tuning of primary amines on the NPssurface and its relative quantification, it is now possible toevaluate and conveniently modulate the molar ratiobetween the linker on the Ab and the amines on the NP,in order to find the optimal condition to conjugate biomol-ecules on activated NPs. The resulting bioconjugateshows that this new characterization has lead to animprovement in the NPs profile in flow cytometry: fluores-cence is brighter and the discrimination is better withrespect to either a classic antibody conjugate or our pre-vious method for NPs bioconjugation.The NPs-Ab conjugate purification process is left

ACTIVATION OF MULTIPLE PATHWAYS BY A NA+/H+ANTIPORTER INHIBITOR IN COLON CANCER CELLS:A FLOW CYTOMETRY ANALYTICAL APPROACHAredia F.1,2, Scovassi A.I.11Istituto di Genetica Molecolare CNR, Pavia, Italy;2Dipartimento di Biologia e Biotecnologie “L. Spallanzani”Universita di Pavia, [email protected]

Drug resistance of cancer cells is still a matter of debate,having been mainly attributed to altered apoptosis and/orautophagy deregulation. Major attention is actually devot-ed to the elucidation of the impact of tumor microenviron-ment on the efficacy of drug treatment, focusing onhypoxia, oxidative stress, pH changes etc. Thus, the useof specific inhibitors of pH modulators could be a tool topotentiate drug toxicity and/or to reverse resistance tochemotherapeutic drugs, possibly via apoptosis and/orautophagy activation. Among the different strategies, theinhibition of Na+/H+ exchangers by the amiloride deriva-tive HMA (5-(N,N-hexamethylene amiloride), a drug thatinduces intracellular acidification, has been applied tounderstand how low pH conditions may influence cancercell growth. We have analyzed HMA effects on differenthuman cancer cell lines, evaluating the impact of HMA oncell survival, DNA damage, cell cycle distribution and acti-vation of different cell death paradigms, i.e. apoptosis,parthanatos and autophagy. We demonstrated that HMA:(i) exerts a cytotoxic effect on colon carcinoma cells; (ii)induces ROS production and accumulation, leading toDNA damage; (iii) is unable to trigger effective caspase-dependent apoptosis; (iv) activates two different forms ofcaspase-independent apoptosis; (v) activates autophagyto counteract cellular stress. Our survey was carried outmainly by flow cytometry by means of different probes:Annexin-PI, tetramethylrhodamine, dichlorofluorescein,etc., thus supporting the use of this technique to beapplied in cancer biology research.

IN VITRO ASSESSMENT OF LOW-LEVEL LASERIRRADIATION EFFECTS ON CELL PROLIFERATIONAND OSTEOGENIC DIFFERENTIATIONBloise N.1,2, Ceccarelli G.2,3, Minzioni P.4, Mantelli M.5,Mazzini G.6, Imbriani M.3,7, Visai L. 1,2,7 1Dep. of Molecular Medicine, 2Center for TissueEngineering (C.I.T.) University of Pavia, 3Dep.of PublicHealth, Experimental Medicine and Forensic University ofPavia, 4Dep. of Industrial and Information EngineeringUniversity of Pavia, 5Pediatric Oncohematology Laboratory,Policlinico San Matteo Foundation, IRCCS, Pavia, Italy6IGM-CNR and Dep. of Animal Biology, University of Pavia,Italy; 7Department of Occupational Medicine, Ergonomyand Disability, University of Pavia and Salvatore MaugeriFoundation, IRCCS, Pavia, [email protected]

It has reported that low-level laser therapy (LLLT) pro-motes bone regeneration and influences the behaviour ofmany cell types. However, the molecular mechanismunderlying this effect is not fully clarified. Human bonemarrow-derived mesenchymal stem cells (hBM-MSCs)have shown to be an appealing source for bone tissueengineering and little is known about their response to



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unchanged: size exclusion chromatography, followed byaffinity chromatography. The combination of this two con-secutive purifications ensures a higher grade of purityand the separation of the bioconjugated species from thesingle unbound components.

AN INNOVATIVE APPROACH TO IDENTIFY CTC CELLS:CULTURE OF PERIPHERAL BLOOD FROM PATIENTSON A 3D SILICON MICROCHIPDanova M.1, Torchio M.1, Aredia F.2,3, Carpignano F.4,Panini N.5, Erba E.5, Surdo S.6, Barillaro G., ScovassiAI.2,3, Mazzini G.2,3 and Merlo S.41Medicina Interna e Oncologia Medica, Azienda Ospedaliera diPavia; 2Istituto di Genetica Molecolare IGM-CNR;3Dipartimento di Biologia e Biotecnologie ‘‘L. Spallanzani’’Università di Pavia; 4Dipartimento di Ingegneria Industriale edell’Informazione, Università di Pavia; 5IRCCS-Istituto diRicerche Farmacologiche “Mario Negri”,Unità di Citometria,Milano; 6Dipartimento di Ingegneria dell’Informazione,Università di Pisa, - [email protected]

Cancer cells have the potential to grow indefinitely and formtumours that are then capable of producing metastasisthrough a series of steps in which cancer cells migrate orflow through different microenvironments (ME). This featurepartially depends on the physical interactions and mechani-cal forces between cancer cells and the ME, regulating largeelastic deformations. We investigated the cell membraneplasticity as an indicator of tumor cell mobility and/or migra-tion, that are two features strongly related to the cellmetastatic potential (MP). We have here exploitedmicrochips, incorporating silicon micromachined structures(SMS), as 3D microincubators. Our SMS consist in periodicarrays of silicon walls, with thickness of a few µm, separat-ed by empty gaps (width of 5 µm), characterized by high ver-tical aspect-ratio. These SMS are able to host either tumorcell lines with different level of MP or mononuclear cells(MnC) from peripheral blood (PB) of cancer patients.Experiments were performed using 4 different human can-cer cell lines, with high (MDA-MB-231, RPMI-7951) or lowMP (MCF-7, CAPAN-1), and MnC separated from peripher-al blood of 4 patients (affected by colon cancers with distantmetastases) as well as of 1 healthy donor (used as a con-trol). After incubation with cells (24-120 hrs), SMS deviceswere washed in PBS, fixed overnight in cold 70% ethanoland stained with FITC/PI. Fluorescence microscopy of SMSpopulated by cells clearly showed that: a) low MP cells main-ly grow on the SMS surface exhibiting nuclei with a typicalround shape); b) high MP cells colonize the narrow emptyspace between silicon walls; stretched nuclei aligned alongthe wall direction clearly prove that they are deeply insidethe extremely narrow gaps of the SMS. These results vali-date our procedure, which is suitable for discriminating cellswith low MP from cells with high MP, thus more aggressive.Then, we applied the assay to identify CTC cells within theMnC population. We found that whereas normal (small) lym-phocytes can sediment inside the silicon gaps, CTCs andother circulating cells are able to actively enter the SMS ashigh MP cells. The less stiff (i.e.. more “plastic”) cytoskele-ton/membrane of CTC cells (potentially metastatic) corre-sponds to their bio-mechanical ability to express their “clini-cal aggressiveness” exhibited also on SMS. Further experi-ments are requested to minimize the interference of lympho-cytes by means of long term (120h) and/or agitated cultureperformed with an orbital mini-shaker.Work partially supported by Fondazione Cariplo, grantno. 2011-0308.

INFLUENCE OF LOW FREQUENCY ELECTROMAGNETICFIELDS ON THE CYTOTOXIC EFFECT OF TEMO-ZOLOMIDEFassina L.1, Capelli E.2, Pasi F.3,4, Lupo G.2, MazziniG.5, Corbella F.3, Nano R.41Dept. of Electrical, Computer and Biomedical Engineering,University of Pavia; 2Lab. of Immunology and Genetic Analysis,Dept. of Earth and Environmental Sciences, University of Pavia;3Dept. of Radiation Oncology, IRCCS Fondazione PoliclinicoSan Matteo, Pavia; 4Lab. of Neuro-Radio ExperimentalBiology, Dept. of Biology and Biotechnology, University of Pavia;5IGM-CNR Histochemistry and Cytometry Section, Pavia, [email protected]

Glioblastoma multiforme (GBM), also known as grade IVastrocytoma, is the most common and malignant glialtumor. Its treatment consists of surgical resection, radio-therapy, and adjuvant chemotherapy with temozolomide(TMZ), a drug able to cross the blood-brain barrier. In par-ticular, TMZ can yield benefits in circa 25% of patients,whereas radiotherapy is controversial for GBM.So, in the last years, other types of physical treatmentshave been investigated to design novel therapeuticapproaches: for example, growing evidences indicatedthat the physiological and pathological mechanisms with-in cells, tissues, and organs can be influenced by low fre-quency electromagnetic fields (EMFs).In particular, at a molecular level, EMF can modulate thestress response (e.g. via the ERK/MAPK cascade),increase the expression of stress-related proteins (e.g.the heat shock proteins HSP70, a family of chaperoneswith cytoprotective roles), and affect the redox status ofthe cells (e.g. rebalancing the redox status).In this work, in order to enhance a toxic effect on GBMcells, we studied the action of TMZ and EMF in two ways:i) TMZ applied for 1 h after EMF pretreatment of 1 h; ii)TMZ and EMF simultaneously applied for 1 h. In terms ofcell survival, clonogenic capacity, expression of HSP70and of its microRNAs, the second simultaneous treat-ment appeared more harmful to cancer cells. Based onthese preliminary observations, the simultaneous use ofEMF and TMZ could improve the adjuvant chemotherapy.

RADIOBIOLOGICAL EFFECTS INDUCED ON NEOPLAS-TIC CELL LINES BY BNCT, NEUTRONS AND GAMMARAYS TREATMENTS Ferrari C.1, Cansolino L.1, Clerici A.M.1, Rovelli C.1,4,Dionigi P.1,6, Mazzini G.2,4, Altieri S.3,5, Ballarini F.3,5,Bortolussi S.3,5, Postuma I.3,5, Protti N.3,5 1Dept. of Clinico-Surgical Sciences, Exp. Surg. Lab.; 2IGM-CNR. Pavia; 3Dept. Physics; 4Dept of Biol.&Biotech,University of Pavia; 5INFN Pavia; 6IRCCS S. MatteoHospital, Pavia, [email protected]

Boron Neutron Capture Therapy (BNCT) is a binary experi-mental radiotherapy able to selectively destroy cancer cellssparing normal tissues. It is based on the combination of twosequential steps, the selective 10B loading of the neoplasticcells followed by the irradiation with low energy neutrons.The highly ionizing particles, α-particles and 7Li ions, gener-ated by the neutron capture in 10-boron, lose their energy inthe range of a cell diameter and therefore, providing that ahigh boron concentration ratio between neoplastic and nor-mal tissues is achieved, they can deliver a lethal dose onlyto target cancer cells. This peculiarity makes BNCT an alter-native therapeutic option for disseminated or infiltrativetumors that cannot be treated with conventional therapies. In BNCT preclinical in vitro studies, the assessment of the



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cell survival curves as a function of the absorbed dosecan give important information about its feasibility anddosimetry. The clonogenic assay is the more acceptedtechnique in radiation biology to determine the radiationsensitivity of different cell lines. For adherent growinglines, the methodology provides that cells can be irradiat-ed either before or after preparation of a suspension ofsingle cells. Main purpose of this work is to investigate if the use of dif-ferent laboratory protocols for in vitro cell exposed to irra-diation can affect their radiosensitivity and has thereforeto be considered in the evaluation and comparison ofinter/intra-laboratories results.Cell survival and cell cycle perturbations were studied onthe rat coloncarcinoma DHDK12TRb and on the ratosteosarcoma UMR-106 cell lines, exposed to Cobalt-60γ-rays, neutrons and BNCT, following two modalities:adherent to culture flask and as cell suspension aftertrypsin detachment. Our findings suggest that the modal-itiy of cell exposure to irradiation influences the cellradiosensitivity. Cells irradiated in the adherent configura-tion are more sensitive, showing lower cell survival andenhanced G2M block than those irradiated as cell sus-pension. In case of adherent growing cell lines, it is there-fore mandatory, to standardize the protocols of irradiationexposure to optimize results and to be able to comparethe survival curves obtained in different laboratories.

DIFFERENT CYTOTOXIC EFFECTS OF CISPLATIN ANDTHE NEW PLATINUM COMPOUND [PT(O,O'-ACAC)(γ-ACAC)(DMS)] ON GLIOBLASTOMA CELL CULTURESGrimaldi M.1, Barni S.1, Mazzini G.2, Veneroni P.1, De PascaliS.A.3, Fanizzi F.P.3, Bernocchi G.1, Bottone M.G.1,21Department of Biology and Biotecnology “L.Spallanzani”, University of Pavia, Italy; 2Institute ofMolecular Genetics of CNR, Pavia, Italy; 3General andInorganic Chemistry Laboratory, Department of Biologicaland Environmental Sciences and Technologies (DiSTeBA),University of Lecce, [email protected]

Glioblastoma multiforme (GBM) is the most frequent pri-mary malignant brain tumor in adults. Despite current treat-ments, this tumor continues to be associated with a lowsurvival rate. Platinum- based drugs, as cisplatin (cisPt)are employed in the chemotherapy of several malignan-cies. CisPt exerts its cytotoxicity interacting with DNA,blocking transcription and translation and leading to celldeath. Since cisPt causes toxicity and drug-resistance, anew platinum compound, [Pt(O,O'-acac)(γ-acac)(DMS)](PtAcacDMS), has been synthesized to overcome theseside effects. PtAcacDMS shows a specific reactivity withsulphur ligands, such as enzymes involved in apoptosis.We investigated the cytotoxic effects induced by bothplatinum compounds on U251 human glioblastoma cellline by means of flow cytometry, electron microscopy andimmunocytochemical expression of apoptotic andautophagic markers. The dose of PtAacDMS was chosenconsidering the effects of different concentrations on thesame cell line. After a short period of exposure (48 h) toboth cisPt or PtAcacDMS, cells underwent apoptosis.Moreover, long-term effects of the drugs have also beenevaluated. Cells were submitted to 7 days in drug-freemedium (recovery), and seeded in normal medium for 5days to evaluate whether they underwent autophagy andwhether it may represent a mechanism of resistance.Interestingly, we found that the cells activated autophagy,but they showed a different cell fate: cisPt-treated cells,after the reseeding period, displayed a morphology very

similar to the control, indicating the protective role forautophagy, which may represent a survival mechanism.On the contrary, PtAcacDMS-treated cells showed apop-totic features together with the activation of the autophag-ic pathway, suggesting that the cells underwent celldeath. Thus, PtAcacDMS may represent an alternativestrategy in the treatment of cancer, given its higher effica-cy and ability to elude drug-resistance.

DETECTION MICROVESICLES AND EXOSOMES INALS AND AD: A WIDE VIEW BY FLOW CYTOMETRY La Salvia S.1, Sproviero D.2, Colombo F.3, PansarasaO.2, Gagliardi S.2, Porretti L.3, Cereda C.2 1Department of Brain and Behavioral Sciences, Universityof Pavia Pavia, Italy; 2Laboratory of ExperimentalNeurobiology, "C. Mondino" National Neurological Institute,Pavia, Italy; 3Clinical Chemistry and MicrobiologyLaboratory, Flow Cytometry and Experimental HepatologyService, Fondazione IRCCS Ca' Granda OspedaleMaggiore Policlinico, Milan, [email protected]

In recent years intercellular transfer of extracellular vesi-cles (EVs), commonly referred to as microvesicles andexosomes, has been considered a new way of cellularcommunication. Microvesicles (MVs) (100-1000 nm)derive from a release membrane process ("blebbing")while exosomes are membrane vesicles of 30 nm to 100nm released from the cell when multivesicular bodiesfuse with the plasma membrane. Microvesicles (MVs) areindicated as important mediators of intercellular commu-nication also in Neurodegenerative Diseases. They trans-fer genetic information via mRNA, non-coding RNA(miRNA, LncRNA), or transcription factors as a possiblespread-pathologic mechanism (Prion-like). Increasedrelease of MVs has been described to be associated tothe acute or active phase of several neurological disor-ders, while cell-to-cell communication exerted by exo-somes in the brain. The aim of this study is to identifymicrovesicles and exosomes from plasma ofAmyotrophic Lateral Sclerosis (ALS) and Alzheimer's dis-ease patients (AD) by flow cytometry. We startedanalysing MVs by using markers of different derivation(leukocyte (CD45), endothelial (CD31),platelet(CD61),erythrocyte (CD235a)) and the apoptotic marker,Annexin V. We analyzed the levels of MPs in plasma ofpatients with AD, ALS and healthy controls. The levels ofannexin-V+ MPs and the CD235a+ Annexin-V + MVsresulted enhanced, respectively of 2 and 3 fold in theplasma from AD patients compared with healthy controls.A higher concentration of Annexin-V+ CD61+ and a slightincrease of CD45+ MVs was detected in the plasma fromALS patients. Annexin-V-+ CD31+ MVs were decreasedin the plasma from both diseases patients. Our prelimi-nary data shows that MPs could have a relevant role inthe disease propagation of AD and ALS and could explainthe spread of the disease from cell to cell.

COMPARISON OF DIFFERENT PROTOCOLS FORHUMAN SPERM CRYOPRESERVATION: ULTRASTRUC-TURAL, MOLECULAR AND CYTOMETRIC ANALYSESOmes C.1, Riva F.2, Savio M.3, Mazzini G.4, Citterio C.2,Marchetti A.5, Sanarica R. C.1, Tinelli C.6, IcaroCornaglia A.2, Casasco M.2, Casasco A.2, Calligaro A.2,Nappi R. E.1,71Dept. Maternal Infant, Ctr. for Reproductive Medicine,Obstetrics and Gynecology Unit, Fondazione IRCCS Policl.S Matteo, Pavia; 2Dept. Public Health, Exp. and ForensicMedicine, Histology and Embryology Unit, UniPV; 3Dept.



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work we report the study of a novel library of syntheticbacterial promoters, regulated by 3OC6-HSL, and their invivo characterization via population-based and single-cellapproaches; finally, we report the use of pre-characteri -zed promoters to tune industrially-attractive metabolicpathways in engineered Escherichia coli.Specific constructs including reporter genes (e.g., GFP)were assembled to indirectly measure the activity of pro-moters. Population-based measures were carried out viamicroplate reader to quantify the average promoter activi -ty in a growing cell population in a parallelized fashion.Since a parameter of interest is cell-to-cell variability, sin-gle-cell analysis via flow-cytometry was crucial to assessthe quality of parts by selecting the unimodal ones withlowest variability. Such variability can be included in pre-dictive mathematical models, together with the promoterdose-response curves, to describe the outcome of inter-connected systems.The studied promoters always showed unimodal fluore scen -ce distribution with a constant CV (55%) and their gradedstrength, spanning a 100-fold activity range, can be useful totune novel metabolic pathways. Pre-characterized promot-ers were used to tune the expression levels of two enzymesinvolved in the fermentation pathway for the production ofbioethanol from lactose-rich dairy wastewater.This work shows the importance of biological compo-nents characterization and the support of single-cellanalysis to evaluate different parts to be used in the bot-tom-up design of synthetic biological systems.

TRAFFICKING OF GABA AND GLYCINE HETERO-TRANSPORTERS IN SPINAL CORD GLUTAMATER-GIC SYNAPSES: A MECHANISM FOR THE ABNOR-MAL GLUTAMATE RELEASE IN AMYOTROPHIC LAT-ERAL SCLEROSISUsai C.1, Milanese M.2, Bonifacino T.2, Fedele E.2,Rebosio C.2, Cattaneo L.2, Benfenati F.3, Bonanno G.21Inst. of Biophysics, Nat. Res. Council, Genoa, Italy, 2Dept.of Pharmacy, Pharmacol. and Toxicol. Unit and Center ofExcellence for Biomed. Res., Univ. of Genoa, Italy. 3Dept. ofNeurosci. and Brain Technol., Italian Inst. of Technol. Genoa,Italy and Dept. of Exp. Med., Univ. of Genoa, [email protected]

Protein trafficking is a crucial process in CNS plasticity.Membrane proteins can move from the cytosol to the plas-ma membrane and vice-versa, exploiting endo/exocytoticevents. It has been proposed that neurotransmitter trans-porters are displaced to and from the plasma membrane inrelation to the exocytotic release of the neurotransmitteritself and that this trafficking has a role in regulating neu-ronal signaling. The impact of synaptic vesicle endo-exocy-tosis on the trafficking of nerve terminal heterotransporterswas studied by monitoring expression and function of theGABA transporter-1 (GAT-1) and of type-1/2 glycine trans-porters (GlyT-1/2) at spinal cord glutamatergic synapses.Exocytosis was induced in wild-type mice, in amphiphy-isin-I knockout (Amph-I KO) mice, which show impairedendocytosis, or in SOD1G93A mice, model of human amy-otrophic lateral sclerosis, that shows an excessive Glu exo-cytosis. Exposure of spinal cord synaptosomes from WTmice to a 35 mM KCl increased the expression of GAT-1and GlyT-1/2 at glutamatergic synapses and enhanced theGABA and glycine-induced glutamate (Glu) release.Preventing depolarization-induced exocytosis normalizedthe excessive GAT-1 and GlyT-1/2 -induced Glu release inWT mice. Impaired endocytosis in Amph-I KO miceincreased GAT-1 expression, the GABA uptake and theGAT-1 -evoked release of Glu in spinal cord synaptosomes.

Molecular Medicine, Immunology and General PathologyUnit, UniPV; 4Dept. Biol. & Biotech., IGM.CNR, UniPV;5Scientific Direction, Fondazione IRCSS Policl. S Matteo,Pavia; 6Scientific Direction, Clinical Epidemiology andBiometric Unit, Fondazione IRCCS Policl. San Matteo,Pavia; 7Dept. Clinical, Surgical, Diagnostic and PediatricSciences, UniPV, Pavia, [email protected]

Sperm cryopreservation has been recognized as a keystrate gy for preservation of fertility in males. Nevertheless,current protocols of semen freezing are neither optimal norstandardized between different labs. In this study we com-pare some protocols of human spermatozoa cryopreserva-tion applying both traditional and improved analysis of spermquality, to define the critical steps of the process and identi-fy possible chance to improve the efficiency of the freezingtechnique. The parameters are based on previous studiesconducted at the PMA Centre of the Fondazione IRCCSPoliclinico San Matteo in Pavia. The studies considered fivedifferent freezing protocols, selecting the best two, in termsof reduction in sperm motility and viability: M1 (Method 1): 30min at + 4°C, 10 min on nitrogen vapors; M2: 2h at +4°C, 10min on nitrogen vapors. Cryopreservation of human sper-matozoa has been related to decreased motility associatedwith impaired velocity and with impaired viability of spermpre-freeze and post-thaw, further investigated with analyticaltests, using semen samples of five healthy and normosper-mic volunteers according to the ethical standards of theHospital. Samples were frozen by two methods, thawedafter 1 month and prepared for tests, to determine whetherthe cooling time to +4°C may affect the procedure. Non-rou-tine analysis were performed using Comet Assay (to assessthe degree of sperm DNA fragmentation), flow cytometry (tostudy the cell morphology by means of correlated scatterpatterns as well as viability/membrane integrity by a coupleof fluorescent probes such as PI/HO33342) and electronmicroscopy (to investigate ultrastructural cell details). Theresults confirmed the presence of a greater number of alter-ations in the frozen samples and no significant differencewas appreciated between the effectiveness values obtainedusing the two different methods. It can be concluded that thetwo different steps of cooling at 4 °C do not cause significantmorphological and functional alterations to the spermatozoa.

CONVERSION OF INDUSTRIAL BIO-WASTE INTOBIOFUELS THROUGH SYNTHETIC BIOLOGYSUPPORTED BY FLOW CYTOMETRYPasotti L.1,2, Zucca S.1,2, Casanova M.1,2, MassaiuI.1,2, Mazzini G.3,4, Micoli G.5, Calvio C.4, Cusella DeAngelis M.G.2 and Magni P.1,21Lab. di Bioinformatica e Biologia Sintetica, Dipt. di IngegneriaIndustriale e dell'Informazione, Univ. degli Studi di Pavia, viaFerrata 5, 27100 Pavia; 2Centre for Health Technologies, Univ.degli Studi di Pavia, via Ferrata 5, 27100 Pavia; 3Istituto diGenetica Molecolare, CNR, via Abbiategrasso 207, 27100Pavia; 4Dipt. di Biologia e Biotecnologie "Lazzaro Spallanzani",Univ. degli Studi di Pavia, via Ferrata 9, 27100 Pavia; 5Centrodi Ricerche Ambientali, IRCCS Fondazione Salvatore Maugeri,via Salvatore Maugeri 10, 27100 Pavia, [email protected]

Synthetic biology is an emerging area of Bioengineering,aimed to develop methodologies for the rational engineer-ing of living systems to design novel high-impact biologicalfunctions in disparate application fields. One of the maingoals of synthetic biology is to realize novel biological func-tions following a bottom-up design, typical of the engineer-ing disciplines, to avoid trial-and-error approaches. In this



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The excessive Glu exocytosis in SOD1G93A mice resultedin increased GAT-1 and GlyT-1/2 expression at glutamater-gic synaptic membranes paralleled by an enhanced GABAand glycine-induced Glu release. Thus, endo-exocytosisregulates the trafficking of GAT-1 and GlyT-1/2 heterotrans-porters at spinal cord glutamatergic nerve terminals. In con-clusion, it can be hypothesized that the excessive GAT-1and GlyT-1/2 -mediated Glu release, in the spinal cord ofSOD1G93A mice, is due to the heterotransporter over-expression at the nerve terminal membrane, promoted bythe excessive Glu exocytosis.

FORWARD SCATTER AS A SENSITIVE PARAMETERTO PERFORM ROUTINE ANALYSIS OF POTENTIALSENSITIZING COMPOUNDSVicini R.1,2, Mazzini G.3, Angelinetta C.2, Pintacuda V.1and Pastoris O.11Dept. of Biology and Biotechnology "Lazzaro Spallanzani",University of Pavia; 2BioBasic Europe s.r.l., via Panizzi 10,Milano: 3Institute of Molecular Genetics ,CNR Pavia, [email protected]

Background. Allergic contact dermatitis is a major part of theoverall safety assessment of new ingredients employed intopically applied cosmetics and drugs. Cosmetic legislation inEurope prohibits the performance of animal testing for bothfinished products and ingredients or combinations of ingredi-ents, thus creating the need for alternative tests. Many testshave been proposed and validated to determine the pro-sen-sitizing activity of a cosmetic product or a medical device invitro. The h-CLAT (human cell line activation test) is undergo-ing to be approved by OECD Guidelines for the Testing ofChemicals: this method evaluates the variation in expressionof two specific membrane antigens induced by sensitizingsubstances. Flow cytometry of specific antigens modulationseem to be the most validated methodology for routine analy-ses. Light scatter signals (FSC, SSC) could also play animportant analytical role being related to cell morphology. It isin fact well known that cells undergoing damages (ie apopto-sis) are characterized by evident morphological changes. Materials and methods. THP-1 cells were cultured in RPMI-1640 medium with 10% FBS, 0.05 mM 2-mercaptoethanoland 1% antibiotic mixture. Cells were seeded at 5x106

cells/ml in a 24 wells plate, cultured for 48 hours at 37°C andthen treated with scalar concentrations (300, 150, 75, 37.5and 18.75 µg/ml) of NiSO4. A set of cells (negative control)received no treatment and has been maintained in the sameculture conditions. After a 24 hours period of incubation withanti-human CD80 (B7-1) FITC-conjugated (dilution 1:20) orAnti-human CD86 (B7-2) PE-conjugated (dilution 1:20), cellswere collected and washed twice in PBS. Flow cytometricanalysis was performed with a Partec PAS II flow cytometerand data collected and elaborated by means of the Flow Maxsoftware. Parallel aliquots of cells were examined with anOlympus BX51 microscope to monitor the cell morphology.Results. We observed a significant change in the forwardscatter of NiSO4 treated cells. Such changes have a clearcorrelation with the concentration of the substance used forthe treatment. These results are supported by the observa-tion of cell morphology.Conclusions. NiSO4, a well-known sensitizing drug, is able toinduce morphological changes in THP-1 cells with a concen-tration-dependent effect, as demonstrated by the significant-ly variations in FSC. This effect is also supported by imagestaken at microscope. This suggests the possible future devel-opment of new, fast, easy and label-free methods based onthe measure of FSC for the analysis of sensitizing sub-stances.

THE ROLE OF IQGAP1 PROTEIN IN TRASTUZUMABRESISTANCE OF GASTRIC CANCERArienti C.,1 Zanoni M.,1 Pignatta S.,1 Zamagni A.,1Cortesi M.,1 Carloni S.,1 Tebaldi M.,1 Tedaldi G.,1Amadori D.2 and Tesei A.11Biosciences Laboratory, Istituto Scientifico Romagnoloper lo Studio e la Cura dei Tumori (IRST) IRCCS,Meldola, Italy; 2Department of Medical Oncology, IstitutoScientifico Romagnolo per lo Studio e la Cura dei Tumori(IRST) IRCCS, Meldola, [email protected]

Gastric cancer is the fourth cause of cancer-related mor-tality in Europe. The failure of standard chemotherapyand surgery has led to the use of targeted therapy. Thephase III ToGA trial showed a significant improvement inthe response rate, PFS and OS of patients with Her-2-positive advanced gastric cancer when trastuzumab wasadded to conventional chemotherapy. Some patients inthis subgroup develop resistance to trastuzumab, but themolecular mechanisms responsible for this resistanceare still not fully understood. Recently, the role of thescaffold protein IQGAP1 in the regulation of HER-2signa ling was highlighted. We studied trastuzumab-induced resistance mechanisms in gastric cancer celllines (3 parental gastric cancer cell lines and 3 clonesresistant to trastuzumab), focusing on the role of IQGAP1protein. We evaluated whether parental cells differedfrom resistant clones in terms of growth kinetics, HER-2/IQGAP1 signaling modulation, and mutational state ofIQGAP1 and ERBB family genes. Flow cytometry analy-sis was used to evaluate the effects of trastuzumab-treat-ment on both parental and resistant cell lines in terms ofcell cycle distribution and apoptosis. Immunophenotypicanalysis was also performed for HER-2 and HER-3 pro-teins. No differences in terms of growth profile were foundbetween parental and resistant cell lines. Only parentalcells responded to trastuzumab treatment, in particularNCI-N87 cell lines, which harbored the highest HER-2expression. A significant overexpression of IQGAP1 pro-tein was observed in all three resistant clones. Thesilencing of IQGAP1 in NCI-N87 confirmed the role of thisprotein in trastuzumab resistance. Sensitivity andresponse to trastuzumab was restored when IQGAP1expression was absent. Our study identified 3 differentmechanisms of resistance to trastuzumab, one asso -ciated with IQGAP1.

FIRST TIME HIGHLY STANDARDIZED CIRCULATINGENDOTHELIAL CELLS EVALUATION IN GEP-NETPATIENTSBocchini M.1-2, Fabbri F.1, Severi S.2, Paganelli G.2 U.O. Lab. di Bioscienze 1U.O. Medicina Radio -metabolica; 2IRST - Istituto Scientifico Romagnolo per loStudio e la Cura dei Tumori, Meldola, [email protected]

Circulating Endothelial cells (CECs) variations have beeninvestigated as markers of cancer progression and ther-apeutic efficacy in solid tumors. Neuroendocrine Tumors(NETs) are relatively rare and very heterogeneous. Inparticular, Gastro-Entero-Pancreatic NETs (GEP-NETs)are often diagnosed when they have already metasta-

Oncology



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sized. A panel of prognostic and/or predictive non-inva-sive biomarkers is still lacking. Currently, nothing isknown about CEC levels GEP-NETs. This study aimed todemonstrate the feasibility of CEC detection in peripher-al blood (PB) of GEP-NET patients providing preliminarydata on their potential role in this clinical settings. Six GEP-NET patient and six healthy donor PB sampleswere collected in EDTA tube and processed withLyotube™ (Becton Dickinson) according to manufacturerinstructions. CECs were evaluated by six-colour flowcytometry. About 20.0 * 106 events per sample wereacquired. A standardized gating strategy was utilized toenumerate CECs. Their phenotype was defined as 7-AAD-, Syto16+, CD45-, CD34bright+, CD146+ andCD309+/-.Median CEC concentration in healthy controls andpatients was 22,9 (Mean 18 ± SD 11,6) and 22,2 (Mean34 ± SD 35,2), respectively. Five patients showed alow/medium grade disease. One patient had a high-gradedisease and a CEC number more than 4-fold higher thanthe median CEC concentration in healthy subjects.The reported results showed for the very first time a new,non-invasive and highly standardized method to evaluateCECs in GEP-NET patients. CEC determination in thisclinical setting is feasible and CECs evaluation in healthycontrols revealed homogeneous and reproducibleresults. Based on these preliminary data, it is tempting tohypothesize that CECs could be a non-invasive marker ofdisease progression capable to discriminate low/mediumgrade disease from high grade GEP-NETs.

MICROALGAE EXTRACTS EXERT ANTIPROLIFERATIVEAND APOPTOTIC EFFECTS ON CANCER CELLLINES A549Bottone C.,1 Carfagna S.,1 Camerlingo R.,2 Miceli R.,2Salbitani G.,1 Pirozzi G.2 1Dipartimento di Biologia, Università di Napoli FedericoII, Napoli, Italy - 2Department of Experimental Oncology,Istituto Nazionale Tumori Fondazione "G. Pascale",Napoli, [email protected];

AbstractIn recent years, microalgae have aroused vivid interest inthe field of biomedical research. We reported the effects oftwo different algal extracts, from the green alga Chlorellasorokiniana and the red alga Galdieria sulphuraria (inautotrophy and heterotrophy conditions) on the adenocarci-nomic human alveolar basal epithelial cells (A549).Cytotoxicity assays on A549 cells have shown a markeddecrease in viability of cells treated with the microalgaGaldieria rather than with Chlorella; in particular extractsfrom heterotrophic cells had larger gains on the treated cellline. In addition, heterotrophic algae show an higher contentof phicocyanin, recognized by the scientific literature, as apigment having excellent pharmacological effects. More,flow cytometry investigation and cell cycle analysis haveshown that Galdieria induces an apoptosis on the cell lineA549. These results indicate that algal extracts fromGaldieria, but in particular from heterotrophic algae, can bean effective advantage on the cell line A549 and that theseunicellular organisms should be studied further for their ther-apeutic potential.

MicroRNAs IN SUPERNATANT OF A LUNG CANCERPRIMARY CELL LINE, ARE INVOLVED IN THEEPITHELIAL TO MESENCHYMAL TRANSITION ANDMETASTASISCamerlingo R.1, Miceli R.1, Sessa G.1, Ceccarelli L.S. 1,D’Agnano I.2, Nardelli M.2, Felsani A.2, Caputo E.3,Rocco G.4, Pirozzi G,11IRCCS-INT Fondazione “G. Pascale” S.C. BiologiaCellulare e Bioterapie S.S Cellule Tumorali e Staminali,Napoli; 2Istituto di Biologia Cellulare e NeurobiologiaCNR Monterotondo, Roma; 3Laboratorio di AnalisiMolecolare del Cancro Istituto di Genetica e BiofisicaI.G.B.-CNR , Napoli; 4IRCCS-INT Fondazione “G.Pascale” Chirurgia toracica, Napoli, [email protected]

The purpose of this study was to analyze the supernatantof a primary cell line of adenocarcinoma (LC 212)obtained in our laboratory.During logarithmic growth, cellssecreted granules detectable by light microscope. We collected the supernatant after 48 hours of, and wetested it on the A549 lung cell line of for 96 hours. The supernatant, put in contact with A549 cells lead tocell death, while if diluted 1:3 with standard medium, itpromoted cell proliferation and, in particular, the EMTprocess. We analyzed the expression of vimentin andCD90, mesenchymal markers and cytokeratin andCD326 epithelial markers using immunofluorescenceassay and flow cytometry. The data obtained demonstrated an up-regulation ofmesenchimal markers expression and a down regulationof epithelial markers expression.Moreover, mass spectrometry analyses, performed onsupernatant, showed the presence of a molecule with amolecular weight compatible with those of miRna.Therefore, we analyzed the supernatant by miRNOmeassay and we found 193 miRNAs with increased expres-sion respect to: hsa-miR-1274a, has-miR-1274b,hsa-miR-191, hsa-miR193b, has-miR-222, has-miR720, has-miR-320, has-miR-574-3p, has-miR-24,has-miR-200among them hsa-miR1274a, has-miR-191, has-miR222,has-miR-200c, has-miR-21, all involved in the EMT.For the first time, these miRNAs are observed in lungcancer.The development of targeted therapies to inhibition ofmiRNA overexpressed in lung cancer and involved in themechanisms of EMT could contribute to improving sur-vival of patients with lung cancer and in particular ofthose with metastatic disease.

FLOW CYTOMETRIC ANALYSIS OF PROLIFERATIONAND INTRACELLULAR OXIDATIVE STATE AS AMETHOD TO MONITOR CHEMORESISTANCE STATUSOF CANCER CELLSColla R., De Ciucis C., Furfaro A.L., Fenoglio D.1, IzzottiA.2, Marinari U.M., Pronzato M.A., Domenicotti C.,Marengo B*.Dipartimento di Medicina Sperimentale, Università diGenova. 1Centro di Eccellenza per la Ricerca Biomedica,Dipartimento di Medicina Interna, Università di Genova;2Dipartimento di Scienze della Salute, Università diGenova - [email protected]

The balance between oxidant-induced cell death and pro-liferation plays a crucial role in determining response tochemotherapy. In order to investigate the molecularmechanisms governing these cell responses, we haveselected a human neuroblastoma cell line resistant to


etoposide (Etopo-R), a standard clinically-usedchemotherapeutic drug. In addition, considering thatetoposide-induced resistance is mediated by redox-modu -lated pathways, glutathione (GSH) and reactive oxygenspecies (ROS) levels were monitored in these cells.Firstly, we observed that Etopo-R cells showed a lowerrate of cell death in comparison with parental cells, whenexposed to high doses of etoposide or doxorubicin, there-by developing a multidrug-resistant phenotype (MDR).Furthermore, as demonstrated by flow cytometry analysisof carboxyl-fluorescein-succinimidyl-ester-positive cells,etoposide treatment reduced the proliferation rate of bothparental and Etopo-R cells and led cells to stay longer inthe older generations respect to the untreated parentalcells. Moreover, cytofluorimetric analysis showed that,after treatment with highest doses of etoposide and dox-orubicin, Etopo-R cells had higher GSH levels and lowerROS production (evaluated as a number of dichlorofluo-rescein positive cells) than parental cells. Collectively, wesuggest that the flow cytometric analyses described canbe applied also in clinical studies for monitoring ROS andGSH in cancer patients in order to predict and control theresponse to anticancer therapy (Grants from CARIGEFoundation 2013, MIUR PRIN20125S38FA and GenoaUniversity).

TRABECTEDIN RESISTANCE: TWO SIDES OF THESAME COINColmegna B., Uboldi S., Panini N., Licandro S.A., BelloE., Frapolli R., D’Incalci M. and Erba E. Department of Oncology, IRCCS Istituto di RicercheFarmacologiche Mario Negri, via La Masa 19, 20156Milan - Italy [email protected]

Multidrug resistance is the major mechanism of chemother-apy failure in many cancer types. Different molecularmecha nisms have been implicated but the most common isthe over-expression of P-glycoprotein (P-gp) and the mul-tidrug resistance–associated proteins (MRP). In order toinvestigate the mechanism of trabectedin (T) resistance, a T-resistant ovarian carcinoma cell line (A2780/T) was develo -ped and characterized in vitro and in vivo. Resistant cell line was obtained by repeated treatmentwith the drug. The characterization was performed bydrug sensitivity assays, molecular studies, flow cytomet-ric analysis (DNA content, cell cycle perturbations, pumpsexpression) and cell sorting. In vivo experiments werealso performed.A2780/T cell line was 6 fold resistant to T as compared toparental cells and maintained the same growth features.Resistant cells did not show the characteristic G2/M blockinduced by T. A2780/T cells were found more sensitive toUV light and platinum (Pt) complexes and this sensitivitywas associated to NER deficiency consisting in the lack ofXPG protein expression. Cross-resistance to doxorubicin(DX) and paclitaxel (PTX) was revealed in T-resi sta nt cells:it was associated to a higher expression of P-gp protein(90% of cells) and could be reversed by using a P-gpinhibitor. A2780/T cells were sorted in P-gp negative andpositive: in vitro the absence of the pump completelyreverse the DX and PTX resistance but cells maintainedpartial resistance to T and the sensitivity to Pt complexes.In vivo studies on P-gp positive cells reproduced the dataobtained in vitro while P-gp negative cells displayedresistance to DX.The finding that resistance to T is associated to the loss ofNER function, with consequent increased sensitivity to Ptcomplexes, and to the acquisition of cross-resistance to DX

and PTX could be useful in the evaluation of the best treat-ment option in patients who have become resistant to T.

MOLECULAR PHENOTYPE OF MELANOMA CELLLINES RESISTANT TO B-RAF INHIBITIONCordaro F.G.1, De Presbiteris A.L.1, Camerlingo R.2,Mozzilo N.2, Pirozzi G.2, Patriarca E.J.1, Ascierto P. A.2and Caputo E.11Institute of Genetics and Biophysics –I.G.B., A. Buzzati-Traverso–, CNR, Via Pietro Castellino, 111, I-80131Naples, Italy; 2Istituto Nazionale Tumori Fondazione G.Pascale, Via M. Semmola, 80131 Naples, [email protected]

It has been reported that B-RAFi improved survival inpatients with melanoma. However, the duration of clinicalbenefits is limited by the development of drug resistance.Therefore, understanding the molecular phenotype of can-cer cells resistant to B-RAF inhibition could contribute todesign efficacious treatments by their targeting. We selectedthree melanoma cell lines for their resistance to B-RAFinhibitor molecule (A375_R, 624.38_R, 397_R) and weexamined their molecular phenotype compared to theirparental ones (A375_S, 624.38_S, 397_S), by FACS analy-sis, immunofluorescence and scratch test. Resistantmelanoma cells exhibited a shape change, as well as agreater cell motility and a perinuclear localization of vimentincompared to the sensitive cells. Furthermore, the FACSanalysis expression of epithelial, mesenchymal and stemcell surface markers (CD326, CD133, CD90 and CD324)revealed a down-regulated expression of CD90 as well asCD324 surface markers in all the B-RAFi resistant cells,compared to the sensitive ones, while both B-RAFi resistantand sensitive cells did not express the stem cell marker(CD133) and the CD326. The reduction of E-cadherin aswell as the perinuclear localization of vimentin together withthe cell shape change in B-RAFi resistant melanoma cells,suggested a mesenchymal cancer cell phenotype of the B-RAFi resistant cells. We also investigated the expression ofOCT4, one among the regulatory core transcription factorsinvolved in the maintenance of stemness in mesenchymalstem cells. It was mainly detected at cytoplasmic level inmelanoma cells sensitive to B-RAF inhibition, while OCT4was trans-located in the nucleus of all three B-RAFi resistantmelanoma cells, suggesting its activation in the resistantcancer cells, supporting the mesenchymal stem-like pheno-type of these cells. Further molecular characterization ofresistant cells will help to identify novel target for develop-ment of novel therapeutic strategies.

AUTOPHAGY, AS SURVIVAL MACHINERY, IS RELATEDTO BORTEZOMIB-RESISTANCE IN BONE MARROWFIBROBLASTS FROM MULTIPLE MYELOMA PATIENTSDesantis V.1, Di Marzo L.1, Vergara D.2, Maffia M.2,Vacca A.1, Frassanito M.A.11Policlinico of Bari, Dept. of Biomedical Sciences andHuman Oncology (DIMO), Section of Internal Medicine andClinical Oncology, Univ. of Bari, Italy; 2Dept.of Biological andEnvironmental Sciences and Technologies, Univ. of Salento,Lecce, [email protected]

Background: Cancer-associated fibroblasts (CAFs) are acell type within the stroma of many tumors, including MultipleMyeloma (MM), which entail with progression and poorprognosis. Since drug resistance of MM cells depends ontumor microenvironment, we analyze the role of CAFs inbortezomib resistance.Methods: Proteomic, phospho-proteomic, Western blot,


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immunofluorescence and flow cytometry studies are per-formed to demonstrate the effect of CAFs on bortezomib-induced apoptosis of MM cells. Results: CAFs from bortezomib-resistant patients are invitro insensitive to bortezomib treatment and protect MMcells from bortezomib-induced apoptosis. The effect isrelated to the ability of bortezomib to induce in borte-zomib-resistant CAFs an increased level of prosurvivalcytokines and growth factors. Proteomic studies suggestthat resistance of CAFs to bortezomib could be related toa cellular stress condition. Indeed, bortezomib treatmentof bortezomib-resistant CAFs induces ROS productionand activates autophagy machinery, demonstrated by anincrease of light chain (LC)3II, a decrease of p62 and p-mTor. In the syngeneic 5T33MM mouse model, borte-zomib treatment prompts an increase of LC3II in CAFs.Using small-interfering RNA to knock-down ATG7 expres-sion and 3-methyladenine autophagy inhibitor reduceCAFs autophagy and restore the bortezomib-susceptibil-ity of MM cells cocultured with bortezomib-resistantCAFs. Finally, TGFβ, acting in autocrine way, activatesprosurvival autophagy and induces CAFs bortezomib-resistance. Blockade of TGFβ pathway by LY2109761induces apoptosis of bortezomib-resistant CAFs restoringthe bortezomib susceptibility of MM cells.Conclusions: CAFs from bortezomib-resistant patients areresistant to bortezomib and protect MM cells from borte-zomib-induced apoptosis. The in vitro and in vivo resistanceof bortezomib-resistant CAFs to drug is associated to theactivation of prosurvival autophagy by TGFβ, which may bethe new target into therapeutic approach of MM.

CINNAMOMUM ZEYLANICUM ANTITUMOR ACTIVITYIN MELANOMA CELLSIannetti I., Giovannini D., Cappelli G., Mariani F. andD’Agnano I.Institute of Cell Biology and Neurobiology – CNR,Monterotondo, [email protected]

Since survival of melanoma patients remain poor (5-yearsurvival below 25%), there is continued need for newtherapeutic approaches. Many studies have shown thatplants are a potential source of natural molecules thancan be used as agent in cancer treatment. Cinnamon is asmall and evergreen plant belonging to Lauraceae fami-ly, native to Asia, whose medical properties are still underinvestigation. In particular, Cinnamomum zeylanicumbesides anti-microbial, anti-mycotic and anti-inflammato-ry properties, have been demonstrated to have an antitu-mor effect. In this study we investigated the anti-melanoma cell activity of Cinnamomum zeylanicumessential oil on an in vitro established melanoma cell line.The results revealed that this agent has anantiproliferative effect as evaluated by cell growth inhibi-tion and cell cycle analysis. Reactive oxygen species(ROS) production was augmented by the treatment withCinnamomum zeylani cum essential oil indicating its pro-oxidant effect in melanoma. In addition, Cinnamomumzeylanicum essential oil was able to improve the effect ofa conventional antitumor drug such as Tamoxifen used atlow doses. Our results strongly support the potential useof this plant in combination cancer therapy.

A NOVEL CELL BLOCK IN CYTOPATHOLOGY FORDIAGNOSIS IN THE TARGET THERAPY ERAMiceli R.1, Scognamiglio G.2, Camerlingo R.1, Marra L.2,Sessa G.1, Ceccarelli L.S.1, Franco R.2 and Pirozzi G.11IRCCS-INT Fondazione “G. Pascale” S.C. Biologia

Cellulare e Bioterapie S.S. Cellule Staminali Tumorali;2IRCCS-INT Fondazione “G. Pascale” S.C. AnatomiaPatologica e [email protected]

The CELL BLOCK (CB) has established itself as a diag-nostic tool since 1947. The cell blocks are micro biopsiesembedded in paraffin. The preparation of cell block hasthe aim of trasforming a cytologic study in histologicalexamination, extending the diagnostic value of cytologyspecimens. The vantages of this technique compared to traditionalcytological smear are: - Reduction of cell overlap and interference due to the

presence of blood and cellular debris; - Keeping the cellular architecture; - Staining with hematoxylin and eosin comparable to that

performed on surgical specimens; - Conservation in time of the material to dispose of it even

in later times; - Possibility to perform additional studies such as IHC and

molecular biology.The increased availability of immunohistochemical investi-gations in recent years has increased the need to proceedwith preparations of CB in the laboratory diagnosisbecause many of today's techniques are evaluated usingthe fine needle aspiration (FNA), whereas in the past wereonly available for the diagnosis biopsies or resections. The aim of our work was to develop a cell block inexpen-sive and easily reproducible, using as a sample asciticfluid of patients with ovarian carcer (stage III-IV).After collecting the ascitic fluid from patients with ovariancarcer, we performed several tests for the choice of theconcentration of agarose to use. The cells were centrifuged and fixed in 10% formalin. Theagarose was dissolved at different concentrations (1%,1.25%, 1.5%) in TAE, TBE, TAE/TBE. After including thecells in agarose blocks obtained were cut and placed in abox for later inclusion in paraffin. The data obtained from the tests of inclusion of cells in 1.25%agarose in TBE/TAE and then dissolved in paraffin show bet-ter results for texture, intensity and number of cells.At the light of the results obtained this technique can beused pair IHC with the advantage of being easily repro-ducible and inexpensive.

TUMOR INITIATING CELLS IN NEUROBLASTOMAARE CHARACTERIZED BY LOW EXPRESSIONLEVELS OF LAMIN A/CMusa C., Nardella M., Guglielmi L., Iannetti I., MarescaG., Amendola D., Porru M., Carico E., Sessa G.,Camerlingo R., Dominici C., Megiorni F., Milan M., BearziC., Rizzi R., Pirozzi G., Leonetti C., Bucci B., Mercanti D.,Felsani A. and D’Agnano I.Institute of Cell Biology and Neurobiology-CNR,Monterotondo (RM), [email protected]

Neuroblastoma is generated and maintained by a smallcell population of undifferentiated cells (1-2% of the total),which are identified as neuroblastoma-tumor initiatingcells and are commonly defined as cancer stem cellsplaying an important role in tumor aggressiveness anddrug resistance. This cell population shares fundamentalbiological properties with normal stem cells including self-renewal, quiescence and differentiation associated withcell-cycle exit, and is considered responsible for therapyfailure in many cancer types. In a previous paper, wehave shown that knock-down of Lamin A/C expression in



37

neuroblastoma cells inhibits cell differentiation andresults in a more aggressive and drug-resistant tumorphenotype. In addition, Lamin A/C is often lost inadvanced tumors during tumor progression. We thenhypothesized that the lack of Lamin A/C is necessary todrive cells toward a stem-like phenotype, thus influencingthe development of tumor-initiating cells in neuroblas-toma. Our results demonstrates that knockdown of LaminA/C triggers the development of a tumor-initiating cellpopulation with self-renewing features in human neurob-lastoma cells. We also demonstrates that the develop-ment of TICs is due to an increased expression of MYCNgene and that in neuroblastoma exists an inverse rela-tionship between LMNA and MYCN expression.

ANTITUMOR ACTIVITY OF TRABECTEDIN IN MV-4-11CELL LINE, A CMML CELLULAR MODEL Romano M.1, Panini N.1, D’Incalci M.1 and Erba E.11IRCCS-Istituto di Ricerche Farmacologiche MarioNegri, Department of Oncology, Flow Cytometry Unit,Milano, Italy [email protected]

Chronic myelomonocytic leukemia (CMML) and Juvenilemyelomonocytic leukemia (JMML) are myelodysplastic -/myeloproliferative neoplasms of childhood and elderlypatients, respectively. Current treatment options for thesepathologies are unsatisfactory, so it is important to identi-fy new effective treatment strategies. Trabectedin wasrecently found to cause selective depletion of monocytesin blood and spleens of tumor-bearing mice. The aim ofthis study was to study the mechanism of action of tra-bectedin using a cellular model that expressed markersof monocytic differentiation, the biphenotypic B-myelomonocytic leukemia cell line MV-4-11. BrdU/DNA flow cytometric analysis showed that in MV-4-11cells trabectedin induced a delay in crossing all the cellcycle phases, particular in the G1 phase, compared withthe G2M block showed in many other cancer cell lines.Trabectedin was also able to induce apoptosis in MV-4-11 cells; d-TdT/DNA analysis showed that the majority ofapoptotic cells were in the G1/S early phases.; it wasfound that the apoptosis seemed to be mediated by theaxis activated TRAIL receptors-cleaved caspase-8,evalua ted by flow cytometric assay. We explored if tra-bectedin was able to induce DSBs on this cell line byimmunocytochemical detection of phosphorylated H2AX,that provides a marker of DSBs. The biparametric flowcytometric detection of γ-H2AX, combined with stainingof cellular DNA to reveal the cell cycle phase, haveshown that, as previously described for other cellularsystem s, trabectedin induced γ-H2AX in a relatively smallfraction of cells. The induction of this DNA damage mark-er started at 2 up to 8 hours after treatment and themajority of the cells were in the S-early phase, while 24hours after treatment most of the apoptotic cells were inthe G1/S phases. The data obtained in this cellular modelare essential to understanding the mechanism of actionof trabectedin in cells derived from patients affected byCMML or JMML.

EPITHELIAL OVARIAN CANCER: COMPARISON ANDPHENOTYPIC CHARACTERIZATION OF OVARY,OMENTUM AND ASCITES SAMPLES BY FLOWCYTOMETRIC ANALYSISSessa G.1, Miceli R.1, Camerlingo R.1, Ceccarelli L.S.1,Carriero M.V.2, Cacciapuoti C.3, Arra C.2, Scaffa C.4,Greggi S.4, Pirozzi G.1

1IRCCS-INT Fondazione “G. Pascale” S.C. BiologiaCellulare e Bioterapie S.S. Cellule Staminali Tumorali;2IRCCS-INT Fondazione “G. Pascale” OncologiaSperimentale; 3IRCCS-INT Fondazione “G. Pascale”Medicina Trasfusionale; 4IRCCS-INT Fondazione “G.Pascale” Chirurgia Oncologica [email protected]

The Epithelial Ovarian Cancer (EOCs) remains the mostlethal gynecologic malignancy. These cancers have arapid growth, metastasize early, and have a very aggres-sive disease course. Unlike most other cancers, EOCsrarely disseminates through the vasculature. However,the majority of patients present widely metastatic diseasewithin the peritoneal cavity. Emerging evidence suggest that the capacity of a tumor togrow and propagate is dependent on a small cellular subset,the Cancer Stem Cells, which can proliferate indefinitelythrough a deregulated cellular self-renewal capacity.The 35 ovarian biopsies (ovary, omentum and ascitessamples), from Gynecologic Oncology Division ofNational Cancer Institute of Naples, were collected fromJuly 2012 to June 2015.The samples were disaggregated and put in culture withdifferent media combinations, in a humidified incubator at37 °C under 5% CO2 atmosphere. The cell suspensionobtained was split in two: one part was frozen with FBSand DMSO and the other was used for flow cytometryanalysis. The markers analyzed were: CD326, CD133,CD90, CD44, CD24, CD10. Moreover a small portion ofthe biopsy was frozen for the biological bank. The cytometric analysis have showed for the ovarianbiopsies a mean expression of 14,2% for CD326, 1,8%for CD133, 3,6% for CD90, 26,1% for CD44, 11,5% forCD24 and 1,3% for CD105. Among all analyzed biopsies, one of the ovarian biopsiesof case number 35 has showed a marked expression oftumor stem cell marker CD133 for excellence of 27%.The expression of the other markers is 2.1% for CD44,58.6% for CD326, 46% for CD24. After putting in culturethis biobsy, we have also obtained the cell line and,because of its instability, we have maintained it in culturefor six steps. All ovarian biopsies show equal expression of markersand that therefore may be used as biopsies representa-tive of the pathology for the study of the markers associa -ted with it and in particular of CD133.



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Frassanito M.A.,35 Fraternali-Orcioni G.,20Fresegna A.M.,12Frezza D.,25Frosali S.,25Furfaro A.L.,34 Gabrielli S.,13,26 Gagliardi S.,31 Gaipa G.,17,21 Gambardella C.,15Gasol J.M.,14Gatti A.M.,15Ghio M.,24 Giorgi D.,8,13,14Giovannini D.,35 Giwercman A.,14Gobbo M.,23Graham S.P.,25 Grasso L.,20Gravetti A.,19Graziano D.,17,19,20,22Greggi S.,37Gregorj C.,20Grimaldi A.,5Grimaldi M.,31 Grosso V.,8,13,14Guarini A.,8Guglielmi L.,36 Hegarty M.,8Iaccarino S.,22Iannetti I.,35,36 Iavicoli S.,12Icaro Cornaglia A.,31 Imbriani M.,29Innocenti I.,18Iorio M.,18Iovane E.,22Izzotti A.,34 Jönsson B.A.G.,14Juris R.,29 Kalli F.,26,27 Kunkl A.,20Kuzmanovic L.,14La Rocca F.,19La Salvia S.,31 Lai S.,10Lanfranchi A.,23Lang F.,15Langdon T.,8Lanuti P.,20Lanza F.,9,18 Laurenti L.,18,19Laurenzana I.,19Lazzari C.,9 Lazzari M.C.,18Lefort T.,14Lenzo G.,26 Leone A.,20Leonetti C.,36 Leter G.,14Licandro S.A.,34 Lo Nigro L.,17Lo Pardo C.,17,19,20,22Lobina M.,10Locatelli F.,26Lolli S.,25Lombardi A. 5Longo N.,27 Longoni P.D.,21

Aiello A.,23 Alberti M.,19Almici C.,20Altieri S.,30Altomare L.,19Altucci L.,5Alzoubi K.,15Amadori D.,33Amalfitano S.,5,11,15,16Amendola D.,36 Angelinetta C.,33 Aracri B.,13Aredia F.,29,30 Arienti C.,33 Arra C.,37Ascierto P. A.,35 Avvisati G.,20Baffelli R.,23Baffone W.,12,13 Balestra C.,12,14Ballarini F.,30Barattini C.,29 Barbaresi M.,20Bardi M.A.,18Barillaro G.,30 Barni S.,31 Barone M.,22Basile F.,17,19,20,22Basso G.,18Battaglia A.,5,24 Bearzi C.,36 Beghin A.,23Bello E.,34 Benfenati F.,32 Berizzi A.,28Bernardi C.,26 Bernocchi G.,31 Bertaina A.,26 Bertoni R.,11Bianchini P.,15Biondi A.,17, 21Bisaga M.,8Bloise N.,29 Bocchini M.,33Bolda F.,23Bonaccorso P.,17Bonanno G.,32 Bonde J.P.,14Bonelli P.,25Bonifacino T.,32 Borino G.,22Bortolussi S.,30 Boselli D.,17Botta C.,10Bottone C., 34Bottone M.G.,31 Brambilla P.,9Bronte V.,28Buccella F.,26 Bucci B.,36 Budillon A.,20Bugarin C.,17Buldini B.,18Buracchi C.,17

Buriani A.,26Cabras A.,23Cacciapuoti C.,27,37 Caivano A.,19Calabrese V.,29 Callieri C.,11Calligaro A.,31 Calvio C.,32Camerlingo R.,34,35,36,37 Campana R.,12,13Campioni D.,18Canonica G.W.,23Canonico B.,9,13,17,26 Canonico E.,9Cansolino L.,30 Cantoni C.,28 Capelli E.,30 Cappelli G.,35 Caputo E.,34,35 Carfagna S., 34Carico E.,36 Caraglia M.,5Carloni S.,33 Carniti C.,21Carpignano F.,30Carriero M.V.,37Casanova M.,32 Casasco A.,31 Casasco M.,31 Casettari L.,12Casotti R.,11,12,14,16Cattaneo L.,32 Catzola V.,24Cavallo D.,12Cavazzini F.,18Cazzaniga G.,21Ceccarelli G.,29 Ceccarelli L.S.,34,36,37 Cecere S.,27 Ceoloni C.,14Cereda C.,31 Cesarini E.,13,26 Chiappori A.,23Cianci R.,25Ciandrini E.,12,13Ciervo A.,12Cioni M.,26 Citterio C.,31 Clerici A.M.,30 Colla R.,34 Colmegna B.,34 Colombo F.,31 Coltrini D.,28 Comini M.,23Contini P.,20,23 Coppola G.,18Corbella F.,30 Cordaro F.G.,35 Corno G.,11Corradini P.,21Cortesi M.,33 Criscuoli M.,19Cristofaro F.,6Crotti M.,9Cucca F.,10Cugia G.,24 Cuneo A.,18Curto M.,26,27 Cusella De Angelis MG.,32

D'Alterio C.,27 D'Arena G.,18D’Agnano I.,34,35,36D’Arena G.,19D’Auria F.,18D’Incalci M.,34,36Dabusti M.,18Danova M.,6,30 De Ciucis C.,34 De Domenico R.,27 De Forni D.,24De Luca L.,19De Palma R.,7 De Pascali S.A.,31 De Presbiteris A.L.,35 De Propris M.S.,8De Vita M.,24 Dei Giudici S.,25 Dei M.,10Del Vecchio L.,19,22Della Chiesa M.,26Della Cioppa P.,22Della Porta M.G.,6Deoni D.,26 Desantis V.,35 Di Cerbo M.,20Di Gennaro E.,20Di Martino G.,9Di Martino M.L.,20Di Martino M.T.,10Di Marzo L.,35 Di Nuzzi P.A.,21Di Sario G.,13Di Terlizzi S.,17Diaspro A.,15Dimaro S.,27Dionigi P.,30Domenicotti C.,34 Dominici C.,36 Doretto P.,20Eleuteri P.,14Erba E.,30,34,36 Fabbri F.,33 Fagioli L.,12Faimali M.,15Falco M.,26 Falda A.,20Falugi C.,15 Fanizzi F.P.,31 Farina A.,8,13,14 Farnesi M.,26 Fassina L.,30 Fattorossi A.,7,24 Fazio G.,17Fedele E.,32 Federici S.,13Felsani A.,7,34,36 Fenoglio D.,23,26,27,34 Ferrari C.,30 Ferrari L.,18Ferrera F.,26 Filaci G.,23,26,27 Fiorillo E.,10Foà R.,8Folli C.,23Fortinguerra S.,26 Franco R.,36 Franzoni G.,25 Frapolli R.,34

Authors’ index



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Lopez-Botet M.,26Lori F.,24 Luchetti F.,13 Lucretti S.,8,13,14,26Ludwicki J.K.,14Lugli E.,9Lupo G.,30 Maffia M.,35 Maglia O.,21Magni M.,21Magni P.,32 Mahadevan N.,27Maiello R.,12Malara N.. 20Mandruzzato S.,28Mangerini R.,20Manna C.,15Mansueto G.,18Mantelli M.,29Manti A.,9,12,13Mappa I.,24 Marcenaro E.,28 Marchetti A.,31Marchini S.,10 Marchisio M.,20Marcovecchio F.,15Marenda B.,26 Marengo B.,34Maresca G.,36 Mariani F.,35Marie D.,14Marinari U.M.,34 Marinelli L.,27 Marini C.,26 Marini M.,20Marino N.,21Marmo R.,25Marongiu M.,10Marotta C.,22Marra L.,36Masetto E.,28 Massaiu I.,32 Massari A.,18Matta J.,28Mattioli M.,22Mazzini G.,29,30,31,32,33Meazza R.,26Megiorni F.,36 Megna M.,20Melioli G.,25Menna M.,29 Mercanti D.,36 Merlo S.,30Miceli R.,34,36,37 Michielotto B.,18Micoli G.,32Milan M.,36 Milanese M.,32 Minzioni P.,29Miscia S.,20 Misso G., 5Molica S.,19Mollace V.,20Morelli G.,22Moretta A.,26,28Moretta L.,26Moretti S.,18Mozzilo N.,35 Muccio L.,26

Muggianu E.,20Mulas A.,10Musa C.,36 Musto P.,18,19Nano R.,30Napolitano M.,27 Nappi R.E.,31 Nardella M.,36 Nardelli M.,34 Nasi G.,26,27 Negrini S.,24 Neva A.,20Nicolussi P.,25Not F.,14Officioso A.,15Oggiano A.,25 Omes C.,31 Orlando A.,25Orrù V.,10Pacchierotti F.,14Paganelli G.,33 Pagliari D.,25Palma F.,26 Palmieri S.,22Palumbo D.,18Pandolfi F.,25Pani G.,6Panico L.,22Panini N.,30,34,36 Pansarasa O.,31 Papa S.,9,12,13,26 Parodi A.,23,26,27 Parolini S.,28 Pascucci B.,6Pashkoulov D.,13Pasi F.,30Pasotti L.,32 Passalacqua G.,23Pastoris O.,33 Patriarca E.J.,35 Patrizi O.,28 Patrone V.,13Paura G.,18Pedersen H.P.,14Pende D.,26Perdonà S.,27 Perrino C.,15Pesce S.,28 Piemonte I.,18Pietrantuono G.,18,19Pignata S.,27 Pignatta S.,33Pilo G.,25Pinna S.,20Pintacuda V.,33Pinton L.,28 Piras M.G.,10Pirozzi G.,34,35,36,37 Piscitelli S.,17,19,20,22Poddesu B.,24 Pollice A.,16Porretti L.,31 Porru M.,36 Porta F.,23Postuma I.,30Pozzuoli A.,28Presbiteris A.L.,35Probert I.,14Pronzato M.A.,34

Protti N.,30 Puppo F.. 24Quartesan R.,26 Raia M.,22Raimondi L.,10Ramoino P.,15Rampinelli F.,28Ravelli A.,9Ravetti J.L.,20Rea G.,6Rebosio C.,32 Reggiani S.,18Riccio A.M.,23Risitano A.M.,22Riva F.,31 Rizzi R.,36 Rizzo A.M.,6Rocco G.,34Rocco M.,22Rogkakou A.,23Romano M.,36 Romanò M.,17Rongioletti F.,26 Rosamilio R.,22Rosati P.,24Rossiello F.,10 Rota R.,10Rotta G.,20Rovelli C.,30Sala S.,21Salbitani G., 34Salerno C.,16Sanarica R.,31 Sanna S.,10Santagata S.,27 Savio M.,31 Scaffa C.,37Scala S.,27 Scalia G.,22Scognamiglio G.,36 Scovassi A.I.,29,30 Serio B.,22Serone E.,25Serra V.,10Sessa G.,34,36,37 Severi S.,33 Sidore C.,10Signorelli A.,26 Silvestri D.,21Simeon V.,18,19Simeone P.,20Sole G.,10Solito S.,28 Songia S.,21Soresina A.,23Spanò M.,14Spina F.,23Sproviero D.,31 Statuto T.,18Steri M.,10Surdo S.,30Tabellini G.,28 Tagliaferri P.,10Tardito S.,27 Tassone P.,10,12Tebaldi M.,33 Tedaldi G.,33 Tesei A.,33 Testa F.,23

Testi M.A.,23Tinelli C.,31Toft G.,14Tommasino C.,22Torchio M.,6,30 Tozzoli R.,20Trano A.C.,12Trino S.,19Trunzo V.,20Uboldi S.,34 Ursini C.L.,12Usai C.,15,32 Vacca A.,35 Vaccaro E.,18Valentino M.,17,19,20,22Valsecchi M.G.,21van Dijk M.A.,16Veglianese P.,11Vendramin A.,21Veneroni P.,31 Ventola A.,29 Ventura M.,10Vergara D.,35 Vergine P.,16Vicini R.,33 Villa C.,17 Villani O.,18Villanova S.,23Virdis F.,10Visai L.,6,29Visconte F.,22Vittoria L.,23Vivier E.,28Volpe A.,22,29 Volpe S.,22Vukich M.,6Whalen M.B.,10Zamagni A.,33 Zamai L.,13,26Zanetti M.,27 Zanoni M.,33 Zanovello P.,28Zappavigna S. 5 Zingone A.,14Zoledziewska M.,10Zorzi G.,26 Zucca S.,32 Zucchi M.,23Zviezdai V.,14



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