PROFESSOR ION CANTACUZINO - roami.ro · PROFESSOR ION CANTACUZINO in 1928 VOLUME 75 - Issue 3-4 July - December 2016 Published quarterly by Institutul CANTACUZINO Spl. Independenþei

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Professor Ion Cantacuzino(1863-1934)

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Editorial Board:Viorel Alexandrescu (Cantacuzino N.I.r., Bucharest, romania)gabriela Anton (Şt. S. Nicolau Virology Institute, Bucharest, romania)Antonios Antoniadis (Aristotelian university of Thessaloniki, greece)Daniela Bădescu (Cantacuzino N.I.r., Bucharest, romania)Adrian Băncescu (Carol Davila u.M.Ph, Bucharest, romania)gabriela Băncescu (Carol Davila u.M.Ph, Bucharest, romania)Coralia Bleotu (university of Bucharest, Faculty of Biology, Bucharest, romania)Jean-Marc Cavaillon (Pasteur Institute, Paris, France)Ana Călugăru (National Society of Immunology, Bucharest, romania)Cornelia Ceianu (Cantacuzino N.I.r., Bucharest, romania)Carmen Chifiriuc (university of Bucharest, ICuB,Bucharest, romania)radu Cojocaru (Ministry of Health, National Center of Public Health, republic of Moldova)Nicolae Corcionivoschi (Agri-Food and Biosciences Institute, Belfast, Northern Ireland)Irina Codiţă (Cantacuzino N.I.r., Bucharest, romania)Lidia Cremer (Cantacuzino N.I.r., Bucharest, romania)Victor Cristea (Iuliu Hațieganu u.M.Ph., Cluj-Napoca, romania)Maria Damian (Cantacuzino N.I.r., Bucharest, romania)Ionica Deliu (university of Piteşti, Piteşti romania)Angel galabov (Bulgarian Academy of Sciences, Sofia, Bulgaria)Steliana Huhulescu (Austrian Agency for Health and Food Safety, Vienna, Austria)Luminiţa Smaranda Iancu (gr. T. Popa u.M.Ph., Iaşi, romania)gabriel Ionescu (Cantacuzino N.I.r., Bucharest, romania)Anca Israil (Cantacuzino N.I.r., Bucharest, romania) Veronica Lazăr (university of Bucharest, Faculty of Biology, Bucharest, romania)Andreea-roxana Lupu (Cantacuzino N.I.r., Bucharest, romania)Emilia Lupulescu (Cantacuzino N.I.r., Bucharest, romania)gina Manda (Victor Babeş N.I.r.D., Bucharest, romania)grigore Mihăescu (university of Bucharest, Faculty of Biology, Bucharest, romania)roxana Moldovan (Victor Babeş u.M.Ph, Timişoara, romania)geza Molnar (Ministry of Health, Bucharest, romania)Alexandra Maria Năşcuţiu (Cantacuzino N.I.r., Bucharest, romania)Monica Neagu (Victor Babeş N.I.r.D., Bucharest, romania)Marian Neguţ (Carol Davila u.M.Ph., Bucharest, romania)Maria Nica (Carol Davila u.M.Ph, Bucharest, romania)Norica Nichita (Institute of Biochemistry, romanian Academy, Bucharest, romania)Marina Pană (Cantacuzino N.I.r., Bucharest, romania)Hervé Pelloux (Centre Hospitalier universitaire, grenoble, France)Mircea Ioan Popa (Carol Davila u.M.Ph, Bucharest, romania)graţiela Pîrcălăbioru (university College Dublin, republic of Ireland)Cristina Purcărea (Institute of Biology, romanian Academy, Bucharest, romania)Alexandru rafila (Carol Davila u.M.Ph., Bucharest, romania)Anca roşeanu (Institute of Biochemistry, romanian Academy, Bucharest, romania)Lila Shundi (Institute of Public Health, Tirana, Albania)Constantin Spânu (National Public Health Center, Chişinău, republic of Moldova)Demetrios Spandidos (Medical School, university of Crete, greece)Crina Stăvaru (Cantacuzino N.I.r., Bucharest, romania)Dan Steriu (Carol Davila u.M.Ph., Bucharest, romania)Cătălina-Suzana Stîngu (university of Medicine, Leipzig, germany)Monica Străuț (Cantacuzino N.I.r., Bucharest, romania)Edit Székely (u.M.Ph Târgu-Mureş, romania)radu Iulian Tănasă (Cantacuzino N.I.r., Bucharest, romania)galina Tseneva (Pasteur Institute, Sankt Petersburg, russia)Codruţa-romaniţa usein (Cantacuzino N.I.r., Bucharest, romania)Alexandru Filip Vladimirescu (Cantacuzino N.I.r., Bucharest, romania)Hans Wolf (regensburg university, germany) Imola Zigoneanu (university of North Carolina at Chapel Hill, uSA)

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INDEXED IN MEDLINE

In memoriam Prof. Ioan Cantacuzino59 THE CAPITAL OF MOLDAVIA AND THE HONOURING OF THE MEMORy OF PROFESSOR IOAN CANTACUzINO

Richard Constantinescu

IMMUNOLOGy

63 THE 46TH ANNUAL IMMUNOLOGy CONFERENCE - ABSTRACTS CONFERENCES, WORKSHOPS,

ORAL PRESENTATIONS, POSTERS, AUTHOR INDEX

97 CLINICAL AND LESIONAL DyNAMICS IN AN INFLUENzA A/PR/8/34-H1N1 AEROSOL INDUCED

INFECTION IN MICE

Ioana Sonya Ciulean, Elvira Gagniuc (Gubceac), Manuella Militaru, Crina Stãvaru

MICROBIOLOGy

109 ISOLATION OF Halobacillus truEpEri DS-1 - A SIDEROPHORE PRODUCING HALOPHILIC BACTERIUM

Leila Mirfeizi, Mohammad Ali Amoozegar, Ahmad Ali Pourbabaee, Hamid Babavalian, Mehrdad Moosazadeh

Moghaddam

115 SUBJECT INDEX

117 AUTHOR INDEX

57

ROMANIAN ARCHIVES OF MICROBIOLOGy AND IMMUNOLOGy

CONTENTS

VOLUME 75 ISSUE 3-4 JULy - DECEMBER 2016

58

Aims and Scoperomanian archives of Microbiology and immunoloy, an internationaljournal dedicated to original research work, publishes papers focusingon various aspects of microbiology and immunology. romanianarchives of Microbiology and immunology is indexed in MEDLINE. Thefrequency of the Journal is currently four issues per year.

Categories of manuscriptsFull-length articles are full-length descriptions of original research (up to 10 printed pages).reviews are comprehensive appraisals of research in a field of currentinterest. All reviews are subject to the normal review process (up to 12 printed pages).rapid communications are brief, definitive reports of highly significantand timely findings in the field (up to 5 printed pages).

Submission of manuscriptsManuscripts and all attached files (tables and illustrations) should besubmitted in electronic form to the Editorial Office, e-mails address:[email protected], [email protected] preferred software is Microsoft Word. In order to speed up theprocess of review, manuscripts should be prepared very carefully.

Cover letterEach manuscript submitted to the Romanian Archives of Microbiologyand Immunology must be accompanied by a Cover letter including anexplicit statement by the corresponding author that:• the manuscript represents an original work, has not been previously

published, and has not been submitted simultaneously for publicationelsewhere.

• the manuscript, as submitted, has been reviewed and approved by allnamed authors and that all authors concur with the submission andare responsible for its content.

• the Cover letter should be signed by the first author and thecorresponding author of the manuscript.

Editorial review and acceptanceAll manuscripts are subject to editorial review by professional peerreviewers (at least two). The acceptance criteria for all manuscripts arebased on quality and originality. The corresponding author of amanuscript is informed within 90 working days after submission that thepaper is accepted for publication in the journal, needs revision or isrejected. Revised manuscripts should be resubmitted as soon as possiblebut not later than 14 days.

Ethical considerationsA paper describing any experimental work with humans should includea statement that the Ethics Committee of the institution in which thework was done has approved it, and that the subjects gave informedconsent to the work.Experiments with animals should be done in accordance with the legalrequirements of the relevant local or national authority. Proceduresshould be such that animals used in experiments do not sufferunnecessarily. Papers should include details of the procedures andanaesthetics used. The Editors will not accept papers where the ethicalaspects are, in their opinion, open to doubt.

Preparation of manuscriptsManuscripts should be submitted in English. American or British spellingcan be used provided that only one spelling style is consistently usedthroughout. Manuscripts must be typewritten on A4 format (210x297 mm), withdouble spacing, margins of 25 mm, on one side only, consecutivelynumbered. Times New Roman font, 12-point size, is required.

Text headings All headings in the text should be set over to the left hand margin, andtext should begin on the next line. Type first level (sectional) headings all in capitals. Second level headings should be typed in small (lower case) letters butwith the first letter of each main word a capital. For third level headings, only the first letter of the first word should bea capital. Underline first and second level headings.

FIRST LEVEL TEXT HEADINGSecond Level Text HeadingThird level text heading

Manuscripts should be divided into the following sections and order: Title page, Abstract and keywords, Introduction, Materials and Methods,Results, Discussion, Acknowledgements, References, Tables, FigureLegends and Figures. 1. Title page contains: title of the paper not longer than 80-100

characters, including spaces and punctuation; full name of the authorsand their affiliation; the author responsible for correspondence willbe marked by an asterisk, and his full address telephone/fax numbers,and e-mail address will be indicated.

2. Abstract must not exceed 250 words and should reflect the contentof the study. Following the abstract, a list of 3-10 keywords is essentialfor indexing purposes.

3. Introduction containing a description of the problem underinvestigation and a brief survey of the existing literature on the subject.

4. Materials and Methods provide sufficient detail to allow the work tobe reproduced.

5. Results. Results should be clear and concise.6. Discussion that enriches but does not repeat Section 3 or 5.7. Acknowledgements (if applicable) containing acknowledgement of

technical help and of financial material support.8. References should be numbered consecutively in the order in which

they are first mentioned in the text. Identify references in text, tables,and legends by Arabic numerals in square brackets (e.g. [1], [2-6],etc.). Please note the following examples:Journals: Allain F, Vanpouille C, Carpentier M, Slomianny MC, Durieux S,Spik G. Interaction with glycosaminoglycans is required forcyclophilin B to trigger integrin-mediated adhesion of peripheralblood T lymphocytes to extracellular matrix. Proc Natl Acad Sci U SA 2002. 99: 2714-2719. Books:Theofilopoulos AN. Immune complexes in autoimmunity. In: BonaCA, Siminovitch KA, zanetti M, Theofilopoulos AN (Eds.) The Mole -cular Pathology of Autoimmune Diseases. Harwood AcademicPu blishers, Switzerland 1993, pp 229-244.

9. Tables with suitable captions at the top and numbered with Arabicnumerals should be collected at the end of the text on separate sheets(one page per Table). Footnotes to tables should be marked with a)b) c) etc and *, **, *** should be reserved for p values. Each tablemust be understood independently of the text. All tables must be citedin the text.

10. Figures (illustrations) Figures should be submitted on separate pagesat the end of the article (new page for each complete figure). Theyshould be numbered in the order of their appearance with Arabicnumerals. Figures should be submitted as TIFF files at a pro perresolution as follows: Graphs at 800-1200 dpi; Photos at 400-800DPI; Color 300-400 DPI. Text in figures should be 8-10 point in size.Each figure must have a separate legend. The legends should notappear under the figures, but be gathered in a separate section(Figure legends). Color figures can only be printed if the author isprepared to pay the cost incurred.

11. Figure legends should be supplied at the end of the manuscript,double spaced, with relevant figure numbers, labeling symbol andexplanation.

Units of measurement, Symbols and abbreviations Symbols for physical units should be those of the Système Internationale(SI) Units. Alternative or non-SI units may be used, but these must bedefined at their first occurrence in the text.

Nomenclature of MicroorganismsBinary names, consisting of a generic name and a specific epithet (e.g.,Escherichia coli), must be used for all microorganisms.

Genetic NomenclatureTo facilitate accurate communication, it is important that standardgenetic nomenclature be used whenever possible and that deviations orproposals for new naming systems be endorsed by an appropriateauthoritative body.

Proofs and reprintsTen reprints of each article and one copy of the journal will be suppliedfree of charge to the first author.

romanian archives of Microbiology and immunology

INSTRUCTIONS TO AUTHORS

59

THE CAPITAL OF MOLDAVIA AND THE HONOURING OF THE MEMORy OF PROFESSOR IOAN CANTACUzINO

Richard Constantinescu1,*1Grigore t. popa university of Medicine and pharmacy, iaşi, romania

*corresponding author: Richard Constantinescu, e-mail: [email protected]

Keywords: Ioan Cantacuzino, Grigore T. Popa, Însemnãri ieşene (Iaşi Chronicles), statuary heritage, St. Spiridon Hospital, Iaşi

ABSTRACTThe text below gives an account of a certain aspect

of how the medical world in the Romanian space hasunderstood to keep alive its historical memory. Themonumental heritage, paintings, medals, stamps andmedical photography may contribute to redefining andstrengthening the identity of the medical profession. Acharacter of a more than national importance, IoanCantacuzino (1863-1934) has been represented in statuesin various cities of Romania, one of which is Iaşi.Romanians are known to be good at organising funeralsand paying immediately posthumous homage to a man.However, they are less skillful in preserving his memory,publishing his work, building a monument, as well asshowing care to the place where he was buried. We admirethe initiative of the people of Iaşi, in the ‘30s-’40s of thelast century, and we wish their example is followed by thecontemporaries also.

REzUMATTextul ce urmează dă seama de un anume aspect al

felului în care lumea medicală din spaţiul românesc aînţeles şi înţelege să-şi întreţină memoria istorică.Patrimoniul monumental, tablourile, medalistica,filatelia şi fotografia medicală pot contribui laredefinirea şi consolidarea identităţii profesiei medicale.Personaj cu o importanţă mai mult decât naţională, IoanCantacuzino (1863-1934) a fost reprezentat statuar înnumeroase oraşe din România, unul dintre acestea fiindIaşul. Se spune că românii se pricep să organizeze binefuneralii şi să omagieze imediat postum un om. Sunt maipuţin iscusiţi în a-i conserva memoria, în a-i publicaopera, în a-i ridica un monument, precum şi în cepriveşte grija faţă de locul în care a fost îngropat.Admirăm iniţiativa ieşenilor din anii ’30-’40 ai secoluluitrecut şi ne dorim ca exemplul lor să fie pilduitor şipentru contemporani.

At 31 years of age Ioan Cantacuzino became ateacher in Iaşi [1] – a substitute professor for theMorphology Department of the Faculty of Sciencesof the University in the capital of Moldavia, between1 November 1894 - October 1896 [2]. He thenunderstood that his mission was more thanpresenting information to the young learners.Despite the fact that he faced financial difficulties asregards the provision of equipment for the laboratoryand the conditions for holding courses, he strived tomake things better, by organising the department,launching public conferences and lectures, roamingthe outskirts of Iaşi with his students and offeringfree medical consultations. “He radiated hisfriendship and embraced the others’ too, and inparallel with the morphology course he taught excathedra, he fostered arts and letters far and wide,being active in the group that gave birth to thebeautiful magazine Viaţa Românească, which hemanaged later”, were the words of Dr. VictorGomoiu (1882-1960) [3].

Today, the House of Corneliu Codrescu, wherehe set up his first animal morphology laboratory, andthe houses on Balş estate where he lived have alldisappeared; only St. Dumitru-Balş Church, wherethe marriage between Ioan Cantacuzino and ElenaBalş was celebrated in 1896, still subtly harboursthose moments within its walls... “I don’t know ifhis figure had already become too big or if Iaşienvironment was too tight” [4], but we know for surethat in two years after his arrival in Iaşi he wouldreturn to Paris. Cantacuzino, “failing to find thosesubtle satisfactions elicited by the sense of a matchbetween his activity and the understanding of thesurrounding world” [5], quits teaching and prefersan inferior job: that of an assistant in the PasteurInstitute, thus he could be in the vicinity ofMetchnikoff.

Ioan Cantacuzino brought joy into the lives ofIaşi people and of the Romanians who were in Iaşiin spring 1917. During World War I, the capital ofMoldavia was the rescue island of thousands of

Romanians. The deaths on the front werecomplemented by those who died of cold, famine,recurrent fever and exanthematic typhus, dubbed as“a dangerous internal enemy” [6]. Dr. Cantacuzinowas appointed director of the General Directorate forPublic Health and made exceptional efforts [7] withthe support of a group of Romanian and Frenchdoctors in curbing the spread of exanthematic typhus[8] and fighting against the epidemics of cholera,typhoid fever and recurrent fever, which werethreatening both the army and the population. In thefunds registry of Iaşi County Directorate of NationalArchives one may consult a report on the workscarried out in the lab of Professor Cantacuzinoduring the Iaşi period: 8 August 1916-1 July 1918[9]; his experimental medicine lab continued toproduce the preparations necessary for theprevention and treatment of communicable diseases.Iaşi was also the place where he founded theMedical-Surgical Society of the Russian-RomanianFront [10].

Two decades after those tragic events IoanCantacuzino returned to Iaşi. He saw his friends andcollaborators again and visited, in a carriage, St.Spiridon Hospital and various sanitary units in thecounty; he also held a conference which manypeople of Iaşi were happy to attend. [11].

When the Professor passed away, the studentsand collaborators dedicated a volume to their master.Grigore T. Popa (1892-1948), the great Romaniananatomist and writer who was dearly devoted toCantacuzino, wrote about that work in Însemnăriieşene magazine, a publication which hecoordinated: “We thus relive, in a panoramic way, agreat pain and an intense emotion. Such a concert ofhigh appreciation and good feelings has never beenproduced until today. Romanian and foreignscholars, politicians, men of letters, colleagues andstudents all joined together spontaneously to expresstheir love for the one who would bodily disappearon 14 January 1934. Cantacuzino’s death meant adramatic surprise and excruciating pain to all whoknew him. He was one of those rare humans amongus who knew how to melt an entire generation in hissoul, lavishly dissipating himself into the hearts ofthose who came in his close proximity. That explainswhy a high school was founded around him whosemembers still retain, almost religiously, the cult ofthe master who used to inspire them. People forgeteverything, and they forget it quickly, while keepingthe memory for the departed alive is a nobleexception” [12].

Two years after the physical demise ofProfesssor Cantacuzino, Grigore T. Popa invites acolleague from the Faculty of Medicine of Iasi, thesurgeon and publicist Professor Paul Anghel (1869-1937) to write a material in which to make asynthesis of the social, educational and scientific lifeof the great departed man [13].

Perhaps one of the warmest, densest and deepesttexts written about Ioan Cantacuzino is thatdelivered by university academic Grigore T. Popa,as published in the volume, after it had been initiallya lecture given at the meeting of the RomanianAcademy – “The Sacred Fire of Science (Dr. I.Cantacuzino)” on 24 September 1937. Whilereviewing this volume, a contributor to the magazinein the capital of Moldavia writes that the author, withan ‘amazing power of evocation’ and ‘in profoundadmiration’, emphasizes the great personality of theRomanian scientist such that, the reviewer says, ‘wecould state that this man, in addition to hisunsurpassed culture and unequalled spiritualqualities, had his share of luck. The luck of beingshortly understood by his students, of being loved,admired and imitated by them to such extent that,with his death, we felt that «an era died»’. This book,‘full of pearls of his own cogitation’ is an interestingreading and, in the eyes of he who introduces him tothe public, ‘would be a wonderful and fundamentalbook for the youth who still believes in the need andpower of culture’14. I will soon publish the text ofProfessor Popa in a book that includes other writingsabout personalities of science and culture, in theseries by author Grigore T. Popa, which I initiatedand coordinate.

An important initiative was the civiceternalization of Professor Ioan Cantacuzino in Iaşi.On the initiative of St. Spiridon Hospital Board ofTrustees and the management of Însemnări ieşene itwas decided to make a statue effigy of ProfessorCantacuzino, in the hospital courtyard. A textannouncing this wish of the people of Iaşi anddetailing the first subscriptions supported thedecision as follows: “This great and understandingman saved for a while the establishments ofSpiridonia centre, offering them the means whichconstitute today an important part of the institution’sinsurance. Under his ministry and on his initiative,the Anti-tuberculosis League was established, whoseincome contributes to building sanatoriums andensuring charity including for the benefit ofSpiridonia. A multilateral scholar, facilitator of greatinitiatives, school creator, organiser and man of vast culture, Professor Cantacuzino remains one the

RIChARD CoNSTANTINESCU

60

most brilliant representative characters in our historyand every institution in this country honours itselfby honouring him. Therefore, we are certain that,before long, our readers will give us the strength toraise him a bust in St. Spiridon Hospital courtyard.Here are the first subscriptions: St. Spiridon Boardof Trustees – 10,000 lei; Professors Al. Slătineanu,Șt. Graçosky, I. Bălteanu, Gr. T. Popa, Al. Ţupa, I.Nicolau, Al. Cosăcescu, C. Ionescu-Mihăeşti, M.Ciucă, A. Ciucă, Gh. Oprescu, D. Combiescu, Gh.Zota – 1,000 lei each; Doctors Gr. Iftimescu,Vasilescu-Popescu, P. Condrea, I. Alexa şi Nasta –1,000 lei each. Total amount – 28,000 lei” [15].Subsequent issues of the magazine [16] announcedthe proposal to raise the bust and gave presentationsof those who had sent money by postal order for thisproject.

In May 1940 the editorial office of Însemnăriieşene magazine announces the closing of thesubscription list and extends its thanks to those whohad donated various amounts of money. It presentsthe last donor and, in order to be transparent all theway through, shows the amount spent for the castingof the bust made by Corneliu Medrea (1888-1964),an artist from Bucharest – 30,240 lei and the amountnecessary to build the base and mount the bust –11,980 lei [17].

The front base reads the following words: ‘Tothe supporter and protector of Spiridonia [St.Spiridon Hospital], Dr. I Cantacuzino, the eternalgratitude from St. Spiridon Board of Trustees; therear base reads the following words engraved instone: “1. By Law of the State Lottery, 20% of theincome awarded to Spiridonia; 2. A grant in thepublic health budget for five clinics of 1,500,000 leiper year; 3. Exemption of Spiridonia from payingtaxes to the State, county, municipality; 4. Planningthe re-apportionment of property to Spiridonia offorestry and plough land from the State reserves”.

The inter-war people of Iaşi were fully aware ofthe value of the personality and work of the greatCantacuzino. The people of our times are neglectingthe memory of the great scholar: his bust base in St.Spiridon Hospital courtyard lies in degradation, andthe space around it is unkempt; a decade ago I savedfrom scrapping an oil painting of Professor IoanCantacuzino and had it restored, framed and shownin the Museum of Medicine History of Grigore T.Popa University of Medicine and Pharmacy of Iaşi,which I was coordinating at that time. Whileformerly in the Communist times and beyond, theimage of Cantacuzino suffered from certaindeformations, caused by the “requisitions” of the

present times, when some monuments enjoyed adegree of consecration in relation to others [18],there is now a pervading disinterest in monumentsrepresenting him and a fading narrative force whichthose of the inter-war period bestowed upon them.

REFERENCES

1. Alexandru Ioan Cuza University of Iasi. The Rector’sOffice in 1860-1944. Archive Inventory, vol. I.,Directorate General for State Archives, Iaşi CountyState Archives Subsidiary, Bucharest, 1985, p. 155.

2. Yearbook of Iaşi University for School Year 1895-1896,Iassy, National Printery, 1896, p. 56.

3. Speech addressed by dr. V. Gomoiu at the meetingcommemorating the Romanian Royal Society for theHistory of Medicine on 19 January 1934, RomanianMedical Movement, an. VII, no 1-2, January-February1934, p. 13.

4. Speech addressed by dr. V. Gomoiu at the meetingcommemorating the Romanian Royal Society for theHistory of Medicine on 19 January 1934, RomanianMedical Movement, an. VII, no 1-2, January-February1934, p. 13.

5. Gr. T. Popa, The Sacred Fire of Science (I.Cantacuzino), Romanian Academy, Memoirs of theScientific Section, series III, tome XIII, mem. 2,Official Gazette and State Printing Works, NationalPrinting House, Bucharest, 1937, p. 25.

6. Ion Agrigoroaiei, Public opinion and state of mindduring the Integration and Great Union War AXISFoundation Publishing House, Iasi, 2004, pp. 50-67.

7. Alin Ciupală, Their Battle. Women of Romania in WorldWar I, Polirom Publishing House, 2017, p. 230.

8. Ioan Scurtu, Politics and Daily Life in Romania in the20th Century and Early 21st Century, Mica ValahiePublishing House, Bucharest, 2011, p. 32.

9. Alexandru Ioan Cuza University of Iasi. The Rector’sOffice in 1860-1944. Archive Inventory, vol. I.,Directorate General for State Archives, Iasi CountyState Archives Subsidiary, Bucharest, 1985, p. 209.

10. Prof. Dr. I. Mesrobeanu, Life and work of professorIon Cantacuzino, In: Ion Cantacuzino, Selected Works.Œuvres choisies, under the editorial work of Prof. Dr.I. Mesrobeanu, Romanian People’s Republic AcademyPublishing House, Bucharest, 1965, p. 27.

11. Richard Constantinescu, Ioan Cantacuzino in IasiCounty, Medical Life, no 46 (1244), 21 November2013.

12. P. Gr., In Memory of Professor Ioan Cantacuzino,Însemnări ieşene, an. I, no 6, 15 March 1936, p. 254.

13. Paul Anghel, Professor Ioan Cantacuzino, an. I, vol.II, no 13-14, 15 July 1936, pp. 22-26.

14. T.A.B., Gr. T. Popa, the Sacred Fire of Science (Dr. I.Cantacuzino), Însemnări ieşene, an. III, vol. V, no 3,1 March 1938, pp. 397-398.

15. A Bust for Professor Cantacuzino in Iasi, Însemnăriieşene, an. III, vol. VII, no 8, 1 August 1938, p. 334.

The Capital of Moldavia and the honouring of the memory of Professor Ioan Cantacuzino

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RIChARD CoNSTANTINESCU

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16. Însemnări ieşene, an. III, vol. VII, no 9, 1 September1938, p. 527, 548; 36490 lei – Însemnări ieşene, an.III, vol. VII, no 10, 1 October 1938, p. 159; 37490 lei– Însemnări ieşene, an. III, vol. VIII, no 11, 1November 1938, p. 348; 37490 lei – Însemnări ieşene,an. III, vol. VIII, no 12, 1 December 1938; 11340 lei– Însemnări ieşene, an. IV, vol. IX, no 2, 1 February1939, p. 357; 21120 lei – Însemnări ieşene, an. IV, vol.

IX, no 3, 1 March 1939, p. 552; 22670 lei – Însemnăriieşene, an. IV, vol. X, no 4, 1 April 1939, p. 169;26670 lei – Însemnări ieşene, an. IV, vol. X, no 5, 1May 1939, p. 386.

17. Însemnări ieşene, an. V, vol. XIV, no 5, 1 May 1940,p. 374.

18. Andrei Pippidi, Of Statues and Graves. For a Theoryof Symbolic History, Polirom Publishing House, Iaşi,2000, p. 7.

63

CP1. MOLECULAR MEDIATORS OF THE IMMUNE RESPONSE AND THE START OF THEIR USE IN THE CLINICAL DIAGNOSIS

ThE 46Th ANNUAL IMMUNoLoGY CoNFERENCE

ABSTRACTSCoNFERENCES

Victor Cristea1,2 Lucian Pop2

1iuliu Haþieganu uMph cluj-Napoca, romania; 2sf. iosif Medical center, cluj-Napoca, romania

The discovery, more than 35 years ago, of thefirst molecules involved in modulating the immuneresponse, brought some clarification on the intimatemechanisms of cell signaling and cooperation.Throughout those years new soluble factors werediscovered having different roles and effects.

As often had happened in immunology, whenthe information explosion had not found enoughtime for the recent acquisitions to settle, to beunderstood and systematized (see HLA, CD,genetics of Ig), the study of those mediatorscontinues today simultaneously in several directions

with clarifications and confusion in equal measure.Justly, the researcher who tries to grub up the

multitude of data that had accumulated, experiencesthe same feelings as an unsuspecting hiker thatsuddenly enters into a “zoo of factors, jungle ofinteractions, swamp of acronyms or desert ofsynonyms” (J.A. Symons, 1995).

In the following we will briefly expose our ownview on these mediators, a classification that weassume and that has as its starting point themolecular criterion beyond the clinical or thetherapeutic.

The sentinel node (SN) is the first station wherea malignant tumor metastasize. The current conceptof SN involves a therapeutic approach of solidtumors in several stages: locating SN by means ofmarking the peritumoral lymphatics, excision of thetumor together with SN, histopathological analysisof SN to identify metastases, excision of the regionallymph nodes (RL) when SN is invaded. Oncologybecomes more conservative and the treatment has abetter chance of success in cases where SN is notinvaded or has only micrometastases,

SN is the seat of antitumor immune responseagainst circulating tumor cells (CTC). Characteristicof CTC attack on immune cells in the SN is inducingimmunosuppression. CTC secrete factors that inhibitresponsiveness to dendritic cells (DC) and T cells,while stimulating regulatory T-lymphocytes withsuppressor role and Th2 cell response.

CTC induce a phenomenon of tolerance totumor antigens (TA), which allows CTC survival,

their multiplication and RL invasion. It is interestingto note that the anti-tumor immune response in RLis more active than the SN response, whichhypothesized that the RL answer would be anindependent process. SN cells (T-CD4+, T-CD8+,DC) were used as effectors of adoptive antitumourtherapy. The method involves activated cellexpansion or activation of resting cells in thepresence of specific TA. By gene recombination onecan get specific clones of antitumour T lymphocyteswith anti-tumor specific chimeric receptor.

At present there is a continuous improvement ofmethodology for investigating the presence of CTCin SN. Histopathological and immunohistochemicalmethods are starting to be replaced by flowcytometry and RT-qPCR, more accurate and lessencumbered by false negative results. SN metastasisdiagnosis becomes a focus of anti-tumor therapytrials, involving surgeons, pathologists,immunologists and oncologists.

CP2. CIRCULATING TUMOR CELLS (II): SENTINEL LyMPH NODE SECRET MISSION

Cornel UrsaciucDepartment of immunology, Victor babeş NirD, bucharest, romania

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Ştefana M. PetrescuDepartment of Molecular cell biology, institute of biochemistry of the romanian academy, romania

Tyrosinase is the source of a major melanomatumor antigen that is recognized by human cytolyticCD8+ T lymphocytes in patients with a growingmelanoma tumor.

This study addresses the basic mechanisms oftyrosinase processing and presentation to T cells inthe absence of N-glycans located distal or within atyrosinase epitope. Using human CD8+ T cell clonesspecific for the HLA-A2 restricted tyrosinaseantigen peptide YMDGTMSQV we show efficientrecognition of melanoma cells transfected withtyrosinase and its N-glycosylation mutants.

However, the single, triple and non-glycosylated mutants lacking the proximal (N337Q)and epitope located N-glycosylation site (N371D)

generated more abundant peptide present at the cellsurface compared to glycosylation mutants affectingdistal sites. They also consistently led to higherCD8+ T cell activation.

We conclude that N-glycans located within andin the vicinity of the epitope impair the ability ofhuman tyrosinase to provide HLA-A2 restrictedantigens for recognition by specific CD8+ T cells.

This report provides evidence of the highefficiency of tyrosinases lacking the epitope locatedN-glycan, as well as non-glycosylated tyrosinase inthe MHC class I presentation. These may bevaluable clues in enhancing the efficiency oftyrosinase based cancer vaccines.

The classical scenario describes how endoge-nous and exogenous antigens are processed in diffe-rent compartments and are also loaded in the MHCI and MHC II grooves in two distinct compartments,and then transported to the cell surface.

Yet, immature dendritic cells seem able toexpress empty MHC II molecules, which are thenloaded with antigens processed extracellularly.Furthermore, the MHC I and II/peptide complexesare presented to CD8+, respectively CD4+ T cells.

However, due to a phenomenon named cross-presentation, the antigens derived from proteins cancross from one cell to another, and from a loadingcompartment to another, hence being presented by

an MHC belonging to a different class to thecorrespondent T cell.

Furthermore, lipids seem to use elementsbelonging to both pathways in order to end uppresented by CD1 molecules.

One way or another, the T cell receptorsrecognize antigens that are presented by self MHCmolecules, in a system known as MHC restriction.

This type of presentation is able to explain mostof the interactions between T lymphocytes and theircellular counterparts, but it is unable to explain themost important mechanism leading to solid graftrejections, that involves the recognition of an antigenassociated with a non-self MHC.

CP4. MHC PRESENTATION: ARE THERE INDEED ANy RULES?

Corina Cianga, Petru Ciangaimmunology Department, Gr. t. popa uMph, iaşi, romania

CP3. IMPAIRMENT OF MHC-I EPITOPE GENERATION By EPITOPELOCATED N-GLyCANS IN THE MELANOMA ANTIGEN TyROSINASE

ABSTRACTS

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Monica Neagu1,2, Andreea Lupu1, Carolina Constantin1

1Victor babeş NirD, immunobiology laboratory, bucharest, romania;2Faculty of biology, university of bucharest, romania

Most cancer immunotherapy approaches such asvaccination, adoptive transfer of anti-tumoral T cells andimmune checkpoint inhibition were pioneered in thisdisease. Immune checkpoint inhibitors based cancerimmunotherapy has recently attracted considerableinterest in the field of cancer therapy.

The relevant immunotherapeutic agents do notdirectly attack the tumor, but boost the body’s immunesystem to recognize and kill cancer cells. Recent effortsutilizing immuno-engineering for local delivery of theseimmune checkpoint antibodies are presented.

This type of immunotherapy was embraced inmelanoma, colorectal cancer, lung cancer, the mostdevastating types of cancers in terms of mortality andincidence. All these types of cancers in order to have anefficient immunotherapy need to be tackled with therecent molecular markers.

Thus as the Diagnosis and treatment of melanoma.European consensus-based interdisciplinary guideline –Update 2016 has shown for melanoma there are severalpoints that should be considered when applyingapproved immunotherapy in melanoma. Thus, mutationtesting of tumour tissue (at least BRAF; NRAS, CKIT)

is a prerequisite for treatment decisions. PD-1checkpoint blockade either as monotherapy or incombination with CTLA-4 blockade should beconsidered as a good option for first-line treatment forall patients with unresectable metastatic melanoma,independently from BRAF status.

When BRAF-inhibitors are considered for BRAFmutated patients, they must be given in combinationwith MEK inhibitors. C-KIT inhibitors should be givento patients that have c-KIT mutant melanomas. This typeof cancer being one example amongst others that needto be approched in applying immunotherapy by usingpanel of specific molecular markers.

These molecular markers can re-orient therapy andcan pin-point the best patient population for an efficienttherapy approach.

Selected references: Garbe et al, Diagnosis andtreatment of melanoma. European consensus-basedinterdisciplinary guideline – Update 2016; Seremet et al.J Transl Med. 14:232, 2016; Wang C et al., Hum VaccinImmunother. 2016 Sep 26:1-4.

Acknowledgement. Grant PN-II -2013-4-1407

A lot of studies are showing thatneuropsychological stress is a powerful factor which candownregulate the immune system and its normalresponse to all foreign substances, including pathogens.It is also established that communication between thecentral nervous system and the immune system involvesthe endocrine system too and bidirectional signalsbetween all parts of this triad. Psychological stressorsdysregulate this network, this condition leading toimmunosupression and a high risk especially forinfectious diseases.

The aim of this presentation is to show theconnection between the central nervous system and theimmune system, the mechanism of action of the stresshormones and their powerful immunosupression effectunder chronic stress condition with a major risk forhealth, underlining the great importance of the anti-stressmeasures, at individual or professional level.

Under normal conditions, our immune systemmaintains a balanced state of health (immunostasis), but

when the organism is exposed to prolonged stress, thesystem breaks down. Our body becomes vulnerable andmanifest not only neurological stress symptoms, but alsolong-term immune system disorders and other illnesses.In fact, it is considered that nearly 70% of all diseasescan be attributed to or influenced by chronic stress.

The stress-induced activation of the sympatheticnervous system and the sympathetic-adrenal medullaryand hypothalamic-pituitary adrenal axes lead to therelease of cytokines. At a molecular level, the immunefunctions are mediated by these messenger molecules,released from a variety of activated cells of the immunesystem - leukocytes or other cells and acting on othertarget cells, with inflammatory or anti-inflammatoryeffect. Adrenergic stress hormones alter the synthesisand release of cytokines and such alterations in theimmune system are influencing its functions. Theexperience of stress affects especially the cellularimmunity, an important specific defense mechanismcontrolling viral infections and even cancer disease.

CP6. INFLUENCE OF NEUROPSyCHOLOGICAL STRESS ON IMMUNITy STATUSVeronica Lazãr

university of bucharest, Faculty of biology – Microbiology & immunology lab., romania

CP5. MOLECULAR MARKERS IN CANCER IMMUNOTHERAPy


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Gina Manda1, Ana-Maria Enciu1,2, Antonio Cuadrado1,3,4

1Victor babeş NirD, radiobiology laboratory, bucharest, romania; 2carol Davila uMph, bucharest, romania; 3uaM, autonomous university of Madrid, Madrid, spain;

4cibErNED, ciber de investigación biomédica en red en Enfermedades Neurodegenerativas, Madrid, spain

A paradigm shift has occurred recentlyregarding the role of reactive oxygen species (ROS)in health and disease: ROS sustain cellularhomeostasis by regulating cell signaling throughredox-mediated mechanisms, and alteration of thetightly controlled redox balance underliescardiovascular and neurodegenerative diseases,cancer, diabetes and obesity.

Clustering of these apparently unrelateddiseases according to their common molecularprofile (“network medicine” approach) opens newperspectives for unraveling pathologic mechanisms,and for developing new targeted therapeuticstrategies.

Recent studies evidenced that redoxdisturbances in Alzheimer’s disease (AD) precedeby many years molecular neurotoxic changes thatproduce cognitive impairment.

Chronic oxidative stress in AD derives fromincreased ROS production (dysfunctionalmitochondria in neurons, enhanced NADPH-oxidaseactivity in glial cells, ROS-producing monocytes andgranulocytes that infiltrate the AD brain), but alsofrom a suppressed endogeous antioxidant response.The reciprocal relation between oxidative,inflammatory and proteotoxic stresses, and their

synergic impact on brain integrity in various stagesof cognitive impairment will be detailed.

Therapies that specifically target oxidativestress through modulation of the endogenousantioxidant system are a promising therapeuticstrategy in redox diseases, including in AD. Apotential approach might be the pharmacologicactivation of the transcription factor Nrf2 which iscritically involved in triggering a plethora ofendogenous cytoprotective mechanisms thatparticipate in redox control, inflammation andproteostasis.

We will present REDBRAIN, a project thatattempts to address these issues in AD. REDBRAINwill design an advanced investigational system forpreclinical and clinical testing of Nrf2 modulatorsand biomarkers of AD. The rational is based on theanalysis of peripheral markers of the molecularsignatures of Nrf2 and nuclear factor-kB in bloodleukocytes, and the functional polarization ofmonocytes, as precursors of microglial cells.

The REDBRAIN project (ID P37_732) is co-financed by the European Fund for RegionalDevelopment through the Operational ProgramCompetitiveness.

CP7. ALzHEIMER’S DISEASE FROM THE REDOX SIGNALING PERSPECTIVE – THE REDBRAIN PROJECT

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Introduction: Body mass index (BMI) and diethave independently been linked to type-2 diabetesmellitus (T2DM) and breast cancer (BC). Currentevidence seems to confirm that common signalingaberrations are implicated in both the increased riskof T2DM and BC. Thus, it is expected that T2DMpharmacotherapy which can reverse or modify thesesignaling aberrations will subsequently affect BCprognosis.

This hypothesis is further supported by evidencethat injectable insulin and insulin stimulating agentsare associated with poorer BC outcomes amongwomen with T2DM while other drugs, most notablymetformin, have had substantially improvedoutcomes. Nonetheless, there is limited evidencesupporting a particular mechanism and to ourknowledge this study was the first to evaluate theinterrelationship between biomarker profiles, T2DMtherapy, and BC outcomes.

Methods: This study was approved by theinstitutional review boards of Roswell Park CancerInstitute (RPCI) and University at Buffalo. All adultwomen diagnosed at RPCI between January 1, 2003and December 31, 2009 were considered for inclusion(n=2194).

Medical records were reviewed to abstractdemographic and clinical parameters as well cancertreatment history and the related outcomes.Subsequently, all subjects that were identified to haveT2DM at diagnosis (cases, n=97) were matched totwo without T2DM (controls, n=194) based on: age,BMI, ethnicity, menopausal status, and tumor stage.All study subjects were required to have treatment andsurgery naïve plasma samples in the RPCI databankand biorepository.

A panel of 33 biomarkers were assessed by eitherELISA or Luminex. Outcome optimized biomarkerthresholds were identified by Kaplan-Meier withrespect to both overall survival (OS) and disease-freesurvival (DFS).

Associations were assessed by Fisher’s exact test,Kruskal-Wallis or Wilcoxon Rank-Sum test, wereappropriate. Multivariate statistical analyses wereperformed to account for age, tumor stage, BMI,estrogen receptor (ER) status, and cumulativecomorbidity. Biomarker correlations were assessed bythe Pearson method.

Results: Utilization of insulin and insulinsecretagogues was associated with ER (-) phenotype(p=0.008 and p=0.043, respectively) and significantlypoorer BC outcomes (p=0.012 and p=0.033,respectively). Insulin use was associated with lowerC-peptide levels and higher IL-6, TNF-α and CRPlevels, elevated CRP and TNF-α were associated withpoorer BC outcomes (p=0.003, MVP=0.210). Insulinand insulin secretagogue users had higher leptin levelsthan controls (p=0.052 and p=0.050). Loweradiponectin levels were observed among non-insulin(p<0.001, MVP=0.006).

C-peptide levels were lower among insulin usersas compared to insulin non-users (p<0.001,MVP<0.001). Multivariate adjustment identifieddifferences in C-peptide levels between insulin usersand controls (p=0.290, MVP=0.001).

Overall, C-peptide levels lower than 0.75 ng/mlwere strongly associated with poorer survival(p=0.007, MVP=0.002).

Among insulin users, C-peptide levels wereinversely correlated with IL-1β and IL-1Ra levelsonly after full adjustment (p=0.012 and p=0.030,respectively). The correlation was not observed in theother treatment groups. By contrast, IL-1β andadiponectin levels were directly correlated amonginsulin users, both before and after adjustment(p=0.021 and p=0.029, respectively), but not in othergroups.

Conclusion: Insulin use was associated with bothelevated leptin, CRP, TNFα, and lower C-peptide aswell as poor BC outcomes. Insulin secretagogue usewas associated with poorer prognosis as well as loweradiponectin levels and higher levels of leptin and CRP.

CP8. PHARMACOTHERAPy, CyTOKINE PROFILES, AND PROGNOSIS IN WOMEN WITH DIABETES MELLITUS AND BREAST CANCER

Zachary AP Wintrob1, Jeffrey P hammel2, Thaer Khoury3, George K Nimako1, hsin-Wei Fu1, Zahra S Fayazi1, Dan P Gaile4, Alan Forrest5, Alice C. Ceacãreanu1,6

1state university of New York at buffalo, Dept. of pharmacy practice, NYs center of Excellence in bioinformatics and lifesciences, 701 Ellicott street, buffalo, NY 14203; 2cleveland clinic, Dept. of biostatistics and Epidemiology, 9500 Euclid

ave., cleveland, oH 44195; 3roswell park cancer institute, Dept. of pathology, Elm & carlton streets, buffalo, NY 14263;4state university of New York at buffalo, Dept. of biostatistics, 718 Kimball tower, buffalo, NY 14214; 5the uNc Eshelman

school of pharmacy, Division of pharmacotherapy and Experimental therapeutics, campus box 7569, chapel Hill, Nc 27599; 6roswell park cancer institute, Dept. of pharmacy services, Elm & carlton streets, buffalo, NY 14263


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Adrian onucantacuzino Nir, bucharest, romania

The long history of vaccination is based on dif-ferent types of vaccines. Although the immunizationprocedures are described from early development ofmedical practices, the modern medical history ofvaccination started in 1798 with the publication ofEdward Jenner experiment which introduced the famous concept of “vaccination”. His method finallyresulted in the eradication of smallpox and definedthe initiation of the concept of “live attenuated” microorganism which has underwent medical andtechnological development since then.

Another definite moment in the vaccinedevelopment was the Pasteur’s rabies vaccine (1885)which had a big impact in human disease fightingwhich defined the concept of immunization with“killed microorganisms”. These two main conceptsled the development of vaccines in the beginning of19th century based on the development ofmicrobiology in the fight against anthrax, cholera,plague, typhoid, tuberculosis, diphtheria, tetanus.From a technological point of view, the last two ofthem defined the “toxoid vaccines” based on aspecific protein and a clear pharmacological“mechanism of action”.

Modern vaccines are essentially based on thesepharmacological concepts and on benefits providedby the development of immunology, molecularbiology and biotechnology. Thus beside liveattenuated vaccines, inactivated vaccines and toxoidvaccines, other vaccine concepts such as subunitvaccines conjugate vaccines, DNA vaccines,recombinant vector vaccines have been developedover the years. Each concept was redefined, a goodexample being the “recombinant subunit vaccines”,based on DNA recombinant technology. Also theavailability of DNA sequences, along with bigachievement in immunology allowed rational designof antigen and introduction of the concept of“reverse vaccinology”. Other face of rational design,“quality by design”, brought benefits in theproduction technology. Last but not least, thedevelopment of adjuvant - an old concept but amodern technology, complements vaccinedevelopment. Cantacuzino National Institute ofResearch approached several of the modern conceptsin vaccine research that are described in thispresentation.

CP9. VACCINE DEVELOPMENT BETWEEN NEW AND OLD CONCEPTS

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Lia Mara Diþuuniversity of bucharest, Faculty of biology – Microbiology & immunology lab., romania

The gastrointestinal tract is a complexecosystem based on the interactions between theepithelium, immune cells and resident microbiota.The three components of the ecosystem have co-evolved, and are interrelated in their physiologicalactivity. Each member of the gastrointestinalecosystem can have a predetermined development,even in the absence of other components as revealedby studies on germ-free animals.

The purpose of this paper is to present someaspects related to the influence of probiotic andparaprobiotic components on the normalfunctionality of the immune system. Probiotics aredefined as live microorganisms that, whenadministered in adequate amount, confer a healthbenefit to the host. Amongst the many benefitsassociated with the consumption of probiotics,modulation of the immune system has received themost attention. Several animal and human studieshave provided unequivocal evidence that specific

strains of probiotics are able to stimulate as well asregulate several aspects of natural and acquiredimmune responses. Different probiotic strains varyin their ability to modulate the immune system andtherefore efficacy of each strain needs to be carefullydemonstrated through rigorously designedexperiments. Our preliminary study suggests that gutmicrobiota, implicit probiotic bacteria, producemolecules that interfere with cytokine signalingpathways of the host organism, suggesting theirinvolvement in the modulation of the host anti-infectious defense ability, mediated by cytokinesable to activate the nonspecific (IL-1, IL-6) orspecific (IFNγ) immune reactions, including:attenuation of the inflammatory response byincreasing IL-6 (which induces acute phase proteinsynthesis); activation of a specific cellular immuneresponse reflected in increasing levels of IFN γ;limiting the inflammatory bowel lesions by loweringthe IL-1α level.

W1. PROBIOTICS AND PARAPROBIOTICS – IMMUNOMODULATORyEFFECTS AND HEALTH BENEFITS

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Patricia Mihãilescu1, Ionica Ceauş1, Claudia Istrate1, Carmen-Michaela Creþu1,2

1Eco-para-Diagnostic srl, bucharest, romania; 2carol Davila university of Medicine and pharmacy, bucharest, romania

Introduction: Parasitic organisms have beenknown since the most ancient times and mentionedby famous medical doctors like Hippocrates. Out ofthem, the most important in terms of significantmaterial and social damages are nematodes andcestodes. The first ones are highly polymorphic andinfest various living environments. Toxocara spp.and Trichinella spiralis, once entered in contact withthe human organism, are causing serious pathologicconditions (e.g., toxocariasis - larva migransvisceralis / ocularis and trichinelosis). Anotherimportant class is Cestoda (fam.Taeniidae) with thevery important representatives Taenia solium andEchinococcus granulosus which cause the two veryserious human diseases: cysticercosis (complicationof Taenia solium infection) and cysticechinococcosis (hydatidosis).

Material and Methods: We performed aretrospective study in the period 01.01.2013 -07.31.2015, in which were included patientsadmitted to Eco-Para-Diagnostics SRL medicalcenter in Bucharest for the following investigations:1598 serum samples tested for specific antibodiesagainst Toxocara canis; 266 for Trichinella spiralis;171 for Taenia solium; 1345 for Echinococcusgranulosus specific antibodies. For these tests wereused commercial diagnostic kits and screeningmethods as ELISA and Western Blot confirmation.

Results: For Toxocara, out of the 1598 serumsamples 1158 were tested by ELISA, and 440confirmed by Western Blot (WB). Of those tested byWB, 155 (35.22%) were positive and 285 (64.78%)negative. Sex distribution was 50.90% men and49.10% women aged between 1-76 years. ForTrichinella spiralis, out of the 266 serum samples216 by ELISA and 50 by WB. ELISA revealed 41

(18.98%) positive samples and 175 (81.02%)negative. From the samples tested by WB, 17 (34%)were positive and 33 (66%) negative. Regarding thedistribution of genders and age groups: 56% weremen, 44% women, aged between 3-62 years. In caseof Taenia solium, from the 98 serum samples testedby ELISA 2.04% were positive and the rest of97.96% negative. From the 73 WB samples 4.10%were positive and 95.9% negative. 32.24% ofpatients were men, 65.76% women, the patientsbeing aged between 1-88 years. ConcerningEchinococcus granulosus, from 1335 serum samples916 were tested by ELISA and 419 by WB. Of theWB tested patients 96 (22.9%) were positive and323 (77.08%) negative. Of the 94 patients (positivein WB), 49 were ELISA positive (52.13%), 19(20.22%), ELISA negative and the remaining 26(27.65%) were not tested. Also 27 cystic materialsamples were examined for testing protoscolexviability, 25 being positive and two negative.Samples were collected during surgery from 25patients (19 women, eight men, aged between 5-74years).

Conclusions: The serology methods for thediagnosis of parasitic diseases are particularlyimportant, especially the Western Blot method(confirmation method with increased sensitivity andspecificity) which is important especially forconfirming the true negative results obtained byELISA, obtained for patients with characteristicimaging diagnosis and clinical symptoms. Acorroboration among several methods of diagnosis,i.e.: serological (ELISA, WB), imaging diagnosisand patient history is needed for an accuratediagnosis.

W2. SEROLOGICAL DIAGNOSIS IN PARASITIC DISEASES: TOXOCAROSIS,TRICHINELLOSIS, CySTICERCOSIS AND CySTIC ECHINOCOCCOSIS

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Lyme disease is the most common infectiontransmitted by ticks in the northern hemisphere andis predominant in temperate regions of NorthAmerica, Europe and Asia. It is a multisystemdisease, inflammatory, determined by the immuneresponse to pathogenic genospecies for humans ofthe complex Borrelia burgdorferi sensu lato (s. l.),which in Europe are transmitted by the bite of Ixodesricinus ticks.

Lyme borreliosis is an inflammatory disease thataffects multiple organ systems: nervous andcardiovascular systems, joints and muscles, but thepathogenesis mechanisms are not completelyunderstood. Borrelia has the ability to invade localtissues and to escape from the host immune defense

mechanisms for a long period of time despite thedevelopment of antibodies in serum and other bodyfluids. Borrelia uses several mechanisms for evasionof immune responses and causes persistentinfections: (1) inactivation of the complementcascade, (2) phase and antigenic variation and (3)the invasion of protective immunological niches.

The infection with B. burgdorferi stimulates thesynthesis of antibodies with a unique and highlyspecific function.

Currently the laboratory diagnosis of variousstages of Lyme disease is based on dynamicevaluation of specific antibodies IgM and IgG byELISA and Western blot assays carried out insuccessive phases.

W3. THE PARTICULARITIES OF THE IMMUNE RESPONSE IN LyME DISEASE

Ani Ioana Cotar, Daniela Bãdescucantacuzino National institute of research, laboratory for vector-borne infections, bucharest, romania

According to international scientific literaturedata, Drosophila melanogaster experimental modelis used to analyze the underlying mechanisms of theimmune response.

These mechanisms can be elucidated by thestudy of experimental infections induced by variousbacterial species of interest such as Pseudomonasaeruginosa and Escherichia coli.

The two methods of inducing experimentalinfections in Drosophila melanogaster are the

pricking and the method of ingestion of bacterialcells, enabling the further analysis of the target genesand extrapolating the results to other mammalianmodels, including humans.

The results obtained in our laboratory haveshown that the effects caused by P. aeruginosainfection given by ingestion are less severe thanthose caused by the infection consecutive to prickingand revealed significant differeces in the expressionlevel of some genes of interest.

W4. UTILITy OF DrosopHila MElaNoGastEr EXPERIMENTAL MODEL FOR THE IN VIVO STUDy OF HOST-INFECTIOUS AGENTS INTERRELATIONS

Mihaela Raluca Mihalache (Radu)1, Alina Neagu1, Veronica Lazãr2,3

1university of bucharest, Faculty of biology, Department of botany and Microbiology, bucharest, romania;2university of bucharest, Faculty of biology, Department of Genetics, bucharest, romania;

3research institute of the university of bucharest - icub, university of bucharest, bucharest, romania

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Gabriela Elena Androsiacsc. Novaintermed, romania

Multicolor flow cytometry is a powerful tool forthe characterization of various cell types using alarge portfolio of conjugated antibodies directedagainst surface, intracellular and secreted markers.

Thus, for a better optimization, some importantaspects should be taken into account, while trying todesign a multicolor panel. Antigen density and itsco-expression on a cell, the correct assignment of

fluorochromes to target antigens and theexperimental controls are able to improve resolutionand to minimize the spillover effects into thepopulation of interest.

The following presentation will guide theresearchers through the rules that should berespected for the optimized selection of multicolorflow cytometry panels.

Flow cytometry has evolved from a laboratorytechnique to a routine tool used in the diagnosis,prognosis and monitoring of hematologicalmalignancies, immune status in patients with cancer,immune deficiency, and allogeneic stem cell andorgan transplants.

Flow cytometry analysis can identify with highaccuracy the distinctive immunophenotype ofmalignancies to select specific therapies and topredict disease evolution. For example, variousCD20+ B cell malignancies are now treated withrituximab (anti-CD20 antibodies) and a subset ofCD52-expressing T cell and B cell malignanciesmay be treated with alemtuzumab (anti-CD52antibodies). Additionally, the screening of residualmalignant cells after oncological therapy is a currentpractice in MRD monitoring.

The flow cytometer also serves as clinicallaboratory platform for assessing the immune statusincluding determination of CD4+ T cell blood counts

after HIV infection as well as classification andprognostication of various primary immunedeficiencies, such as autoimmunelymphoproliferative syndrome or severe combinedimmune deficiency. Flow cytometric quantificationof specific immune cell subsets in an allogeneic stemcell graft and of immune reconstitution followingallogeneic bone marrow transplantation serves aspredictors of survival posttransplant.

Flow cytometry can also be used for cell therapyapproaches to quantify the extremely rare CD34+endothelial progenitors in cord blood. Last but notthe least, flow cytometry might offer a flexibleplatform for liquid biopsy approach in patients withepithelial cancers.

All of these translational applications of flowcytometry in the clinical practice became reality dueto the innovative approaches in the reagentsdevelopment that assured standardization andreproducibility.

W6. FLOW CyTOMETRy APPLICATIONS FOR CURRENT CLINICAL PRACTICE

Bianca Gãlãþeanu1, Ariana hudiþã1, Mirela Şerban1, Carolina Negrei2, Codruþa Vagu3, C. Şerban3, Anca Gheorghe3, Marieta Costache1,

1Department of biochemistry and Molecular biology, university of bucharest, bucharest, romania;2Faculty of pharmacy, carol Davila university of Medicine and pharmacy, bucharest, romania;

3Fundeni clinical institute, laboratory of Hematological analysis specialized for Medullar transplant, bucharest, romania

W5. NEW INSIGHTS FOR MULTICOLOR PANEL DESIGN

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Mihaela Surcel1,2, Radu-Ionuþ huicã1,3, Adriana Narcisa Munteanu1,Ioana Ruxandra Pîrvu1, Dan Ciotaru1, Cornel Ursaciuc1

1Dept. immunology, Victor babeş NirD, bucharest, romania; 2Faculty of biology, university of bucharest, romania; 3carol Davila uMph, bucharest, romania

B lymphocytes are involved in the humoralimmune response, causing the antibody secretionafter contact with antigen (Ag). Activation and blasttransformation of B lymphocytes results in antibodysecreting plasma cells. The secretedimmunoglobulin (Ig) are identical as specificity withAg-specific Ig receptor on activated B-cellmembrane.

It can be determined in peripheral blood morecategories of B cells CD45+CD19+CD20+following the developments or contact Ag: immature(transitional) - CD10+sIg++, naive (which did notprime with Ag) – sIg+, memory (results after contactwith Ag and specific humoral immune response) -CD27+sIg+/-, plasma cells (in the course of passagefrom the lymph nodes to bone marrow) -CD38+++sIg-. These categories have certain rangesof values, varying with age and disease situation.There are some subpopulations derived from theabove (“border B”) and may occur temporarily inperipheral blood according to the conditions of theimmune response: activated B lymphocytes(CD10+CD21+) and regulatory B cells(CD24+CD38+). Subpopulation identification wascarried out by an 8-color method at FACSCanto II

cytometer (BD) after processing cells with labeledmonoclonal antibodies: antiCD45-BV510, anti-CD19-PerCP-Cy5.5, antiCD20-APC-H7,antiIgD-FITC, antiIgM-APC, antiCD10-PE-Cy7,antiCD27-BV421, antiCD38-PE.

The results showed variations between Bsubpopulations shared in adults and children. Totalnaive and immature B cells percentages were higherin children, while plasma cells and memory Blymphocytes were higher in adults.

Evaluation of B lymphocyte subpopulation ofperipheral blood is useful mainly in B cell secondaryimmunodeficiency disorders (ID), infectioussyndromes, autoimmune diseases, type II diabetesmelitus, graft-versus-host syndrome. The primary ID(humoral or combined) and B lymphocyteproliferation (leukemias, lymphomas,myeloproliferative syndrome) are not recommendedfor determination as described above, since it doesnot provide suggestive indications because of majordisturbance in distribution, markers and categoriesof peripheral B lymphocytes.

Work supported by grant of the RomanianNational Authority for Scientific Research PN16.22.03.04.

W7. MULTICOLOR ASSESSMENT OF B LyMPHOCyTE SUBPOPULATIONS IN PERIPHERAL BLOOD

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The optimal treatment of end–stage organfailure is transplantation, since it enhances bothquality of life and survival. Immunology hasprovided a rationale for the development of clinicaltransplantation. The scientific basis oftransplantation means the understanding of themolecular and cellular events which lead to graftrejection or acceptance. Matching HLA alleles andminor histocompatibility complex between donorsand recipients prevents rejection episodes.Differences between HLA alleles stimulate rejection.

Typing MHC alleles polymorphisms andrevealing their role in immune recognition is theevery day challenge of transplant immunologists.Also the clinical relevance of HLA polymorphismsis specifically assessed for each type oftransplantation.

Our evidence-based experience in solid organtransplantation kidney, liver and heart) at Fundeni

Clinical Institute shows that both completeimmunological assessment of the recipient priortransplantation and careful follow up are the keys tosuccessful outcome of the transplanted patient.Clinical examples of evidence-based case reports aregiven within this lecture. Molecular high resolutiontissue typing used in Fundeni Clinical Instituteallows detailed and comprehensive HLA results sothat the best matched donor is always chosen.

In hematopoietic stem cells transplantationHLA matching at eight digits is very important andactually dictates the clinical status post transplanta-tion. In bone marrow transplantation, not only MHCis essential to be perfectly matched but also theminor histocompatibility complex is important to bematched. Graft versus host disease could be causedonly by a limited number of mismatched minor HLA alleles. Transplant immunology is complex andchallenging but this is the beauty of it!

W8. TRANSPLANT IMMUNOLOGy IN SOLID ORGAN TRANSPLANTATIONAND HEMATOPOIETIC STEM CELLS TRANSPLANTATION

Ileana Constantinescu1,2

1carol Davila university of Medicine and pharmacy, bucharest, romania;2Fundeni clinical institute, bucharest, romania

When an HLA-matched donor is not availablewithin the family in the national register of volunteerunrelated donors could be found a matched donorfor the patient.

Our Romanian volunteer donor register forperipheral hematopoietic stem cells has improvedsignificantly the access to bone marrowtransplantation for many patients diagnosed with awide range of haematological malignancies.

Our debate concentrates on the challenge offinding a suitable HLA matched donor withinRomanian families and the need of more extensivetesting in all the regions of our country.

We have notified some problems based on ourexperience: the lack of policies concerning HLAtyping and testing strategies; the lack of donorassessment, follow up and outcome; in our countrythere are no cord blood banks developed yet.

W9. WHAT IS THE IMPACT ON BONE MARROW TRANSPLANTATION OF THE ROMANIAN VOLUNTEER DONOR REGISTER

FOR PERIPHERAL HEMATOPOIETIC STEM CELLS?

Ana Moise1,2, Daniela Nedelcu2, Mihaela Sora2, Adela Toader2, Larisa Ursu2, Ileana Constantinescu1,2

1carol Davila university of Medicine and pharmacy, bucharest, romania;2centre for immunogenetics and Virology, Fundeni clinical institute, bucharest, romania

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WoRKShoPS

Daniela Nedelcu2, Ana Moise1,2, Adela Toader2, Mihaela Sora2, Larisa Ursu2, Ileana Constantinescu1,2

1carol Davila university of Medicine and pharmacy, bucharest, romania;2centre of immunogenetics and Virology, Fundeni clinical institute, bucharest, romania

Long-term immunosuppression helps to preventgraft loss but it can often be associated withworsening of pre-existing conditions or newcomplications. Close monitoring of calcineurininhibitors levels is mandatory for improving qualityof life and post transplant survival rate. Calcineurininhibitors remain the corner stone of mostimmunosuppressive regimens. Tacrolimus is theagent of choice in our transplant centre at FundeniClinical Institute. Several studies have documentedits advantage over cyclosporine in preventing acuterejection, promoting allograft function and reducingcardiovascular factors (hypertension andhyperlipidemia). Along with the calcineurin

inhibitors it is common the use of corticosteroids,sirolimus, everolimus, antithymocyte globulin,daclizumab, basiliximab, alemtuzumab.

Some new immunosuppressive drugs are underclinical trials: betalacept, alefacept, CP690, 550 (aJAK 3 kinase inhibitor), AEB071 (a protein kinaseC inhibitor).

There are four issues of debates: inductiontherapy (ATG, alemtuzumab); corticosteroid freeregimens; minimizing adverse reactions; newimmunosuppressive drugs.

We present our experience and our opinionrelated to immunosuppression both in solid organtransplantation and in bone marrow transplantation.

W10. IMMUNOSUPPRESSIVE DRUGS: WHICH WAy TO GO?

HLA alleles’ matching is crucial for solid organtransplantation but there are also other essentialfactors equally important for the prevention of acuterejection.Recipient selection criteria are essential.The management of immunized recipient iscompletely lacking and this aspect affects theoutcome post transplantation. The problem of denovo anti HLA antibodies is also a challenge.

Selection of immunosuppressive drug regimenscould be problematic in some cases and should bemore personalized according to the needs of eachpatient.

Other issues in debate are: avoidance of positivecrosshatches, HLA typing strategies, screeningstrategies for anti –HLA antibodies.

In our experience, evidence based reviews haverevealed the effectiveness of HLA matching inimproving outcomes. But HLA polymorphisms arenot always clinically relevant for a specific type oftransplantation.

Therefore HLA matching is not the only factorin preventing acute rejection episodes posttransplantation.

W11. IS HIGH RESOLUTION HLA TyPING ABLE TO RESOLVE ACUTEREJECTION EPISODES POST SOLID ORGAN TRANSPLANTATION?

Larisa Ursu1, Ana Moise1,2, Mihaela Sora2, Adela Toader2, Ileana Constantinescu1,2



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Cancer represents a major cause of morbidityand mortality for renal transplant recipients. Theaverage of cancer onset at ten years post renaltransplantation is about 15 %.

There are several issues mentioned in thisdebate: the aspects related to pre-existing cancers;transmission of cancer from donors; de novocancers; immunosuppressive therapy; geneticdeterminants of renal transplantation outcome.

At the moment we do tumour marker screeningin order to prevent de novo cancers but also thetransmission of cancer from donors.

Immunosuppressive therapy is one of the mostimportant causative factors. The risk of tumour isclearly related to the intensity of

immunosuppression. Post transplant neoplasiasincrease with time. Viral infections can favourneoplastic tissue development and can be involvedin a number of different types of cancers (mostlymph proliferative disorders are B cells lymphomaand are caused by EBV).

Genetic predispositions have been found to beassociated with a higher incidence of skin cancer.

Epigenetic and whole genome sequencing couldprovide more in-depth knowledge of the geneticinfluences on renal transplant outcome.

Long term renal allograft survival has notchanged over the last decade and there is still a lotof scientific work to do in order to prevent canceronset post transplantation.

W12. ARE WE ABLE TO PREVENT NEOPLASIAS POST KIDNEy TRANSPLANTATION?

Ileana Constantinescu1,2, Ana Moise1,2, Larisa Ursu2


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oRAL PRESENTATIoNS

oRAL PRESENTATIoNS

Lucian Pop1, Victor Cristea1,2

1sf. iosif Medical center, cluj-Napoca, romania; 2iuliu Haþieganu university of Medicine, cluj-Napoca, romania

Cytokines are a group of heterogeneous molecules(about 100 types), with molecular weight between 15and 30 kDa, of glycoprotein or protein nature, whichare secreted by a cell and binds to specific, high-affinity, membrane receptors, displayed on the surfaceof other cells or even on the cell that has secreted them.Within the immune response, cytokines are recognizedas mediators of immunity, inflammation, proliferationand differentiation of cell lines.

In a series of scientific papers (parts of completedPhD theses) we have tried, since 1997, to see what is

the behavior of the main pro and anti-inflammatorycytokines in the onset and progress of diseases and iftheir activity is different in the control groups.

Studies have been conducted on patients withchronic hepatitis B or C, cirrhosis (versus non alcoholicsteatohepatitis), atopy and chronic urticaria, allergicrhinitis, periodontal disease, breast cancer, rheumatoidarthritis, bullous pemphigoid, septic shock, anestheticshock. The most significant results are presented anddiscussed in order to enrich the disease diagnosisthrough the use of these new “markers”.

PO1. MOLECULAR MEDIATORS OF THE IMMUNE RESPONSE

Ana-Maria Enciu1,2, Maria Dudãu1,2, Elena Codrici1, Daniela Ionela Popescu1, Simona Mihai1, Laurenþiu Anghelache1,3, Radu Albulescu1,4, Cristiana Tãnase1,5

1Victor babeş NirD, bucharest, romania; 2carol Davila uMph, bucharest, romania; 3usaMV bucharest, romania;4iccF bucharest, romania; 5titu Maiorescu university, bucharest, romania

Introduction: amyloid precursor protein (APP) isthe central component of the amyloid cascade - themain pathogenic link of Alzheimer’s disease. Itsphysiological role is not well known, since researchefforts are mainly concentrated on the pathologicenzymatic cleavage of the protein. Recently, presenceof dimers was highlighted in cell cultures transfectedwith APP. To date, there is no data on the existence ofthese dimers in tissues.

Aim: Identification of APP dimers or oligomersin cerebellar cortex of normal laboratory mice, withoutknown central nervous system pathology.

Material and Methods: Biological material:cerebellum cortex, from C57BL6 male mice, agebetween 3 and 9 months. Methods: ultracentrifugationand separation of cell membranes, protein extraction,non-denaturing electrophoresis followed by transferand immunoblotting.

Results: Using standardized protocols for non-reducing SDS PAGE electrophoresis (which preservesdisulphide bonds) we could not reveal the presence of

dimers reported in the literature to occur in in vitrosystems. However, we were able to obtain a protocoloptimized for isolation, extraction, electrophoresis andimmunoblotting of molecular complexes containingAPP. When used on cerebellar cortex, this workingprotocol identified a macromolecular complex, of about600 kDa, 3 times higher than the expected dimer size.

Conclusions: Using an adapted protocol of non-denaturing electrophoresis, we have identified an APPbased macromolecular complex. The next steps are toidentify the composition of this protein complex, to seewhether it is formed of APP oligomers or it is aheterocomplex. Special emphasis will be put oninvestigating the existence of specific membranecoating proteins, such as caveolin-1 in the compositionof this complex.

Acknowledgement: This work was supported bya grant of the Romanian National Authority forScientific Research and Innovation CNCS-UEFISCDI,project number PN-II-RU-TE-2014-4-1534 and PN16.22.04.01.

PO2. A NON DENATURING ELECTROPHORETIC METHOD FORIDENTIFICATION OF OLIGOMERIC FORMS OF AMyLOID PRECURSOR

PROTEIN IN THE CEREBELLAR CORTEX OF LABORATORy MICE


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Introduction: The NK cells and some Tlymphocytes express killer Ig-like receptors (KIR).Some of these receptors play activatory roles, othersare inhibitory receptors. The number of Ig-likedomains and the size of their cytoplasmic tailscontribute to the receptor’s denomination. 16 humanKIR genes are known so far, as well as a number ofallelic variants. KIR2DL4, KIR3DL2, KIR3DL3and KIR3DP1 are considered the so called“framework genes” as they are present practically inall individuals.

Materials and Methods: A hundred non-related individuals were characterized for their KIRgenotypes using Sequence Specific Primers (SSP)from four different manufacturers: BAG, Olerup,InnoTrain and Invitrogen. All these subjects werehealthy volunteers from various counties of Moldova(mostly Bacău and Iaşi), who registered as donorsfor the Romanian Stem Cell National Registry.

Results: The KIR genotypes that we were ableto characterize allowed us to calculate and furtheradjust the frequencies of these genes in theRomanian population. Furthermore, a particulargenotype could be evidenced: 2DL2; 2DL3; 2DL5B;2DS1; 2DS2; 2DS3; 3DL2; 3DL3; 2DP1; 3DP1,which, remarkably, lacks 2DL4. This result wasconfirmed with kits from three distinctmanufacturers.

Conclusions: The www.allelefrequencies.netdatabase reports only ten KIR2DL4-negativegenotypes, which demonstrates a very strongpositive selection favoring the KIR frameworkgenes. The individual we were able to characterizeis thus one of the very few reported worldwide.Furthermore, the data generated by this studyprovided a more accurate image upon KIR genes andgenotypes frequencies in Romania.

PO3. KIR2DL4 NEGATIVE GENOTyPE – A VERy RARE FEATURE

Daniela Constantinescu1,2, Corina Cianga1,2, Camelia Mihãilã2, Petru Cianga1,2

1immunology Department, Grigore t. popa university of Medicine and pharmacy iaşi, romania;2laboratory of immunology and Genetics, st. spiridon Hospital, iaşi, romania

The green barley total extract Natural SOD waspatented by Cantacuzino Institute during the 80s asa food supplement with antioxidant (SOD-like andPeroxidase-like) activity.

Aiming to identify and assess the scavengingproperties of our product, we tested the antioxidantcapacity against peroxyl and DPPH radicals (ORACand DPPH methods).

We also demonstrated that the methods we usedwere suitable for a more complex and more rigorousquality control protocol (including acceptanceranges).

The obtained data offer information about thequality of the raw material, the influence oftechnological steps on antioxidant capacity and thevariability between different batches. Stabilitystudies were performed in order to establish theoptimal conditions for packaging and storage. Ourresults, in correlation with previous data, contribute

to further studies aiming to demonstrate thebioavailability of antioxidant compounds in NaturalSOD product (extract – gut microbiota interaction,absorbtion and metabolization pathways) as well asother beneficial compounds (vitamins,oligoelements).

Its complex antioxidant capacity (bothenzymatic-like and scavenger) and other potentialbeneficial effects recommend the total extractNatural SOD as a high-quality antioxidantsupplement, an efficient energizer and a potentialanti-inflammatory agent.

Green barley extracts (e.g. Natural SOD),traditionally used to maintain and improve thehealthy state, could be the main ingredient of newproducts with complex effects (such as antioxidantand hepatoprotective activities).

This work was supported by PN16390204/2016.

PO4. NATURAL SOD PRODUCED By CANTACUzINO INSTITUTE: FROM TRADITION TO INNOVATION

Andreea-Roxana Lupu1,2, Lidia Cremer1, Adrian onu1, Cristina Cercel3

1cantacuzino Nir, immunomodulation Group, bucharest, romania; 2Victor babeş NirD, immunology laboratory,bucharest, romania; 3university of bucharest, Faculty of biology, bucharest, romania

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Single-cell analysis has been called “the newfrontier in Omics” [Wang et al., 2010]. Accuratecharacterization of samples with high cellularheterogeneity and its effect in complex phenomenasuch as stem cell differentiation and cancerdevelopment may only be achieved by analyzingsingle cells. Large-scale whole transcriptome studiescan be performed to survey heterogeneity anddiscover novel cell populations. The technologyrelies on the separation of cells from a cellularsuspension in a microfluidic system after they wereselected either by microaspiration, flow cytometryor laser capture microdissection. Separated cells arefurther automatically lysed and the content of eachsingle-cell is released for analysis in an individualreaction chamber. Single-cell technology consists ofa unique approach that allows the analysis of DNA,RNAs (mRNA, miRNA, ncRNA) or proteins in thesame single cell. Particularly for single-cell geneexpression profiling, end-point application is RT-qPCR.

The immune system is an extremely complexnetwork of cells with a vast number of interactionsamong them. Immune monitoring evaluates thetypes and functions of the immune cells involved inthe evolution of an immune response (activation and

differentiation of different cells subsets, includingmacrophages, T cells, B cells, etc). To address thechallenges for single-cell analysis in immunemonitoring, single-cell modern technology is neededto (1) facilitate the isolation of single cells, (2)identify phenotypical and functional variations, (3)define cell-cell specific interactions, (4) enablemultidimensional analysis and quantitativeextraction of information. For functional systemsimmunology, single-cell measurements would helpdefine statistical network models that may improvethe knowledge of specific mechanisms that governthe human response to disease [Yalcin et al., 2015].Additionally, the transcriptomics and genomics ofantigen-specific T cells can be more definitivelyelucidated. Single-cell technology should providenew resolution for defining signatures of immuneresponses to pathogens and therapeuticinterventions.

Selected references:[1] Wang D., Bodovitz S., Trends Biotechnol.

28 (2010) 281–290. [2] Yalcin A., Yamanaka Y.J., Love J. C., Single-

Cell Analysis, Methods in Molecular Biology 853(2012) 211-230.

PO5. SINGLE-CELL TECHNOLOGy - PRINCIPLE AND APPLICATIONS. IMPORTANCE AND CONTRIBUTION FOR IMMUNOLOGy

Sorina Dinescu, Marieta CostacheDepartment of biochemistry and Molecular biology, university of bucharest, romania


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Introduction: Cancer therapeutic strategies arebased on the targeted cytotoxic therapies as well as theown resources of the organism by modulating anti-tumorimmune response.

Materials and methods: We used 8-10 weeks oldwild type C57BL/6 mice. We employed animal models ofB16F10 mouse melanoma and also cutaneous carcinoma.Mice were monitored every week to evaluate tumorgrowth. On day 21, after tumor cell inoculation,populations and sub-populations of lymphocytes (CD3,CD4, CD8, CD19, NK1.1) were evaluated by flowcytometry.

Results: Evaluation of peripheral blood lymphocytepopulations such as the spleen and tumor-bearing miceshowed alteration of their distribution in comparison tohealthy mice. A significant decrease in the percentage ofT lymphocytes (CD4+ and CD8+) and NK cells wasobserved in peripheral blood of carcinoma-bearing mice.These changes were accompanied by a significantincrease in the percentage of B cells. On the other hand,in melanoma-bearing mice, we found only a significantdecrease in the percentage of NK cells and TCD4+

lymphocytes. In the spleen, in the cases of both tumortypes, a significant decrease of NK cells and Blymphocytes percentage was observed. Moreover, astatistically significant increasing of the percentage ofTCD4+ lymphocytes was documented. For TCD8+lymphocytes, we found only a statistically significantpercentage reduction in carcinoma-bearing mice.

Conclusions: Lymphocyte populations aredifferently distributed in peripheral blood and spleen oftumor-bearing mice. Tumors induce the decrease in thepercentage of T lymphocytes and NK cells in theperipheral blood, but induce the increasing in thepercentage of TCD4+ lymphocytes in the spleen. Theseare possible due to TCD4+ lymphocytes sequestration insecondary lymphoid organs.

Moreover, at least numerically, a compensatorymechanism between TCD4+ and B lymphocytes has beenrevealed.

Work partially supported by the grant of theRomanian National Authority for Scientific Research PN16.22.04.04.

PO6. ANALySIS OF LyMPHOCyTE POPULATIONSFROM TUMOR-BEARING MICE

Gheorghiþa Isvoranu1, Bogdan Marinescu1, Laurenþiu Anghelache1, Mihaela Surcel1,2, Cornel Ursaciuc1, Cãtãlin Gabriel Manole1, Gina Manda1

1Victor babeş National institute of pathology, animal Husbandry, bucharest, romania;2university of bucharest, Faculty of biology, bucharest, romania

Introduction: Proteomics studies from differentfluids of patients with systemic sclerosis identified anincreased number of candidate proteomic biomarker forthis disease. Among these, reticulocalbin family memberswere identified. They are chaperons associated with theprotein synthesis apparatus of the fibroblast. The aim ofthe study was to confirm upon an independent cohort ofsystemic sclerosis patients reticulocalbin 1 and 3 asbiomarkers for systemic sclerosis.

Material and methods: 40 patients with systemicsclerosis were recruited. Alongside 20 healthy controls.Serum levels of reticulocalbin 1 and 3 were analyzedusing commercial ELISA kits.

Results: Reticulocalbin 1 and 3 were identified inserum of both healthy controls and systemic sclerosis

patients. Moreover, serum levels of reticulocalbin 1 and3 were correlated. Although there was not a statisticallysignificant difference between serum levels ofreticulocalbins between healthy controls and systemicsclerosis patients there was a subgroup of six patients thathad higher serum reticulocalbin levels. They share asimilar pattern, having higher prevalence of digital ulcers,telangiectasias, calcinosis cutis, esophageal hypomotilityand diffuse subset of disease.

Conclusions: Reticulocalbins were confirmed asbiomarkers for systemic sclerosis. They seem to be usefulespecially for patient stratification rather than for thediagnosis of systemic sclerosis.

PO7. CONFIRMATION OF RETICULOCALBIN 1 AND 3 AS PROTEOMIC BIOMARKERS FOR SySTEMIC SCLEROSIS

Paul Bãlãnescu1,2, Anca Bãlãnescu1,3, Eugenia Bãlãnescu2, Cristian Bãicuş1,2, Gheorghe Andrei Dan1,4

1carol Davila uMph, bucharest, romania; 2immunology laboratory, colentina clinical Hospital, bucharest, romania; 3alessandrescu-rusescu iNsMc, bucharest, romania;

4internal Medicine Department, colentina clinical Hospital, bucharest, romania

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Recent data reveal the fact that between the twopathophysiological states - inflammation and chronicdiseases such as cancer - there is a symbioticrelationship, which involves mechanisms forconfounding, such as oxidative stress, hypoxia,expression of transcription factors / in particularnuclear factor B (NF-kB) /, the presence ofnumerous factors such as chemokines, cytokines,growth factors, prostaglandins, angiogenic factors,etc. Apart from genetic predisposition, epigeneticchanges accumulated in the body throughout lifeinvolve major effort to ensure the body’shomeostasis.

In individuals with inflammatory, immunesystem it is disturbed and becomes persistentinflammatory response, involving many types ofimmune and non-immune cells. Basal inflammationcan be induced by several factors, such as: a) thereactivation of latent viral infections; b) Toll-likereceptor expression decreased with reduced capacityfor recognition of pathogens (PRRS); d) decreasedability of immune cells migration; f) increasedoxidative stress; g) adaptive immunity decline; h)deficiencies in the production of cytokines, growthfactors, with the shift from Th1 to Th2 cytokineprofile and especially i) altering of the interactionbetween mesenchymal stem cell and the immunesystem. Prolonged, professional to asbestos,constantly associated with functional morpho-functional changes in the lung, comes witharguments in this regard. The purpose of this paperis to demonstrate the protective effect of thegemmotherapy remedies Buxus and Thuia on the

immune and oxidative response, altered by chronicexposure to asbestos. In vivo experiment wasperformed on 100 rats, Wistar line, divided into thefollowing groups: 1) Control-group (C); 2) Buxus +Thuia –group (B + T); 3) Asbestos group (As); 4)As+B+T- group. Asbestos was administered byintratracheal instillation. B + T were administeredorally. A half of each group was sacrificed after 30days and the other part after 180 days.Bronchoalveolar lavage was performed to achievealveolar macrophages (Ma). To investigate theimmune response, the preparation of spleenlymphocytes (Ls) and their cocultivation with Mawere carried out. The following parameters wereevaluated: 1. Incorporating 3HTdR- test; 2. IL-1-test; 3. TNF-test; 4. Chemiluminescence test for freeoxygen species; 5. Phagocytosis assay.Administration of the As was associated with alteredimmune and oxidative response and after 180 days,histo -pathological changes, respectively, atypicalcells were pointed out, that indicate development ofa lung cancerous process. Partial reversibility of theparameters in the B + T + As - group suggests aprotective effect of the gemmotherapy remedies.

In conclusion, inflammatory and immunologicalreactions play an important role in cancerdevelopment. An effective treatment must interfereseveral of the patho-physiological pathways, that gointo the development of pathological process.Prevention and early treatment of asbestos-inducedinjury in people at high risk consist in a complex andalternative treatment, that has to includegemmotherapy and a proper diet.

PO8. INFLAMMATION AND CANCER, PATHOPHySIOLOGICALSTATES WITH INTRICATE MECHANISMS, IN WHICH THE IMMUNE RESPONSE IS CENTRAL. BENEFICIAL EFFECT OF GEMMOTHERAPy

Didi Surcel1, M. Surcel2, S. Toader2, M. Butan3, Simona Niþu4, Carmen Ponoran4

1blue life Medical center, cluj-Napoca, romania; 2iuliu Haþieganu university of Medicine, cluj-Napoca, romania;3center of the public Health, cluj-Napoca, romania; 4plant Extract, cluj-Napoca, romania


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IgG4-related disease (IgG4-RD) is a rare systemicfibro-inflammatory disorder. The evaluation for IgG4-RDshould include a comprehensive clinical history, physicalexamination, and selected laboratory investigation, alongwith appropriate imagistic studies. A wide variety oforgans can be involved in IgG4-RD. Confirmation of thediagnosis by biopsy is important for the exclusion ofmalignancy and other disorders that may mimic IgG4-RD.The serum IgG4 level is generally elevated above theupper limit of normal (>135 mg/dL) and tends to increasewith the number of organs involved and to decrease aftertreatment with glucocorticoids. The hallmarks of IgG4-RD are lymphoplasmacytic tissue infiltration of mainlyIgG4-positive plasma cells and small lymphocytes, whichmay be accompanied by fibrosis, obliterative phlebitis,and, in the majority of patients, elevated serum levels ofIgG4. IgG4 does not bind complement and therefore doesnot generate a significant inflammation. IgG4 can bind toFcy receptor I (CD64), which presents on monocytes,

macrophages and neutrophils. IgG4 production iscontrolled by T helper 2 cells (interleukins 4 and 13) thatmediate allergic responses and IgE production. Findingsconsistent with both an autoimmune disorder and anallergic disorder are present. The contribution ofautoantibodies to IgG4-RD remains unclear. IgG4-RD hasfeatures of an allergic disorder: abnormal high Th2cytokines (cell messenger proteins) in tissues, raised IgEand increased T-reg lymphocytes in blood and raisedeosinophil count in 40% of patients. IL-10 and TGF-β areknown to support IgG4 production and are at elevatedlevels in IgG4-RD. Increased tissue levels of Th2-cytokines (IL-4, IL-5 and IL-13) were found in IgG4-RDwhich is characterized by both systemic and localizedIgG4 production. It is unclear whether these antibodiesare acting as pathogenic antibodies or are just a bystanderphenomenon. Glucocorticoids, azathioprine,mycophenolate mofetil, methotrexate and rituximab aretherapeutical options.

PO9. THE SPECTRUM OF IGG4-RELATED DISEASESManole Cojocaru1,2, Inimioara Mihaela Cojocaru3,4, Bogdan Chicoş2

1titu Maiorescu university, Faculty of Medicine, bucharest, romania; 2Dr. ion stoia clinical center for rheumatic Diseases, bucharest, romania; 3carol Davila university of Medicine and pharmacy, bucharest, romania;

4Department of Neurology, colentina clinical Hospital, bucharest, romania

IgG4-related intracranial hypertrophicpachymeningitis (IgG4-RIHP) is an uncommon chronic,fibrosing, inflammatory disorder that causes thickeningof the dura mater of the brain. A 56-year old womanexperienced diffuse headache started three months beforeadmittance. The headache was daily, constant, bilateral,affecting the fronto-temporal regions and it was notinfluenced by posture. On neurologic examination shepresented deep-tendon reflexes brisk bilaterally.

The cerebral MRI revealed dural thickening andnodularity of cerebral hemispheres including the falx andtentorium (T1w and T2w hyposignal) with contrastenhancement (T1w) and left transverse sinus occlusion.CSF examination had shown mild lymphocytosis.Extensive infectious work-up was negative. Work-up forcollagen vascular diseases and vasculitides was negative.The results of coagulation testing were negative. Serumangiotensin converting enzyme (ACE) level was normal.The screening for neoplasia or for lymphoproliferative

disorder was negative. A treatment with anticoagulantsand symptomatics was started. Because headache wasincreasing, a second MRI was performed two monthslater; it had shown worsened thickening.

The patient presented high serum IgG4 level. Thehistologic examination of dura mater biopsy had revealedlymphoplasmacytic inflammation, with fibrosis with adiffusely storiform pattern, and infiltrating macrophages.The immunohistochemical staining revealed the presenceof IgG4 positive plasma cells and mild meningothelialhyperplasia.

The final diagnosis was: IgG4-related intracranialhypertrophic pachymeningitis with mild meningothelialhyperplasia Left transverse sinus thrombosis.

High dose corticotherapy was begun with clinic andimagistic improvement. The biopsy of dura mater withIgG4 immunostaining confirmed the diagnosis. IgG4-RIHP should be seen as a possible cause of chronic dailyheadache.

PO10. IGG4-RELATED INTRACRANIAL HyPERTROPHIC PACHyMENINGITIS REVEALED By CHRONIC DAILy HEADACHE

Inimioara Mihaela Cojocaru1,2, Daniela Ştefãnescu2, Vlad Ştefãnescu2

1carol Davila uMph, bucharest, romania; 2Department of Neurology, colentina clinical Hospital, bucharest, romania

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MicroRNAs play a role more and more studiedin myogenesis and autoimmunity in recent years.Improving the understanding of microRNAsfunction, and their target discovery could lead toelucidation of the pathogenesis of idiopathicinflammatory myopathies (IIM). There are cleardifferences in the microRNAs profile in patientswith IIM compared to healthy controls. It isincreasingly obvious that microRNAs can be usedas biomarkers in diagnosis, or as targets fortherapeutic intervention.

The microRNAs study can be performed byqRT-PCR methodology or microarray on samplesfrom muscle tissue (muscle biopsies from patientswith different types of IIM), blood (circulatingmicroRNAs), or even hair shafts. From the multitudeof microRNAs modified in inflammatory muscle

pathology, those significantly altered in patientscompared to control samples are selected, eitheroverexpressed or downregulated. It was observedthat microRNA-1, -133a, -133b, -206, -208, -208b,-486, -499, -146b, -221, -155, -214, -222 havemodified levels in IIM adult patients, associated withthe autoimmune response, but also with certainmuscle-specific regulatory factors (in myogenesis,regeneration). In children with untreated juveniledermatomyositis there is a significant decrease inmicroRNA-10a and -10b, which target regulatorygenes of proinflammatory cytokines.

In conclusion, microRNAs may be used undercertain conditions as diagnostic markers, and couldrepresent future therapeutic targets to enablepersonalized treatment for patients with IIM.

PO11. MICRO-RNAS ASSOCIATED WITH INFLAMMATORy MyOPATHIES:DIAGNOSTIC BIOMARKERS AND THERAPEUTIC TARGETS

Emilia Manole1,2

1Victor babeş NirD, Molecular biology Department, bucharest, romania;2colentina university Hospital, research center, bucharest, romania

Since Gregor Mendel’s discoveries regardingheredity, there was a tremendous searching fordeciphering those molecular characteristics definingeach individual. Nowadays, rapid progress leads topersonalized medicine as a newest interface forimproving diagnosis, prognosis, and patientmanagement. In dermatology and dermatopathologydomains, molecular technologies help in redefiningclinic management of skin wounds; therefore besttherapeutic targets could be selected in cutaneousneoplasias, infections, inflammatory skin conditionsor wound healing. The process of woundregeneration is vital for whole organism, and theincreasing number of patients suffering of chronicwounds triggers worldwide the development of newstrategies and tools for monitoring various cutaneousconditions. The regenerative approach involves bothclinical (bedside) and preclinical assessments

(bench) where molecular technologies hit bothidentification and quantification of key biomoleculesunderlying chronic wounds intimate mechanisms.The primary source in providing valuable dataregarding different analytes of interest was ELISAmethod, further the techniques assortment wasenlarged by immunohistochemistry, tissue array,protein array and more recently cellular arrays thatcombine protein microarray with in vivo confocalmicroscopy. All these tools allow a rigorousevaluation of wound condition alongside withinnovative biomaterials, cellular therapies andmolecular therapies that achieve the regenerativeprocess completion. It is increasingly obvious thatinnovative technologies are flexible tools indermatology and dermatopathology assessments.

Financed by project PN-II-PT-PCCA-2013-4-1386.

PO12. INNOVATIVE TECHNOLOGIES IN CHRONIC LEG ULCER WOUNDS MANAGEMENT

Carolina Constantin1, Andreea-Roxana Lupu1, Monica Neagu1,2

1Victor babeş National institute, immunobiology laboratory, bucharest, romania;2Faculty of biology, university of bucharest, romania

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Introduction: Despite thorough riskstratification and the addition of novel therapeuticmodalities, roughly half of the patients diagnosedwith acute myeloblastic leukemia (AML) relapse inthe first two years following diagnosis.

Meticulous evaluation of immunophenotypic,molecular and cytogenetic features and rigorousminimal residual disease (MRD) monitoring arerequired to identify a subclass of high risk patientseligible for allogeneic stem cell transplantation afterfirst remission. Nonetheless, novel prognosticfactors are much needed for a more accurate riskstratification and personalization of therapeuticstrategies.

The functional integrity of the MHC II antigenpresenting machinery and the expression of B7 co-stimulatory molecules on leukemic myeloblasts havebeen correlated with favorable outcomes in AMLpatients. However, immune-related prognosticfactors are not currently taken into account whenmanaging AML.

Matherials and methods: We examinedperipheral blood and bone marrow samples from 4patients with newly diagnosed AML. Leukemic

myeloblasts were investigated by flow cytometry forthe expression of CLIP, CD74, HLA-DM and B7family molecules (CD80, CD86, CD273, CD274,CD275, CD276, B7-H4). CD4 positive bone marrowT cells were examined for the expression ofstimulatory or inhibitory receptors of the B7molecules (CD152, CD278, CD279).

Results: A new algorithm of investigationdirected towards the antigen presenting capacity ofleukemic myeloblasts and their co-stimulatory or co-inhibitory properties in newly diagnosed AMLpatients is presented. Based on our preliminary data,we speculate that a functional antigen presentingmachinery and the expression of co-stimulatorymolecules on leukemic cells might facilitate immunesurveillance.

Conclusions: An individualized and improvedunderstanding of the immune evasion strategies ofAML blasts will be crucial in defining the role ofnovel therapies (monoclonal antibodies, chimericantigen receptor T cells, bi-specific T cell engagers,microenvironment modulation) in the first-linetreatment of AML.

PO13. ANTIGEN PRESENTATION PATTERNS IN NEWLy DIAGNOSEDACUTE MyELOID LEUKEMIA PATIENTS

Ion Antohe1, Angela Dãscãlescu1, Mihaela Zlei2, Cãtãlin Dãnãilã1, Petru Cianga3

1Haematology Department, Grigore t. popa university of Medicine and pharmacy, iaşi, romania;2immunophenotyping Department, regional oncology institute, iaşi, romania;

3immunology Department, Grigore t. popa university of Medicine and pharmacy, iaşi, romania

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Ioana Sonya Ciulean1, orhan Raşid2, Jean-Marc Cavaillon2

1cantacuzino Nir, cellular and Molecular immunity, bucharest, romania;2institut pasteur, Département infection et Epidémiologie, unité cytokines & inflammation, paris, France

Introduction. Natural killer (NK) cells havebeen shown to play a key role in the inflammatorycascade leading to systemic inflammatory responsesyndrome (SIRS). However, little is known aboutorgan-specific features of NK cells activation duringconditions like SIRS. The aim of this study was toinvestigate how NK cells isolated from differentcompartments behave in other environments, afterlipopolysaccharide (LPS) exposure.

Materials and methods. Adoptive transfermodels were performed using CD45.2 and CD45.1congenic mice. Lungs and spleens were harvestedfrom CD45.2 donor mice, NK cells were enrichedby negative selection and transferred by intra-venous(IV) or intra-peritoneal (IP) injection into recipientCD45.1 mice. Endotoxemia was induced by IPinjection of 10 mg/kg LPS. Surface CD69 andintracellular IFNγ expression in lung, spleen andperitoneal NK cells was determined by flowcytometry at 3 and 6 hours (h) post LPS.

Results. In the early stages of endotoxemia, IVtransferred NKs did not reach the peritoneum, whileby IP injection cells seemed to be trapped in theperitoneal cavity. Compared to local peritoneal NKs,transferred spleen and lung NKs are hyporesponsivein the first 3 h, but these differences disappear by 6h post LPS. Similar observations were made inrecipient spleens and lungs at 3h post LPSadministration, with transferred cells behaving liketheir organs of origin in the new environment, butreaching the same level of activation by 6 h postLPS, regardless of their origin.

Conclusion. Our results suggest thatcompartment-specific microenvironmental factorsinfluence resident NK cell responses and possiblymodulate responsiveness of newly recruited NKcells.

Acknowledgements. Ioana Sonya Ciulean’swork at Institut Pasteur was supported by the EFIS-IL Short Term Fellowship.

P1. COMPARTMENTALIzATION OF NK CELL RESPONSES DURING EARLy STAGES OF ENDOTOXEMIA


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Introduction: Celiac disease (CD) is a chronicintestinal inflammation resulting in villous atrophyoccurring in genetically predisposed individuals inresponse to the dietary ingestion of gluten. A widespectrum of clinical phenotypes is present, rangingfrom classical gastrointestinal manifestations to onlyatypical signs. The diagnosis of CD is readily esta-blished in those who, while consuming gluten-con-taining diet, have positive serology and a duodenalbiopsy with obvious celiac histology. HLADQ2/DQ8 tests are increasingly considered as asolid support in the diagnostic algorithm of CDmainly for its negative predictive value (~100%)since CD is highly unlikely when DQ predisposingalleles are absent.

Case presentation: We report the case of a 45-year old female patient who was admitted to ourclinic for abdominal pain, watery diarrhea (3-5bowel movements/day, including nocturnal stools)and weight loss, symptoms of insidious onset severalmonths prior admission. Laboratory investigationsshowed iron deficiency anemia. Fecal studies werenegative for bacteria and parasitic infections and

thyroid function tests were in the normal range.Abdominal ultrasound, colonoscopy and scansshowed no abnormalities and anesophagogastroduodenoscopy was performed.Congestion of duodenal folds was identified withpartially flattened villi (Marsh 3a) without presenceof Helicobacter pylori infection. Totalimmunoglobulin A, anti-tissue transglutaminase(tTG) and anti-gliadine (AGA) IgA antibodies levelswere measured. A positive result was obtained withmild elevation of AGA IgA titer of 29 U/ml (positivevalues>18U/ml). HLA DQ2 and DQ8 genotypingwas performed and HLA-DQA1*01, *01: 01 andHLA-DQB1*05, *06 were detected in this patientmaking the diagnosis of CD improbable.

Discussion: Although the patient presented withsigns and symptoms of malabsorption, partial villousatrophy and mild elevation of AGA antibody titer,CD is highly unlikely when DQ predisposing allelesare absent. In this case, the genetic testing showeddiscriminatory value by virtually excluding a CDdiagnosis.

P2. CLINICAL IMPLICATIONS OF MOLECULAR HLA TyPING AMONGSUSPECTED ADULT CELIAC DISEASE PATIENTS: CASE REPORT

Roxana Maxim¹, Anca Trifan¹,², Alina Plesa¹,², Irina Ciortescu¹,², Petru Cianga¹,³, Carol Stanciu¹1Grigore t. popa university of Medicine and pharmacy, iaşi, romania; 2institute of Gastroenterology and Hepatology, iaşi, romania; 3Department of clinical immunology and Genetics, iaşi, romania

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The development of new effective strategies forobesity treatment has become a global priority dueto the obesity epidemic and its main role indeveloping insulin resistance, metabolic syndromeand type II diabetes. As excessive adipogenesis isone major cause underlying the development ofobesity, targeting this key process is a moderntherapeutic strategy for obesity management. Lipiddroplet associated proteins lie at the core of lipolysisregulation, perilipin being the most important markerof mature adipocytes.

Therefore, designing novel compounds that caninhibit adipogenesis may lead in preventing and po-tentially treating obesity and its related diseases. Fla-vonoids are natural compounds that present asignificant potential in improving metabolic abnor-malities through their positive role in glucose home-ostasis and insulin secretion. Their association withtrace elements can increase the biological effects offlavonoids through a synergistic mechanism basedon the capacity of various trace elements to increasethe efficiency of insulin. In this context, the aim of

our study was to investigate the in vitro anti – adi-pogenic potential of an original chromium complexwith 5 – hydroxyflavone upon adipogenic commit-ted adipose – derived stem cells (hASCs) by scree-ning perilipin expression.

The anti – adipogenic potential of the chromiumcomplex with 5 – hydroxyflavone was assessed onhASCs, during 3 weeks of its administration in anadipogenic medium. Perilipin expression wasevaluated both through quantitative (flow cytometry)and qualitative (fluorescence microscopy) methodsat 7, 14 and 21 days after adipogenic induction. Flowcytometry histograms revealed that the perilipinexpression is significantly decreased in the presenceof the chromium complex with primuletin. Theobtained results were confirmed by theimmunofluorescent labeling of perilipin afterexposure to the novel synthetized complex.Consequently, the chromium complex with 5 –hydroxyflavone can be further employed in in vivostudies on animal models.

P3. CHROMIUM COMPLEX WITH 5 – HyDROXyFLAVONE REMODELSLIPID DROPLETS By DECREASING THE PERILIPIN EXPRESSION

Ariana hudiþã1, Bianca Gãlãþeanu1, Mirela Şerban1, Sorina Dinescu1, Valentina Uivaroşi2, Marieta Costache1

1Department of biochemistry and Molecular biology, university of bucharest, bucharest, romania;2Faculty of pharmacy, carol Davila university of Medicine and pharmacy, bucharest, romania


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Introduction. Neuroendocrine tumors (NETs)are neoplasms arising mainly from thegastrointestinal tract, pancreas and bronchial tree.Histopathological and immunohistochemicaldiagnosis and classification of NETs play a role inestablishing the therapeutic management of thesetumors. Effective methods of diagnosis are neededfor the rigorous selection of cases with greaterlikelihood of therapeutical response. RT-PCR is amore accurate quantitative method which canreplace the routine immunohistochemical testing.

Materials and methods. We studied tumoraland peritumoral fragments fixed in formalin andembedded in paraffin from 26 patients diagnosedwith NET (pulmonary, gastrointestinal andpancreatic) by histopathological andimmunohistochemical techniques. In order to assessgene expression, a PCR-array method (Qiagen) wasused, which identified relative expression of 18genes of interest by comparing the tumor with theperitumoral tissue, the normalization being carriedout by means of two reference genes.

Results. In cases of pulmonary NETs, VGF,MGMT, SSTR1 genes were strongly overexpressed,

GAST, SYP, CCND1 genes were moderatelyoverexpressed, while MTOR, INS, CHGA, MKI67,SSTR5, SLIT2 had a low overexpression. Ingastrointestinal NETs, CHGA, SSTR1 genes weremoderately overexpressed, GRPR, mTOR, SSTR3,SSTR5 genes had a low overexpression, SSTR2,MKI67 were downregulated. In pancreatic tumors,GAST, VGF, TYMS genes were moderatelyoverexpressed, SSTR3 and SSTR1 had a lowoverexpression and MGMT, SLIT2 genes weredownregulated. Somatostatin receptor (SSTR)overexpression represents a predictive marker for thetherapy with somatostatin analogues and theexpression of somatostatin receptors, especiallySSTR1, is positively correlated with the patientsurvival rate.

Conclusions. Identification of the gene profilemay represent a sensitive tool for diagnostic andtherapy in NETs. A better survival rate of patientswith SSTRs overexpression justifies the use of thesereceptor genes as prognostic markers in NETs.

This work was supported by project PN-II-PT-PCCA-91/2012 – RENET.

P4. DIAGNOSTIC, PROGNOSTIC AND THERAPEUTIC TARGETS IN NEUROENDOCRINE TUMORSMaria Dobre1, Maria Victoria Comãnescu1, Florina Vasilescu1,2

1Victor babeş National institute of pathology, bucharest, romania;2Dr. carol Davila central university Emergency Military Hospital, bucharest, romania

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Langerhans cells are a major subset of skin dendriticcells localized in the epidermis, which have an importantrole in the pathogenesis of many inflammatory diseasesof the skin, being also involved in maintaining peripheraltolerance against antigens expressed in the skin. Eversince Paul Langerhans described the epidermal dendriticcells, several studies aimed to analyze the morphologyand location of this type of cells in different conditions ofthe skin using techniques such as histology, electron mi-croscopy, immunohistochemical staining methods.

One of the most anticipated drawback of theseprocedures was distortion of the tissue. Therefore, in anattempt to enhance clinical diagnosis, confocalmicroscopy was developed as an in vivo skin-imaging

technique which allows visualization of superficial dermisand epidermis with high resolution, much like classichistology.

In this study we examined in vivo basal cellcarcinoma lesions using a reflectance laser confocalscanning microscope, and further we compared thefeatures found with the histological examination results.

In addition, we also proceed our investigation withimmunohistochemical tests for S100, CD1a and Langerinproteins for the same skin lesions previously examined.

Confocal microscopy turned out to be a promisingmethod of visualization of Langerhans cells in variouspathological conditions. However, further studies areneeded in order to improve this technique.

P5. NEW VISUALISATION TECHNIQUE OF EPIDERMAL LANGERHANS CELLS

Alexandra Victoria Ion1, Mihaela Adriana Ghiþã2, Iulia Solomon1, Sabina Zurac3, Daniel Boda2,4, Constantin Cãruntu2,4

1Elias Emergency university Hospital, Department of Dermatology and allergology, bucharest, romania;2carol Davila university of Medicine and pharmacy, Dermatology research laboratory, bucharest, romania;

3carol Davila university of Medicine and pharmacy, colentina university Hospital, Department of pathology, bucharest,romania; 4prof. N. paulescu National institute of Diabetes, Nutrition and Metabolic Diseases,

Department of Dermatology, bucharest, romania

The polycistronic miRNA cluster (oncomiR-1)produces a single 800b primary transcript (pri-miRNA)that yields six mature miRNAs: miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92a. The miRs belongsto 4 different families, namely miR-17/miR-20a (seedAAAGUG), mir-18a (seed AAGGUG), miR-19a/19b(seed GUGCAA), and miR-92a (seed AUUGCA). Thecluster inhibits gene expression through interaction ofmiRs with 3′ untranslated regions (UTRs) of messengerRNAs (mRNAs). The biological properties of the clusterare the result of the cooperative actions of each member.

The cluster is expressed in HSCs and the miR levelsincrease during the stem cell commitment to thegranulocyte-monocyte progenitors. Although manystudies have demonstrated the important contribution ofthe oncomiR-1 in leukemia and hematopoiesis, there are

no published data regarding the role of oncomiR-1 inhematopoietic stem cell (HSC).

To study the cluster role in HSC homeostasis, wehave knocked-out the oncomiR-1 by crossing thetransgenic Mx1-Cre mouse with a mouse line that carries“flox” sequences inserted within the cluster gene. TheMx1 is very active in HSC and we demonstrated that thecluster miRs are completely absent after Cre induction.

We tested the stem cell properties such as self-renewal, pluripotency and differentiation, both insteady-state and post-transplant hematopoiesis. Thephenotype of the KO mice shows that the HSC functionsare altered when oncomiR-1 is ablated.

This work was supported by a grant of the Ministryof National Education, CNCS – UEFISCDI, projectnumber PN-II-ID-PCE-2012-4-0395.

P6. THE MIR CLUSTER ONCOMIR-1 REGULATES THE HEMATOPOIETIC STEM CELL HOMEOSTASIS

Valeriu Bogdan Cismaşiu1, Laurenþiu Anghelache1, Andreea oana Urs1, Florina Gisela Gãinã1, Mihaela Surcel1,2, Cristina Mariana Niculiþe1, Bogdan Marinescu1, Radu-Ionuþ huicã1,4, Ioana Mãdãlina Fenyo3

1Victor babeş National institute of pathology, bucharest, romania; 2Faculty of biology, university of bucharest, romania;3Nicolae simionescu ibpc, bucharest, romania; 4carol Davila university of Medicine and pharmacy, bucharest, romania

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Introduction: Recent published data on NK cellactivation in cancer patients indicate that severalimportant parameters, such as tumour capacity tomodulate NK function and NK cells phenotype should beconsidered for NK-based therapy. Here we show that in amelanoma-bearing mouse model NK cells from the spleenare percentually reduced and express different phenotypicfeatures comparing with NK cells of healthy mouse.

Materials and methods: We used 8-10 weeks oldC57BL/6 mice. Melanoma lesions were induced bysubcutaneous inoculation of B16F10 cells. Normal valueswere established in healthy, age-matched mice. 21 daysafter, the spleens were harvested and the flow cytometryanalyses of NK cells were made. Stained cells wereanalyzed with a FACSCanto II flow cytometer usingDIVA software.

Results: Experimental data show a statisticallysignificant reduction of the percentage of NK cells inmelanoma-bearing mice in comparison with healthy

animals. Also, we found a reduction of mature NK cellsubsets, an increase of gp49R+NK cells and decrease ofCD122+NK cells in melanoma-bearing mice. Analysis ofNK cell subsets, defined by the differential expression ofa combination of CD27 and CD11b, indicated asignificant difference in the distribution of NK cell subsetswith the mature subset being dominant in the healthymice. Increase of gp49R expression on NK cells, that isan inhibitory receptor, may facilitate tumor immuneescape. The blocking of inhibitory receptors may proveto be an effective strategy for elimination of cancer cells.NK cell death could be caused by the reduced (or failed)expression of CD122 (receptor subunit that confers IL-15responsiveness).

Conclusions: Our study has provided new insightsinto NK cell phenotype in tumors and new approaches tocancer immunotherapy.

Work supported by grant of the Romanian NationalAuthority for Scientific Research PN 16.22.04.04.

P7. PHENOTyPIC CHARACTERISTICS OF NATURAL KILLER CELLS FROM MELANOMA-BEARING MICEGheorghiþa Isvoranu1, Mihaela Surcel1,2, Bogdan Marinescu1,

Laurenþiu Anghelache1, Radu-Ionuþ huicã1,3, Cãtãlin Gabriel Manole1

1Victor babeş National institute of pathology, bucharest, romania; 2Faculty of biology, university of bucharest, romania;3carol Davila university of Medicine and pharmacy, bucharest, romania

Introduction: Prolonged macrophage inflammatoryresponse can lead to rejection or impaired function of theimplant, therefore material surface must be modified toallow proper integration. The aim of this study was toexamine the effect of modified material surfaces withimmunododulatory active components (lactoferrin-Lf andhydroxypaptite-HA) and biocompatible copolymer (Co,polyethylene glycol-polycaprolactone) on cellularbehavior and cytokine release by human monocyticleukemia THP-1 cells.

Materials and methods: THP-1 cells grown onbiomaterials were differentiated to macrophages withPMA (phorbol-12-myristate-13- acetate) and treated ornot with lipopolysaccharide (LPS). This in vitro cellularmodel allows analysis of early events in inflammationgiving information about mechanisms involved inbiomaterial-associated inflammation.

Cells were characterized for viability and byfluorescence microscopy for attachment and spreading.The release of tumor necrosis factor (TNF-α) in theabsence or presence of LPS was analyzed using an ELISAassay technique.

Results: Immunofluorescence images of stainedactin and vinculin proteins of THP-1 cells cultured onmodified surfaces showed an elongated morphology ofmacrophages adhered on hybrid Co-HA-Lf coatedsurfaces that might be associated with a certain state ofactivation. LPS treatment reduced the number ofmetabolically active cells on Co-HA-Lf modified surfacescompared to unmodified surfaces. Low levels of secretedproinflammatory cytokine TNF-α were obtained in thecase of LPS treatment from cells adhered on hybridcoatings. However, no proinflammatory cytokinesecretion was detected without LPS stimulation in noneof the analyzed surfaces.

Conclusions: Our results suggest that hybridcoatings modulate recruitment, viability, morphology andinflammatory behavior of macrophage cells onbiomaterial and thus having the potential of minimizingthe host inflammatory response after implantation.

Acknowledgements: The research was supported bythe Romanian Ministry of National Education, CNCS –UEFISCDI, under the project PN-II-PT-PCCA 239/2014and Romanian Academy project 1/2015-2016.

P8. MACROPHAGE RESPONSE ON HyBRID MODIFIED SURFACES

Mãdãlina Icriverzi1,2, Livia Sima1, Valentina Dincã3, Anişoara Cîmpean2, Anca Roşeanu1

1ibar, ligand-receptor interaction Departament, bucharest, romania; 2ub, Departament of biochemistry and Molecularbiology, bucharest, romania; 3iNFlpr, photonic processing of advanced Materials Department, Mãgurele, romania

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Introduction and Objectives: Discoid lupuserythematosus (DLE) is an inflammatory chronic skindisease clinically characterized by well demarcatederythematosquamous infiltrated plaques, which showa predilection for sun-exposed areas.

Even though the clinical diagnosis is based ontypical lesions, because of their clinical similarities,DLE can be confused with other erythematosquamousskin diseases. Thus, clinical diagnosis of DLE shouldbe often sustained by histopathological examination.To enrich the diagnosis of DLE, in vivo confocal laserscanning microscopy (CLSM) was proposed as a non-invasive imaging technique which allows theevaluation of skin lesions with microscopic resolution,close to conventional histopathology.

Materials and Methods: We used a reflectancelaser confocal scanning microscope with a wavelengthof 785 nm to examine in vivo the skin lesions deep to

superficial dermis. In addition, for histopathologicalexamination we took a 3mm punch biopsy fromerythematosquamous skin lesion previously examined.

Results: Correlating the features found in CLSMwith histopathological features of the same DLElesions, we noticed that criteria such as interfacedermatitis, epidermal atrophy, perivascular andperiadnexal inflammatory cell infiltration were foundin both methods of investigation. One major drawbackof CLSM technique has been the depth of imaging,especially in the diagnosis of DLE that involve thedeeper dermis.

Conclusions: In the light of our study, we couldestablish the value of CLSM in the diagnosis of DLEas a promising non-invasive dermatological exa -mination method which needs a better standardizationand application in clinical practice.

P9. CORRELATIONS OF HISTOPATHOLOGy AND iN ViVo REFLECTANCECONFOCAL MICROSCOPy IN DISCOID LUPUS ERyTHEMATOSUS

Iulia Solomon1, Mihaela Adriana Ghiþã2, Alexandra Victoria Ion1, Sabina Zurac3, Daniel Boda2,4, Constantin Cãruntu2,4

1Department of Dermatology and allergology, Elias Emergency university Hospital, bucharest, romania; 2Dermatology research laboratory, carol Davila university of Medicine and pharmacy, bucharest, romania;3Department of pathology, carol Davila university of Medicine and pharmacy, colentina university Hospital,

bucharest, romania;4Department of Dermatology, prof. N. paulescu National institute of Diabetes, Nutrition and Metabolic Diseases,

bucharest, romania

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Introduction. Cutaneous melanoma (CM) is amelanocytic malignant tumor and is the most severeskin neoplasia. Although it represents only 4% of allskin cancers, it is responsible for 80% of deaths,melanoma being one of the most metastasizingneoplasms. CM generally affects relatively youngpopulation, the average age of diagnosis being 50 yearsold. Over 90% of cases of melanoma can be treatedsuccessfully if detected and treated in early stages,justifying the interest in identifying and describing theprognostic markers and evolution.

Materials and methods. Lymphocytespopulations and subpopulations of peripheral bloodwere quantified: total T cells (CD3+), B cells (CD3-

CD19+), NK cells (CD3-CD16+ and/or CD56+), Thelper cells (CD3+CD4+), T supressor/cytotoxic cells(CD3+CD8+) and regulatory T cells(CD4+/CD25+/FOXP3+), for a group of patients withCM. Also the percentages of lymphocytes secreting IL-2, IFN-γ and TNF-α from peripheral blood weredetermined. The samples were analized by flow-cytometry (BD FACSCanto II, software BDFACSCanto and BD FACSDiva 6.1), and the valueswere reported to those obtained in a group of normalsubjects.

Results. The main registered changes were thedecreasing percentage and absolute number of T-CD8+lymphocytes in 83%, respectively 61% of tested

patients. T-CD4+/T-CD8+ ratio was increased to 60%of cases, due to the decrease of the T-CD8+

lymphocytes percentage. T-reg values were elevated in53% of cases; 63% of patients with increased T-regpresented decreased percentage of T-CD8+lymphocytes. For intracellular cytokines, the results areexpressed as percentage of secreting lymphocytes fromall lymphocytes. We observed significant changes inthe proportion of lymphocytes involved in the IFN-γsynthesis (17±7% of cases, and 9±3% in controls,p<0.02) and in TNF-α synthesis (33±9% of cases,compared with 22±11% in controls, p<0.05).

Conclusions. CM cellular profile suggests a trendof dissociation of antitumor immune responseefficiency. Firstly, the decreasing of T-cytotoxic cellsand the increasing of regulatory T cells diminish thecytotoxic activity. On the other hand, the increasing ofpercentages of the proinflammatory cytokinessecretory cells (IFN-γ and TNF-α) may be relative,being the consequence of increasing the share of T-helper cells, indicating the turning trend of antitumouractivity from cytotoxicity to inflammation.

The results are part of the doctoral thesis titled”The immunological exploring of the inflammatorysyndrome in autoimmune and malignant tumoral skinpathology (psoriasis, cutaneous melanoma)” (PhDcandidate Mihaela Surcel).

P10. CHANGES OF T LyMPHOCyTES AND CyTOKINES SECRETIONIN CUTANEOUS MELANOMA

Mihaela Surcel1,2, Radu-Ionuþ huicã1,3, Adriana Narcisa Munteanu1, Ioana Ruxandra Pîrvu1, Dan Ciotaru1, Cornel Ursaciuc1, Monica Neagu1,2

1Department of immunology,Victor babeş National institute of pathology, bucharest, romania;2Faculty of biology, university of bucharest, romania;

3carol Davila university of Medicine and pharmacy, bucharest, romania

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The present study is a complex interdisciplinaryapproach that aims to develop pharmaceutical productswith significant antitumor action and minimal side effects.The clinical use of Cisplatin (CisPt) might be limited bytumor drug resistance and adverse effects, therefore othermetal agents that possess anti-tumor and anti-metastaticactivities such as ruthenium (III) compounds (Ru-nf, Ru-oflo, Ru-levo), could be considered as alternative drugs.Studies were extended to ruthenium compoundsassociated to cyclodextrins in order to improve theirpharmacokinetics. In order to evaluate the in vitrocitotoxicity of the new synthetized compounds andcontinuously monitor the viability of LoVo human colonadenocarcinoma cells, the real time cell analysis (RTCA)was used by xCellingence system (ACEA Biosciences),comparing it to cytotoxicity evaluation by colorimetricassay with MTS. The technique allowed to pinpoint theoptimal time points and concentrations for conducting

endpoint assays such as flow-cytometry approaches. Thebiological effect of Ru compounds+cyclodextrins onLoVo cells was asssessed by evaluation of apoptosis usingAnnexin V/FITC and propidium iodide (PI) doublestaining, followed by flow-cytometry analysis with BDFACS CantoII flow-cytometer. Treatments with Ru-levo,Ru-nf, Ru-oflo + HPbetaCD induced apoptosis in 26.6%,23.4%, and 24.6% respectively, of the LoVo cells,compared to Cis-Pt induced apoptosis of 23.3% cells.Results showed that association of Ru-nf and Ru-oflo withHPbetaCD induced a significant increase of apoptoticevents compared to single treatments (15% and 9%,respectively). Since chemotherapy of solid tumors is stilllimited by the lack of selectivity of oncolitical drugs andrecurrence of resistant tumors to treatments, theassociation between chemotherapeutical drugs and newpharmaceutical compounds might be of great benefitinducing an increase in anti-tumor response.

P11. BIOLOGICAL EFFECTS OF RUTHENIUM (III) COMPOUNDSASSOCIATED TO CyCLODEXTRINS ON LOVO CELLS

Mirela Mihãilã1, Camelia hotnog1, Marinela Bostan1, Viviana Roman1,Valentina Uivaroşi2, Lorelei Irina Braşoveanu1

1Ştefan s. Nicolau institute of Virology, center of immunology, bucharest, romania;2carol Davila university of Medicine and pharmacy, Faculty of pharmacy, inorganic chemistry, bucharest, romania

Malignant lesions arising in the pharynx are initiallymainly asymptomatic, aggressive, and frequently invadeand migrate to distant organs, making them difficult totreat. Current treatment for pharynx cancer is using achemotherapeutic agent – Cisplatin - which induces sometoxic effects at the renal and bone marrow levels.

That prompted us to investigate the in vitro effectsof Ganoderma lucidum (GL), an oriental medicalmushroom (which contains many biologically activecompounds such as polysaccharides, triterpenes andimmunomodulatory proteins) on cell proliferation andapoptosis of FaDu human pharynx carcinoma cellcultures, treated or not with cisplatin.

Cells were treated with varying concentrations of GLin the presence or absence of cisplatin, and CellTiter 96Non-Radioactive Cell Proliferation assays were thenperformed to evaluate the effect of single or associatedtreatments on FaDu cell growth. The cell cycle phasedistribution, apoptosis process and molecular markers

expressions (p21 or Bax) in FaDu cells were assessedusing flow cytometry approaches.

Results showed that GL extract had a stronginhibition effect of cell proliferation on FaDu tumor cellcultures. Cell cycle analysis showed that the growthinhibition effect was associated with G2/M arrest and up-regulation of p21 antigen expression. In addition, GLinduced apoptosis of FaDu tumor cells and an increase ofthe pro-apoptotic Bax protein expression.

Furthermore, the treatment with GL significantlyamplified the apoptosis induced by cisplatin in FaDu cells.Taken together, these findings suggest that GL exerts anti-tumor effects on FaDu tumor cells and enhances thesensitivity of FaDu tumor cells to cisplatin.

In conclusion, the effects induced by GL on cancercells are due to activation of various cellular andmolecular mechanisms, which might suggest the use ofthis compound as an adjuvant to the classical pharynxchemotherapy.

P12. MODULATION OF CHEMOTHERAPy RESPONSE By NATURALCOMPOUNDS IN HUMAN PHARyNX TUMOR CELLS

Georgiana Gabriela Petricã-Matei1,2, Viviana Roman1, Mirela Mihãilã1, Camelia hotnog1, Lorelei Irina Braşoveanu1, Marinela Bostan1

1center of immunology, stefan s. Nicolau institute of Virology, bucharest, romania; 2Departament of cytogenetics, personal Genetics - Medical Genetics center, Discipline of Medical Genetics, bucharest, romania

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The way a person responds to medication depends on acomplex of factors, including uptake, bioavailability,activation, deactivation, metabolism and clearance of thedrug and the deactivated metabolites. Thiopurines,substituted analogs of purines, are widely used for thetreatment of certain types of leukemia, organ rejection andinflammatory disorders. They are activated and metabolizedby the enzymes of the degradation and inter-conversionreactions of purine metabolism. As such the enzymes activityis indicative for their efficacy and safety. It has been welldocumented that a genetic defect in one of these enzymesmay result in an altered response to purine or pyrimidinederived medication. The study of such alterations in themetabolism of therapeutic drugs is known aspharmacogenetics. Thiopurine S-methyltransferase (TPMT,EC2.1.1.67) is one critical enzyme involved in themetabolism of thiopurine drugs (such as tacrolimus). Geneticfactors influence how thiopurines are handled: metabolitesmay accumulate or are converted into less efficient or more

toxic compounds. It has been proven that polymorphisms inthe gene encoding TPMT, resulting in its decreased activityare associated with adverse drug reactions during thiopurinetherapy, and a person can be adversely affected if commondoses of thiopurines are prescribed. Biochemical monitoringof the metabolites resulted from thiopurine reactions beingdifficult, the genotyping of the gene encoding for TPMT isconsidered the most informative for the drug efficiency andpersonalization.

Two methods have been approached on 6 healthyindividuals for TPMT genotyping of its single nucleotidepolymorphisms and haplotypes: Next GenerationSequencing (NGS) on ThermoFischer Ion Torrent systemand High Resolution Melting Analysis (HRMA) on Arkrayi-densy system and their efficiency as diagnostic tools hasbeen estimated. The results are commented in terms of thesignificance of the TPMT*2 and *3 genotypes for drugsdoses recommendations in the case of eventual thiopurinetreatments.

P13. PHARMACOGENETIC APPROACH OF IMMUNOSUPPRESSIONTHERAPy: TPMT GENOTyPING METHODS FOR EFFICIENT

TREATMENT BASED ON THIOPURINE DRUGS

Maria Iacob1, Adriana oprea1, Ileana Constantinescu2, Natalia Cucu1,3

1university of bucharest, Faculty of biology, Department of Genetics, Epigenetics laboratory, bucharest, romania;2carol Davila university of Medicine and pharmacy, clinical Hospital Fundeni, Department of immunogenetics,

bucharest, romania; 3association for Epigenetics and Metabolomics, bucharest, romania

Proliferation of mammalian cells is tightly regulated bymultiple environmental influences, adhesion to extracellularmatrix (ECM), cell-cell adhesion and soluble factors likecytokines. Adhesion represents a main component of cellmigration and recognition involved in proliferation,differentiation and apoptosis, immune and inflammatoryresponse. Formation and invasion of tumors are closelyassociated with decreased dependence on adhesion neededfor growth and survival. Biomarkers like mucins, EpCAM,E-cadherin and VEGF are considered as potentialimmunotherapeutic targets because they play an importantrole not only in releasing cells from the primary tumors andin invasion processes, but also in angiogenesis. Therefore,our study focused on the possible modulation of antigenexpression of several biomolecules associated to coloncancer cell lines with different degrees of proliferation (LoVoand SW1116) by cytotoxic drugs (5-Fluorouracil) and/ornatural compounds (resveratrol, curcumin). Treatment doseswere established after real time cell analysis (RTCA) byusing xCELLigence system. Levels of ICAM-1, VCAM-1,

E-cadherin, VEGF or endoglin antigen expression wereevaluated by sequential treatment of cells with specificmonoclonal antibodies and secondary FITC-labeledpolyclonal antibodies, followed by flow-cytometry analysis.In addition, the biological effects of stimuli treatment wereevaluated by flow-cytometry approaches in terms ofproduction of reactive oxygen species (ROS) and modulationof apoptotic process, in the presence or absence of anti-VEGF treatment with Avastin.

Data obtained showed a differential expression andfunctional behavior of TAA associated to colon cancer cellssuggesting their possible involvement in regulating theinteractions between tumor cells and host immune system.Adjuvant mAb-mediated immunotherapy may provide aneffective method to prevent or reduce the spread of tumorcells. The results obtained will further be used in elaboratingnew anticancer immunotherapeutic strategies focused onanti-TAA mAbs with biological functions in order todiminish the primary tumor and control/eliminate themetastases.

P14. FUTURE PERSPECTIVES FOR ADJUVANT TREATMENT OF COLONCANCER: BIOMOLECULES OF POTENTIAL CLINICAL USE

Camelia hotnog1, Mirela hirt1, Marieta Panait2, Marinela Bostan1, Maria Iuliana Gruia2, Lorelei Irina Braşoveanu1

1Ştefan s. Nicolau institute of Virology, center, of immunology, bucharest, romania;2alexandru trestioreanu institute of oncology, bucharest, romania


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AAlbulescu Radu 77Androsiac Gabriela Elena 72Anghelache Laurenţiu 77, 80, 89, 90Antohe Ion 84

BBădescu Daniela 71Băicuş Cristian 80Bălănescu Anca 80Bălănescu Eugenia 80Bălănescu Paul 80Boda Daniel 89, 91Bostan Marinela 93, 94Braşoveanu Lorelei Irina 93, 94Butan M. 81

CCavaillon Jean-Marc 85Căruntu Constantin 89, 91Ceacăreanu Alice C. 67Ceauş Ionica 70Cercel Cristina 78Chicoş Bogdan 82Cianga Corina 64, 78Cianga Petru 64, 78, 84, 86Ciortescu Irina 86Ciotaru Dan 73, 92Cişmaşiu Valeriu Bogdan 89Ciulean Ioana Sonya 85Cîmpean Anişoara 90Codrici Elena 77Cojocaru Inimioara Mihaela 82Cojocaru Manole 82Comănescu Maria Victoria 88Constantin Carolina 65, 83Constantinescu Daniela 78Constantinescu Ileana 74, 75, 76, 94Costache Marieta 72, 79, 87Cotar Ani Ioana 71Cremer Lidia 78Creţu Carmen-Michaela 70Cristea Victor 63, 77Cuadrado Antonio 66Cucu Natalia 94

DDan Gheorghe Andrei 80Dănăilă Cătălin 84Dăscălescu Angela 84

Dincă Valentina 90Dinescu Sorina 79, 87Diţu Lia Mara 69Dobre Maria 88Dudău Maria 77

EEnciu Ana-Maria 66, 77

FFayazi Zahra S. 67Fenyo Ioana Mădălina 89Forrest Alan 67Fu Hsin-Wei 67

GGaile Dan P. 67Găină Florina Gisela 89Gălăţeanu Bianca 72, 87Gheorghe Anca 72Ghiţă Mihaela Adriana 89, 91Gruia Maria Iuliana 94

HHammel Jeffrey P. 67Hirt Mirela 94Hotnog Camelia 93, 94Hudiţă Ariana 72, 87Huică Radu-Ionuţ 73, 89, 90, 92

IIacob Maria 94Icriverzi Mădălina 90Ion Alexandra Victoria 89, 91Istrate Claudia 70Isvoranu Gheorghiţa 80, 90

KKhoury Thaer 67

LLazăr Veronica 65, 71Lupu Andreea-Roxana 65, 78, 83

MManda Gina 66, 80Manole Cătălin Gabriel 80, 90Manole Emilia 83

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Marinescu Bogdan 80, 89, 90Maxim Roxana 86Mihai Simona 77Mihalache (Radu) Mihaela Raluca 71Mihăilă Camelia 78Mihăilă Mirela 93Mihăilescu Patricia 70Moise Ana 74, 75, 76Munteanu Adriana Narcisa 73, 92

NNeagu Alina 71Neagu Monica 65, 83, 92Nedelcu Daniela 74, 75Negrei Carolina 72Niculiţe Cristina Mariana 89Nimako George K. 67Niţu Simona 81

OOnu Adrian 68, 78Oprea Adriana 94

PPanait Marieta 94Petrescu Ştefana M. 64Petrică-Matei Georgiana Gabriela 93Pîrvu Ioana Ruxandra 73, 92Pleşa Alina 86Ponoran Carmen 81Pop Lucian 63, 77Popescu Daniela Ionela 77

RRaşid Orhan 85Roman Viviana 93Roşeanu Anca 90

SSima Livia 90Solomon Iulia 89, 91Sora Mihaela 74, 75Stanciu Carol 86Surcel Didi 81Surcel M. 81Surcel Mihaela 73, 80, 89, 90, 92

ŞŞerban C. 72Şerban Mirela 72, 87Ştefănescu Daniela 82Ştefănescu Vlad 82

TTănase Cristiana 77Toader Adela 74, 75Toader S. 81Trifan Anca 86

UUivaroşi Valentina 87, 93Urs Andreea Oana 89Ursaciuc Cornel 63, 73, 80, 92Ursu Larisa 74, 75, 76

VVagu Codruţa 72Vasilescu Florina 88

WWintrob Zachary A.P. 67

ZZlei Mihaela 84Zurac Sabina 89, 91


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CLINICAL AND LESIONAL DyNAMICS IN AN INFLUENzA A/PR/8/34-H1N1 AEROSOL INDUCED INFECTION IN MICE

Ioana Sonya Ciulean1*, Elvira Gagniuc (Gubceac)2,3, Manuella Militaru2, Crina Stãvaru1

1cantacuzino National institute of research, bucharest, romania; 2university of agronomic sciences and Veterinary Medicine of bucharest, Faculty of Veterinary Medicine bucharest, romania; 3synevovet laboratory, bucharest, romania

REzUMATÎn acest studiu sunt investigate dinamica leziunilor din

gripă şi corelaţiile stabilite între modificările histologice şisimptomatologia exprimată de şoarecii infectaţi.

Femele adulte BALB/c au fost infectate prin nebulizarecu virus gripal adaptat A/ PR/8/34-H1N1 in doză moderatã(10-3 PR8) sau letală (10-1 PR8). Au fost realizate înregistrărizilnice ale semnelor clinice şi a greutãţii corporale; iar la 24,48, 72 de ore şi 5 zile post-infecţie (pi) a parametrilorrespiratori (volum inspirator (TV); minut volum (MV) şifrecvenţă respiratorie (f)). La 2, 3, 5, 7 şi 10 zile pi, au fostdeterminate nivelurile serice ale metaloproteinazei 9(MMP9) totale, factorului de creştere şi transformare β1(TGF- β1) şi s-au recoltat organe (pulmoni, suport ososnazal-corneţi nazali; cord şi encefal) în vederea realizăriiexamenului histopathologic.

Doza infectantă moderată nu a determinat pierderi îngreutate, iar scorul clinic de suferinţă a fost minim.Comparativ, simptomatologia exprimată în infecţia letală secorelează direct cu dinamica leziunilor. Pletismografiacorporală nu a detectat modificări importante aleparametrilor respiratori investigaţi în niciuna dintre infecţii.Nivelul seric de MMP9 total a fost mai mare în infecţialetală, pe când nivelul de TGF-β1 a rămas în intervalulgrupului control în cazul ambelor doze infectante. Au fostîntâlnite modificări histologice mai severe ale structurilorcavităţilor nazale şi ale pulmonilor la indivizi infectaţi cudoză letală, comparativ cu cei infectaţi cu doză moderată.Denaturări ale muşchiului cardiac şi ale encefalului au fostidentificate în ambele loturi experimentale.

Rezultatele sugerează corelarea leziunilor microscopicecu simptomatologia exprimată în cazul infecţiei letale.Infecţia gripală moderată determină importante leziuniorganice, însă discrete din punct de vedere clinic.

*corresponding author: Ioana Sonya Ciulean, Cantacuzino National Institute of Research (NIR), Cellular and Molecular Immunity Laboratory, 103,Splaiul Independentei, 050096, Bucharest, Romania, [email protected], 004/0727364445

Keywords: Influenza A, mouse model, clinical and lesion dynamics

ABSTRACTThis study aimed to investigate the dynamics of

influenza lesions and the correlation established betweenthe symptomatology expressed by infected mice andhistological alterations.

Adult female BALB/c mice were infected by aerosolexposure to the mouse adapted influenza A/PuertoRico/8/34-H1N1 in a mild (10-3 PR8) or lethal (10-1 PR8)dose. Disease evolution was monitored by dailyrecordings of body weight, clinical signs, while breathingparameters (tidal volume (TV); minute volume (MV) andbreathing frequency (f)) were determined at 24, 48, 78hours and on day 5 post infection (pi). On days 2, 3, 5, 7and 10 pi, serum matrix metalloproteinase 9 (MMP9) andtransforming growth factor beta1 (TGF-β1) levels wereevaluated. Organs (lungs, nasal conchae-cavities, heartand brain) were harvested and processed forhistopathological examination at the same time points.

While mild infection seemed clinically inapparentwith no body weight drop and a very low suffering score,lethal infection symptomatology was directly correlatedwith lesion dynamics. In both infection groups, nosignificant alterations were visible by whole-bodyplethismography. Compared to mild infection, serum totalMMP9 levels were higher in lethal infection, while TGF-β1 remained within control values in both infectiongroups. Histologically, more severe modifications of thenasal components and lungs were seen in lethal infectioncompared to mild infection. Alterations of the heartmuscle and brain were detected in both infection groups.

Our results indicate a clearer reflection ofmicroscopic lesion in symptomatology at a higher virusdose. Although clinically discrete, mild infection causesimportant microscopic organ lesions.

INTRoDUCTIoN

With important repercussions in public healthand a strong socio-economic impact, influenza stillremains among the most frequent causes ofmorbidity and mortality in humans. This explains theincreased research done in the latest years toinvestigate the pathogenesis and pathology of this

disease, but also towards developing newtherapeutics and prophylaxis strategies. In humans,while clinical manifestations of influenza are wellknown, a correlation of these symptoms withinternal lesions is hard to comprehend as pathologystudies have emphasized on the material provided

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from autopsies [1-4]. In addition to the highvariability of influenza histopathology, humanstudies mostly provide microscopic alterationsspecific for late-stage or lethal outcome of thedisease [5-8]. Moreover, secondary bacterialinfections, which are extremely common in humans,complicate histopathological appearance, makinglesion dynamics hard to establish [9, 10]. In thiscontext, animal models play an essential role indecoding disease pathologycal dynamics. The mouseis the most extensively used host in pathogenesis andimmunogenicity research because of the similaritiesto human viral pneumonia pathology, but also forpractical reasons like high breeding rate, small size,low husbandry cost and variety of available strains[11]. Because mice are not natural hosts forinfluenza viruses, there is a need for prior adaptationof the strain used for infection [12-14]. Also, thesymptomatology displayed by infected animals isnon-specific [11, 15-17], which makes diseaseevaluation difficult. In addition to this, influenzapathogenicity in mice has been shown to be bothvirus and host dependent [18]. That is why whenusing murine models aspects like choosing the rightinfectious dose, clinical evaluation parameters, theright moment to apply different treatments or collectsamples, become crucial for the outcome of theexperiment. In the attempt to facilitate a rigorousexperimental set-up and in depth understanding ofdisease pathogenesis which could lead to futureinfluenza diagnosis and anti-viral treatments, thispaper discusses lesion dynamics in an aerosolinduced influenza PR8 murine model and thecorrelation with disease symptomatology.

MATERIALS AND METhoDS

Animals and InfectionThe protocol used in the study was approved by

the Internal Ethics Committee of Cantacuzino NIR(CE 18/05.02.2013).

Female, 6-8 weeks old, specific pathogen freeBALB/c mice were obtained from Cantacuzino NIRanimal facility, Bucharest, Romania. Before enteringthe experiment, the animals were conventionalizedfor 1 week. Mice were housed in polycarbonatecages with wire-grid lids (Techniplast, Varese, Italy)and wood-chip bedding (Lignocel 3/4 S, JRS,Rosenberg, Germany). Untreated, 8-mm pelleteddiet (SB 780, Cantacuzino NIR, Bucharest,Romania) and tap water were provided ad libitum.Mouse adapted PR8 virus, influenza A/PuertoRico/8/34 - H1N1 was kindly provided by

Cantacuzino NIR from the institute’s collection.Mice were infected by aerosol distribution of viralparticles, using a nebulizer adapted to a whole-bodyplethysmography system (PLY 310, EMMS,Hampshire, UK). One ml of allantoic fluid contai -ning 3584 hemagglutinin units was used to obtainedserial dilutions of the viral suspension. Mice (n=4)exposed to saline constituted the control group.Experimental groups were infected by nebulizationwith the PR8 virus using a lethal infection dose (10-1

PR8; n=6) and a mild infection dose (10-3 PR8;n=10). Mice were placed conscious and unrestrainedin the 4 contention chambers of the plethys mographicsystem, one mouse per chamber. One ml of the viralsuspension was placed in the nebulizer, the machinewas set on and the obtained aerosols were deliveredthrough a tubular system to the contention chambers.A final volume of 0.25 ml of corresponding viralsuspension was received by each mouse.

Clinical Follow-Up and Paraclinical Evaluationof Disease

Mice were weighed and clinical signs ofsuffering were monitored daily. Symptoms used fordaily clinical scoring are presented in Table 1.

Breathing parameters were determined at 24,48, 72 hours and on day 5 post infection (pi) using awhole-body plethysmography system (PLY 310,EMMS, Hampshire, UK). Assessed breathingparameters were measured on conscious andunrestrained mice and are presented din Table 2. Thesystem was calibrated prior to each recordingsession. In order to prevent carbon dioxideaccumulation in the chambers, a bias flow supplywas used during the recordings. Before inducinginfection, recordings were made on the same miceduring a protocol of 20 minutes (10 min-acclimatization period; 10 min- recording time) anda median baseline was established for eachparameter. Breathing parameters were recordedusing a 15 min protocol (5 min-acclimatizationperiod; 10 min- recording time).

ELISA. Commercial ELISA kits DuoSet ELISADevelopment System (R&D Systems, Abingdon,UK) were used according to the manufacturer’sinstructions to determine serum total MMP9 andTGF-β1 levels on days 2, 3, 5, 7 and 10 pi.

Histopathological analysis. Mice wereanesthetized (Ketamine 100 mg/kg and Xylazine 10mg/kg) and euthanized by exsanguination asfollows: lethal infection group on days 2, 3 and 5 pi,mild infection group on days 2, 3, 5, 7 and 10 pi,

Clinical and lesional dynamics in an influenza A/PR/8/34-h1N1 aerosol induced infection in mice

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control group on days 3, 5, 10 pi. Two mice perinfection group, respectively one for the controlgroup were sacrificed on the mentioned time points.In order to prevent animal suffering, a humaneendpoint was established and defined as 25% bodyweight loss following infection. A completenecropsy was performed on all animals immediatelyafter euthanasia. Special attention was given to themacroscopic aspect of vital organs. The nasalcavities and conchae, tracheae, lungs, heart and brainwere harvested, fixed in 10% formaldehyde for 24h,embedded in paraffin wax, sectioned at 4μm andstained with haematoxylin and eosin (H&E).Samples consisting of nasal cavities and conchaerequired a decalcification step which was conductedusing Osteodec O (Bio-Optica, Italia) in a volumeratio specimen/decalcifier 1:100. Histopathologicalscoring was performed by microscope evaluation ofthe slides with the optic microscope BX41 Olympus,Germany. Image acquisition and analysis were madewith an adapted Olympus DP25 camera and usingthe Olympus Cell^b soft. For lungs, nasal cavitiesand conchae examination, the establishedhistological scoring system is presented in Table 3.

Statistical analysis. Data are expressed as mean± SD. Differences between groups were assessedusing one-way ANOVA and Student’s t test, andp<0.05 was considered statistically significant.

RESULTS

Clinical Manifestation of the Two InfectiousDoses

Both lethal and mild infection groups weremonitored daily for clinical observations. On the onehand, lethal infection induced approximately 1g(about 5% of initial body weight) loss within the first72h pi, a tendency which continued until day 5 piwhen survivors reached the humane endpoint andwere euthanized (Fig. 1A). While, on the other hand,no significant changes in body weight dynamicswere observed in the mild infection group (Fig. 1A).As a component of the clinical score, body weightdrop indicates the degree of suffering. Loss ofappetite and lethargy are clear indicators of sufferingon behalf of the mice. Both of these signs contributeto inanition and body weight drop. Table 4 presentsthe frequency of clinical signs occurring withinexperimental groups, and indicates acute and moresevere symptoms in lethal than mild infection. Lethalinfection was clinically visible at 48h pi and induceda growing clinical score which reached its peak onday 5 pi (Fig. 1B). By day 3 pi, 4 out of 4 remainingmice with lethal infection have lost more than 5% oftheir initial body weight, while displaying othersigns of suffering (Table 4). The most commonclinical signs on day 3 pi were changes in generalappearance (4 out of 4 mice), accompanied byruffled fur (4 out of 4 mice), hunched posture (4 out

Table 1. Clinical signs and score used for mouse monitoring

Table 2. Selected breathing parameters for measurements

of 4 mice) and dyspnoea (4 out of 4 mice) (Table 4).By day 5 pi, surviving mice showed a completeimage of the disease and lost more than 25% ofinitial body weight. In contrast to lethal infection,mild infection seemed to be clinically inapparent,with a clinical score which was not different fromthe control group (Fig. 1B). On days 5 and 7 pi, asmall outbreak of clinical signs was seen in thisgroup. Two out of the six remaining mice began todisplay clinical signs of illness such as a modifiedgeneral appearance, accompanied by ruffled fur and

5% loss of body weight on day 5 pi (Table 4). Byday 7 pi, one out of 4 remaining mice showed moresevere signs like hunched posture, lethargy anddyspnoea (Table 4). Dehydration is a symptom thatcould be added to the list of signs used in assessinginfluenza evolution. In this study, dehydration wasnot mentioned on the main symptoms list (Table 1).As was found to affect only mice with lethalinfection (Table 4), dehydration was scored underthe “clinical signs which were not mentioned above”category.

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Table 3. Histological scoring system used for lungs, nasal cavities and conchae examination

Table 4. Distribution of clinical signs at different points in time

Paraclinical FindingsBreathing parameters. Major changes were

found at 24h pi in investigated parameters in allinfected mice. In both infection groups, tidal volume(TV) reported a significant drop at 24h pi (Fig. 2A).After this alteration, the TV values returned withincontrol range. Breathing frequency (f), in oppositionto TV, showed an increase in value at 24h pi (Fig.2B). By day 5 pi, f values reached the control rangein the mild infection group, while in the lethalinfection group, values tended to decrease. Minutevolume (MV) is a complex parameter, composed ofTV and f. The fluctuations dynamics of MV is notsignificant and this might be due to TV and f tryingto balance each other (Fig. 2C).

ELISA. A greater serum total MMP9 level wasfound in lethal compared to mild infection. The

maximum level of MMP9 was reached on day 3 pi,with values returning within control range by day 5pi (Fig. 3A). Despite the displayed variability, mildinfection does not modify serum MMP9 levels to asignificant level (Fig. 3A). Both lethal and mildinfections do not seem to alter TGF-β1 serum levels(Fig. 3B).

Histological analysis of selected organsExcept for the lungs, no gross lesions were

found during necropsy in any other organ, in allinfected mice. Different degrees of pulmonaryhyperaemia were seen throughout disease evolution,with severe interstitial bronchopneumonia producedby both infectious doses. Compared to the controlgroup (Fig. 4A), pulmonary irregular checkerboardpattern was found in the lungs of the lethal infection


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Fig. 1. Body weight and clinical signs dynamicsBALB/c mice were either exposed to saline (control group); 10-1 PR8 virus, lethal dose (PR8 10-1) and 10-3 PR8 virus,mild dose (PR8 10-3). Mice were weighed and monitored daily for clinical signs of disease. (A) Body weights are represented as mean and standard deviations, (B) Clinical signs were gathered and quantified as clinical scores of suffering,represented as mean per group(**p<0.01)

Fig. 2. Breathing parameters dynamicsBALB/c mice were exposed to saline (control group); 10-1 PR8 virus, lethal dose (PR8 10-1) and 10-3 PR8 virus, mild dose(PR8 10-3). Breathing parameters like (A) tidal volume, (B) breathing frequency and (C) minute volume were measuredat 24, 48, 78h pi and on day 5 pi. Data is represented as percentage out of a baseline established on the same mice, priorinfection (*p<0.05).

group on day 5 pi (Figs. 4C and D), and day 7 pi inmild infection (Fig. 4B). This aspect ischaracteristics of interstitial bronchopneumonia andis created by the alteration of lung consolidation andnormal foci.

With no visible macroscopic lesions,histological analysis of the nasal cavities and

conchae revealed important changes of this segmentin both infectious doses. Small quantities ofinflammatory infiltrate were found on day 2 pi inlethal infection, but gradually accumulated by day 5pi. Epithelial destruction was visible through the lossof cellular cilia and/or epithelial denudation (Fig.5B) and was constant throughout the experiment,while exudate was present in very small andinconstant quantities. On the other hand, in mildinfection inflammatory infiltrate was in smaller andfluctuating amounts, epithelial destruction wascharacterised by focal loss of cilia or denudation andexudate was present in very small amounts (Fig. 5C).The total histopathological score was higher in lethalthan in mild infection, with a maximum ofinflammatory infiltrate, epithelial destruction andexudate reached on day 3 pi for the severe influenzaform, days 3 and 10 respectively for mild infection(Fig. 6A).

Lung pathology was clearly dependent on theinfectious dose as lesion severity was greater inlethal than in mild infection throughout diseaseevolution. In mice nebulized with a lethal PR8 dose,lung inflammatory infiltrate appeared to be in bigamounts starting with day 2 pi and organised itselfaround bronchioles and blood vessel creating a“cuffing“ pattern (Fig. 5E), an aspect whichremained at a constant level throughout theinvestigation period. Destruction and desquamationof the bronchiolar epithelium was important on day2 pi and became more severe by day 3 pi, aphenomenon accompanied by the accumulation ofexudate in the bronchiolar lumen and alveoli (Figs.5E and 6B). In mild infection, the degree ofperibronchiolar and perivascular cuffing was lower

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Fig. 3. Serum total MMP9 and TGF-β1 levelsBlood was collected on day 2, 3, 5, 7 and 10 pi to determine serum levels of (A) total MMP9 and (B) TGF-β1. BALB/cmice were either exposed to saline (control group); 10-1 PR8 virus, lethal dose (PR8 10-1) and 10-3 PR8 virus, mild dose(PR8 10-3). Data is represented as mean per group (**p<0.01).

Fig. 4. Interstitial bronchopneumonia in lethal and mild influenza infection

Next to the control lung (A), gross pulmonary aspect onday 7 pi in mild infection (B) and day 5 pi in lethal infec-tion (C and D) consists of an admixture of lung consoli-dation and normal foci which resembles an irregularcheckerboard. Mice in control group were exposed tosaline, while PR8 lethal and mild doses were used in in-fected groups.


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Fig. 5. Microscopic aspects of affected organsMice were exposed to influenza PR8 or saline by nebulization and sacrificed at the indicated moments pi. Organs ofinterest were process and stained with H&E for analysis. Saline nebulized mice served as control: (A) nasal cavity, X400and (D) lung, X100. (B; E; H) histological aspects from lethal infection; (C; F; G; I) histological aspects from mild influenzainfection. (B) X400, day 3 pi, nasal cavity, severe destruction of the respiratory epithelium and polymorphonuclear exudateaccumulated in the cavity; (C) X400, day 5 pi, nasal cavity, missing cell cilia and focal regions of epithelium denudation;(E) X100, day 3 pi, necrotic and desquamated bronchiolar epithelium, exudate in the lumen, peribronchiolar and perivas-cular cuffing of mononuclear cells; (F) X100, day 5 pi, thickened interalveolar septum and perivascular inflammatorycuffing found in the mild infection lung; (G) X200, day 2 pi, cardiac muscle fibres with hyalinosis; (H) X100, day 5 pi,cerebral cortex, multifocal mononuclear infiltrate; (I) X100, day 10 pi, cerebral cortex, multifocal mononuclear infiltrate.

than in lethal infection (Fig. 5F). The inflammatoryinfiltrate was present in small amounts on day 2 piand started to accumulate gradually by day 5 pi.Epithelial destruction followed a similar path, whileexudate was in almost undetectable amounts in thefirst 3 days pi (Fig. 6B). The generalhistopathological score was higher in lethal than inmild infection, with a maximum of inflammatoryinfiltrate, epithelial destruction and exudate reachedon day 3 pi for the lethal influenza form, day 5 pirespectively for the milder infection (Fig. 6B). Thedynamics of microscopic modifications determinedby both viral loads follows an ascending-descendingpath which does not correlate with gross lesionsfound in the lungs.

Microscopic alterations were also found intissues such as the heart muscle and cerebral cortex.

While the myocardium showed signs of multifocalincipient muscular degeneration in the lethalinfection group on days 2 and 3 pi, mild infectionbrought the lesions by day 10 pi to a more advancedstage, that of muscular hyalinosis (Fig. 5G).Furthermore, focal and multifocal mononuclearinfiltrate was found in the cerebral cortex of micefrom both test groups. Four out of 6 mice with lethalinfection, respectively 4 out of 10 mice with mildinfection displayed focal inflammatory infiltrate inthe first 24-48 hours of infection, which multipliedby day 10 pi (Figs. 5H and I).

DISCUSSIoN

With the present study, we offer insight onlesion dynamics in influenza mouse models and PR8

pathogenicity. Symptomatology expressed by thelethal infection group recreates a clearer reflectionof internal lesions than the clinical picture seen inmild infection. Body weight drop, although a non-specific symptom, is the most commonly used signof distress in mice [11, 12, 19]. Weight loss can beused to monitor the installation and evolution ofdisease, but can also indicate the degree of sufferingas it is generally used to establish the humaneendpoint [20, 21]. Our data suggest that thisparameter is useful only in high dose PR8 infections,while lower doses do not determine bodyweightdrop. Furthermore, the mouse is known not todevelop human-like flu symptoms, but to ratherdisplay a non-specific list of signs like lethargy,hunched posture, inanition and ruffled fur [11]. Inaddition to this drawback that makes diseaseevaluation difficult, we have found that a mild virusdose determines an asymptomatic infection, while ata lethal virus dose symptoms appear in the first 48hpi and worsen by day 5 pi. This course of infection

evolution is common in influenza mouse models [11,22, 23]. A clinical sign which was not displayed inthe list from Table 1 on the account of being lesscommon, but falls into the “not mentioned above”category is dehydration. While easy to spot and avery good indicator of the degree of animal distress[24], dehydration is condoned in influenza mousemodels. Without causing additional stress to themouse, just by looking at it or by assessing skincrease elasticity and withdraw speed after restraint,the presence and degree of dehydration can bedetected [25]. In our study, this symptom wasspotted only in severely affected mice (Table 4 -clinical signs not mentioned above).

Paraclinical investigation like breathingparameters determinations did not bring newinformation in the process of disease dynamicsevaluation. Significant modifications were seen at24h pi in both infection groups, but further variationswere insignificant. It is possible that changes seen at24h pi were due to pulmonary load after

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Fig. 6. Histopathological scores of the nasal cavities, nasal conchae and lungs Mice were sacrificed on day 2, 3, 5 pi in the lethal infection group (PR8 10-1); respectively day 2, 3, 5, 7 and 10 pi in themild infection group (PR8 10-3). Microscopic lesion of the (A) nasal cavities, nasal conchae and (B) lungs were quantifiedas histopathological scores taking into account the degree of inflammatory infiltrate, epithelial destruction, presence andquantity of exudate (Table 3). Data are representative of 2 mice per moment in each test groups. The control group scorerepresents the average from 3 mice, sacrificed on days 3, 5 and 10 after nebulization.

nebulization, as viral allantoic fluid was diluted insaline and administered by aerosols. It is known thatlung absorption of inhaled particles depends on theproperties of the particles in question like: dispersionof particles, concentration, osmolarity, density, pHand substances used as vehicles [26]. Moreover,while tidal volume dropped, breathing frequencyincreased, probably to counterbalance the firstreaction, and as a global effect of this act, minutevolume, a composed parameter of the mentionedtwo, did not suffer significant variations. Influenzapathology can be attributed to the combination ofviral pathogenicity, worsen by the viral clearanceactivity of the host innate immune response [27].During the acute inflammatory phase, extracellularmatrix undergoes successive breakdown andremodelling processes operated by neutralproteinases like matrix metalloproteinases (MMPs)[28]. MMP9 is involved in the process ofextracellular matrix remodelling in bothphysiological and pathological conditions [29]. Inthe lung, MMP9 is not expressed in homeostaticconditions, but may be released upon inflammation[30]. In influenza infections, local lung MMP9 levelswere found at their highest between days 6-9 pi [31].In our study, similar to literature findings, MMP9levels were elevated, but at serum level. This growthin serum total MMP9 was seen only in lethalinfection, where it reached its peak on day 3 pi andcorresponded with the highest histopathologicalscore recorded in the lungs. We also determinedTGF-β1 serum levels. TGF-β1 is a cytokine knownto have multiple functions in proliferation,differentiation, and other cell activities [32]. Inrespiratory diseases like viral infections andallergies, TGF-β1 expression was shown to beincreased in vitro, in 24h human bronchial epithelialcell cultures [33, 34]. In contrast, TGF-β1 displayeda latent activation in vivo, in mouse serum [35]. Ourdata, opposed to literature findings, indicateunaltered TGF-β1 serum levels in both infectiongroups, at all investigated time points.

Organ analysis revealed inconstant macroscopicalterations, found only in the lungs of infectedanimals. Pulmonary irregular checkerboard pattern,an aspect characteristics of interstitialbronchopneumonia and created by the alternation oflung consolidation and normal foci, was found in thelungs of mice form both infection groups. Similar toour observations, macroscopic lung hyperaemia andconsolidation was described in a comparativevirulence study of two mouse-adapted variants of

influenza A virus strain, in which infected micedisplayed lesions in the last stage of disease that didnot correspond with their microscopic reflexion [36].Compared to lung destruction, the nasal cavitieswere far less affected by the virus. While in humansvirus multiplication occurs in the upper respiratoryepithelium [37, 38] it was demonstrated that in micevirus proliferation is concentrated in the lowerrespiratory tract [39-41]. In addition to this, by usingthe nebulizer for aerosol distribution and infectioninduction, a method which mimics natural infection[42, 43] nasal epithelium lesions were minimal,perhaps due to the nebulizing speed which mighthave directed the virus deep into the airways. Thedose dependent characteristic of organ lesiondynamics described in the present article was alsoreported in mice intranasally infected with PR8,when a greater virus dose determined a highermortality rate and symptoms to develop quicker thanin a milder infection [22]. The lung is the primaryinvestigation site when it comes to assessinginfluenza pathology. Depending on the virus strainand dosage used, histologically detectable lunglesions appear early in disease evolution and quicklyincrease in severity [22, 23, 44]. Focal lung lesionswere described at 24-72h pi and evolved to a diffusecharacteristic in the last stages of influenza, in dyingor dead mice when viral pneumonia progresses todiffuse alveolar damage (DAD) [22]. Althoughlesion dynamics in our study was similar to thatdescribed by Fukushi et al., hyaline membrane wasnot found in the alveoli, so as the climax of diseaseevolution in both infection groups was not acomplete DAD picture. Lesion reflection inexpressed symptomatology, quantified through theclinical score, was significantly increased in lethalinfection (Figs. 1B and 6B). While in mild infection,the highest histopathological score did coincide withthe two small outbreaks of clinical signs, thisincrease was not statistically significant (Figs. 1Band 6B). In humans, multiple organ failure has beenreported in H5N1 [45, 46] and pandemic H1N1 [47]infections, with lesions being found in the myocardiaand the brain of dead patients. Similar to humans,A/PR/8/34 nebulized mice were found to displaydegenerating and necrotic myofibers starting withday 3 pi [48]. These lesions represent incipient signsof myocarditis, as does the hyalinoses found in ourstudy, in the heart of both lethal and mild infectedmice starting with day 2 pi. In the brain, the presenceof inflammatory infiltrate in infected animals isassociated with HPAI viruses [49, 50] but neural


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degeneration and inflammation has been reported inA/PR/08/1934 mouse models [51], therefore theinfiltrate seen in the cortex of infected mice from ourstudy could be an effect of the viral infection.

In conclusion, the present study underlines thedose depend characteristic of flu manifestations inmice and shows that although clinical inapparent,mild infection causes multiple organ microscopiclesions. This observation could be of use for studiesin which the survival of the animals and theconcomitant existence of characteristic influenzalesion are essential. Whole-body plethysmography,although a method broadly used in lung pathologystudies, did not seem to have the sensitivity to detectchanges in PR8 infected mice. Furthermore, serumdetermination of total MMP9 could be a goodpredictor of the degree of lung alteration ininfluenza. The observations made within this studymay contribute to mouse influenza PR8 modelrefinement.

ACKNoWLEDGEMENTS

This study was supported by the PN 09220201grant funded by the Ministry of Research andEducation.

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ISOLATION OF Halobacillus truEpEri DS-1 - A SIDEROPHORE PRODUCING HALOPHILIC BACTERIUM

Leila Mirfeizi1, Mohammad Ali Amoozegar1*, Ahmad Ali Pourbabaee2, hamid Babavalian3, Mehrdad Moosazadeh Moghaddam4

1Extremophiles lab, Department of Microbiology, college of science, university of tehran, tehran, iran; 2Department of soil science, university college of agriculture & Natural resources, university of tehran, Karaj, iran;

3applied Virology research center, baqiyatallah university of Medical sciences, tehran, iran;4applied biotechnology research center, baqiyatallah university of Medical sciences, tehran, iran

REzUMATPentru capturarea şi solubilizarea fierului,

multe organisme utilizează un sistem eficientconstând din compuşi cu o înaltă afinitate pentrufier, numiţi siderofori. Având în vedere importanţametaboliţilor produşi de microorganismelehalofile şi a aplicaţiilor biotehnologice aleacestora, scopul acestui studiu este izolareabacteriilor halofile producătoare de siderofori dinsoluri agricole aflate în regiunea Eshtehard Karaj,Iran. Iniţial, s-a realizat izolarea bacteriilor din sol,urmată de separarea bacteriilot halofile de celehalotolerante, obţinându-se şapte izolate.Producerea de siderofori a fost măsurată utilizândteste universale (testele O-CAS şi CAS), cuajutorul cărora au fost identificate şase izolatehalofile producătoare de siderofori, dintre care s-a selectat tulpina DS-1, având cea mai înaltăproducţie de siderofori. S-au efectuat testespecifice pentru identificarea caracterului chimical sideroforului, iar rezultatul final al analizeimoleculare a genei codante pentru ARNr 16S aconfirmat faptul că izolatul halofil aparţineagenului Halobacillus, având o similaritate de99,8% cu H. trueperi DSM 10404T.

*corresponding author: Mohammad Ali Amoozegar, Extremophiles Lab, Department of Microbiology, College of Science, University of Tehran, Tehran, Iran. Email: [email protected]

Keywords: halophilic bacteria, hydroxamate siderophore, O-CAS

ABSTRACTTo sequester and solubilize ferric iron many

microorganisms utilize an efficient system consistingof compounds with high iron affinity, calledsiderophores. By considering the importance of themetabolites produced by halophilic microorganismsand their biotechnological applications, the aim of thisstudy was the isolation of siderophore producinghalophilic bacteria from agricultural soils ofEshtehard Karaj region of Iran. Initial isolation of soilbacteria using serial dilutions was performed,followed by separation of halophilic bacteria fromhalotolerant ones, which resulted in seven isolates.Production of siderophores was measured usinguniversal tests including O-CAS plates and CASassay solution. By exploiting the CAS solutionmethods, six siderophore producing halophilicisolates were identified, among which strain DS-1,with the highest siderophore production was selected.Specific tests were performed for identifying thechemical nature of siderophore and the final result ofmolecular analysis of 16S rRNA gene confirmed thatforegoing halophile isolate belonged to Halobacillusgenus with 99.8% similarity to H. trueperi DSM10404T.

INTRoDUCTIoN

Iron is an essential element required for keybiological processes, including amino acid synthesis,oxygen transport, respiration, nitrogen fixation,methanogenesis, citric acid cycle, photosynthesis,and DNA biosynthesis. However, obtaining ironpresents challenges for the majority ofmicroorganisms because of the insolubility of iron(III) at physiological pH in aerobic environments

which severely limits the availability of this essentialnutrient [1]. Therefore, microorganisms have todevelop strategies for solubilization and uptake ofthis mineral iron. This is accomplished bybiosynthesis and excretion of nonporphyrin,nonprotein iron-containing or iron-bindingcompounds [2], called siderophores, which act asextracellular solubilizing agents for iron fromminerals or organic compounds under conditions of

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iron limitation [3] and to make it available to themicrobial cell [4]. Typically, microbial siderophoresare classified as catecholates, hydroxamates, and α-carboxylates, depending on the chemical nature oftheir coordination sites with iron [5, 6].

Halophilic bacteria with high salt necessity forgrowth and production of metabolites such assiderophores have been less studied. In this study weinvestigated siderophores production and chemicalcharacterization of siderophores in halophilicbacteria.

MATERIALS AND METhoDS

Microorganisms and growth conditionsIsolation was accomplished by serial dilution

and plating technique on 12% moderate halophile(MH) medium containing (g/L in distilled water):NaCl, 101; MgCl2, 7; MgSO4, 9.6; CaCl2, 0.36;KCl, 2; NaHCO3, 0.06; proteose peptone, 5; yeastextract, 10; glucose, 1; and agar 15, pH adjusted to7.5 by 1M Tris.

Separation of halophilic isolates from halo -tolerant ones was done by cultivation of each isolateon minimal medium (5 g/L yeast extract) containing0 and 20% NaCl. The isolate which was able to growon 0 and 20% salt was considered halotolerant andthat which was only able to grow on 20% salt wasconsidered halophilic, the latter isolates being notable to grow on salt free medium [7, 8].

Siderophore detectionQualitative detection of siderophore (plate

assay) method [9]: strain DS-1 cultivated on 12%MH medium with some modifications, such as

glucose replaced with calcium lactate 8 g/L, theamount of yeast extract and peptone reduced up to 5and 1.5 g/L respectively.

Yeast extract and peptone were deferrated byextraction with 3% (w/w) 8-hydroxyquinoline inchloroform solution for 24 h [10] as well allglassware objected to acid wash by HCl 6M.

Whereas all halophilic isolates were Gram-positive, two-layer plate method was used in whichcultivated basal MH medium was covered bychrome azurol sulfonate (CAS) agar medium despiteneutral portion of secondary layer was omitted.

After inoculation all plates were incubated at30°C for 4 days, then each plate overlaid bysecondary layer by 15 ml chrome azurol sulfonate(O-CAS method) [11], and finally incubated at 30°Cagain. After two to three days, halo formation by thecolor changing from blue to orange was observed(Fig. 1A).

Determination of siderophore in solutionCAS assay solutionFor preparation 100 ml of CAS assay solution

6 ml volume of 10 mM hexadecyl trimethylammonium bromide (HDTMA) solution was placedin a 100 ml volumetric flask and diluted with water.A mixture of 1.5 ml iron (III) solution (1 mM FeCl3.6H2O, 10 mM HCl) and 7.5 ml 2 mM aqueous CASsolution was slowly added under stirring. A 4.307 gquantity of anhydrous piperazine was dissolved inwater and 6.25 ml of 12 M hydrochloric acid wascarefully added, this buffer solution (pH 5.6) wasrinsed into the volumetric flask which was then filledwith water to afford 100 ml of CAS assay solution(10). The solutions were stored in polyethylene

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Fig. 1. DS-1 on 12% salt MH medium, the color change of chrome azurol S medium from blue to orange indicatessiderophore releasing in medium (A). CAS assay solution, proportion of 1:1DS-1 supernatant, non-inoculated MH mediumas negative control, and desferoxamine as positive control with CAS solution from left to right respectively (B).

A B

bottle in the dark. Siderophore produced by isolatesvia fermentation was examined. One lop of eachstrain was inoculated into 50 ml modified 7.5% MHin 250 ml flask, for five days and shaken at 150 rpmat 30°C. The cell suspensions were centrifuged at8000 rpm for 10 min and 1ml of supernatant mixedwith 1ml CAS assay solution. Absorbance at 630 nmwas measured after 1 h of incubation at roomtemperature (Fig. 1B).

Siderophore units are defined as [(Ar-As/Ar)]×100 = % siderophore units, which related toabsorbance of reference (Ar) and absorbance ofsample (As). Percentages of siderophore units lessthan 10 were considered as negative and in this caseno change in the blue color of CAS solution wasobserved [12].

FeCl3 testTo 0.5 ml of culture filtrate, 0.5 ml of 2%

aqueous FeCl3 solution was added and examined forthe appearance of orange or reddish brown colorwhich was positive indication of siderophoreproduction [13]. The results are shown in Fig. 3.

Specified functional group of siderophoreTo determine hydroxamate nature of

siderophores Csaky test was applied [14]. Catecholnature of siderophores was determined by Arnowtest [15] and Shenker method [16] used to identifythe carboxylate siderophores. In all cases noninoculated MH medium was selected as negativecontrol, for hydroxamate type desferoxamine and forcatecholate nature catechol were positive controls(Fig. 2).

Salt Stress on Siderophore excretionSalt stress has a significant effect of halo

diameter of siderophore on CAS agar medium

(Fig. 4A). Iron free MH medium was prepared withdifferent total salt concentration ranging from 0.5 to20% and inoculated by 100 µl of DS-1 suspensionin three repeat and after growth overlaid accordingto O-CAS technique [11]. The siderophore haloswere evaluated every 48 h of incubation at 30°C fortwo weeks (Fig. 4B).

Molecular identification Total DNA was extracted according to modified

Marmur method [17]. PCR reaction was performedusing 27F (5´-AGAGTTTGATCMTGGCTCAG-3´)and 1492R (5´-GGTTACCTTGTTACGACTT-3´)universal primers with the following condition:Initial denaturation at 94 °C (5 min); 25 cyclesincluding denaturation at 94 °C (1 min), annealingat 56 °C (1 min) and extension at 72 °C (90 seconds);final extension at 72 °C (10 min). The purified PCRproduct was cloned using Thermo Scientific Ins TAclone PCR Cloning Kit and extracted plasmid wassequenced by an ABI 3730/3730XL DNA sequencerat Bioneer (Daejeon, South Korea). Identification of

Isolation of Halobacillus trueperi DS-1 - a siderophore producing halophilic bacterium

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Fig. 3. FeCl3 test, no change in color was observed in comparison to positive control

(desferal) in response to FeCl3 reagent

Fig. 2. Identification of siderophore's functional group by positive Csaky test (A) and negative Arnow test (B)

A B

phylogenetic neighbors was initially perfomed byBLAST [18] and mega BLAST [19] programs. Thealigned 16S rRNA gene sequences of DS-1 withmost closely related type strains were analyzed usingMEGA version 6 program [20].

RESULTS AND DISCUSSIoN

Sampling was performed on nine agriculturalsoils by cultivation wheat, barley and cotton alsofarm fallow and salty soil surrounding the farms. Thesoil samples after measuring EC [21] showedvarying degrees of salinity [22], including non-salt,low salt, moderate salt, high salinity, and extremelyhigh salinity (data not shown here). Among 90isolates which were obtained by isolation techniqueseven were identified as halophile isolates. Thesemoderate halophile isolates were obtained frombarley, fallow and saline soils mean while strain DS-1 was isolated from soil sample of fallow farm.Strain DS-1 due to the better performance ofsiderophore production on O-CAS and in CASsolution methods in the same conditions from sevenisolates was selected.

All Gram-positive isolates were able to produceorange halo on O-CAS agar plates by differentdiameters, but in solution medium siderophore unitpercentage which produced by SS-7 was less than10% so considered as nonsiderophore producerhalophile isolate (Table 1).

As can be seen in (Fig. 2) Csaky test bydisplaying hydroxamate nature of siderophorethrough chemical methods for siderophorediagnostic tests was thrived. In all tests negativecontrol was non inoculated medium after addingreagents, desferoxamine mesylate for Csaky test and

catechol for Arnow test were instances for positivecontrol.

FeCl3 test for all isolates was negativefurthermore non-suitability of FeCl3 test forassaying haloarchaeal siderophores was reportedbefore [23].

In salt concentration between 2.5-7.5% thehighest siderophore production was observed,accordingly, this concentration range is as optimalamount of salt that DS-1 strain has the highestproduction capability. Generally, strategies whichhalophilic prokaryotes employed in saline environ -ments for survival include accumulation of highconcentrations of inorganic ions in the cytoplasmused by the aerobic archaea and also anaerobicbacteria [24], and organic solutes that are eitherproduced by the cells or accumulated from themedium. Osmoprotectants which are known ascompatible solutes include amino acids, sugars,polyols, betaines and ectoines as well as derivativesof some of these compounds [25]. It can be inferredthat with increasing salinity bacterial cells can usesiderophores as a source of compatible solute. Sothese compounds are accumulated or trapped in thecell and used as a strategy to deal with salinity. Thismay just be temporary and if iron tension to the cellsbecomes more serious, with more energyconsumption other compounds embed as compatiblesolute.

The 16S rRNA gene sequence analysis showedthat strain DS-1 with 1572 nucleotides belonged toHalobacillus genus and was closely associated withH. trueperi DSM 10404T (99.8 % similarity value).To date, few studies have investigated siderophoreproduction in halophilic bacteria. Gascoyne et al.

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Fig. 4. The difference in the haloes diameter of siderophore produced by strain DS-1: by increasing the percentageof salt in the medium, diameter of halo decreased significantly. Plates contain 5% and 20% salt MH medium fromleft to right (A). Ranges of salinity and its effect on haloes (B).

A B

Isolation of Halobacillus truperi DS-1 - a siderophore producing halophilic bacterium

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[26] report siderophore production in an alkalineenvironment while Dave et al.[23] recently detectedsiderophore production in a number of halophilicArchaea. Siderophore production was identified inthe moderately halophilic, alkaliphile Halomonascampisalis [27].

Strain DS-1 was deposited in the open culturecollection of microorganisms of IRAN underaccession number IBRC- M 10964.

ACKNoWLEDGEMENTS

We thank Iranian Biological Resource CenterIBRC for technical assistance in molecularidentification and University College of Agricultureand Natural Resources Karaj, as well as SoilBiotechnology Laboratory for cooperating with thisproject.

REFERENCES

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2. Neilands JB. Microbial iron compounds. Ann. Rev.Biochem 50 (1981): 715-31.

3. Jorge CH. Genetics and molecular biology ofsiderophore-mediated iron transport in bacteria.Microbiological reviews 53.4 (1989): 517-530.

4. Neilands JB. Siderophores: structure and function ofmicrobial iron transport compounds. Journal ofBiological Chemistry 270.45 (1995): 26723-26726.

5. Günther W. Structural and stereochemical aspects ofiron transport in fungi. Biotechnology advances 8.1(1990): 207-231.

6. Engy A., Holmström SJM. Siderophores inenvironmental research: roles and applications.Microbial biotechnology 7.3 (2014): 196-208.

7. Babavalian H. Isolation and identification ofmoderately halophilic bacteria producing hydrolyticenzymes from the largest hypersaline playa in Iran.”Microbiology 82.4 (2013): 466-474.

8. Babavalian H. Comparison of bacterial biodiversityand enzyme production in three hypersaline lakes;

Urmia, Howz-Soltan and Aran-Bidgol.” Indian journalof microbiology 54.4 (2014): 444-449.

9. Pal RB., Gokarn K. Siderophores and pathogenecityof microorganisms. Journal Bioscience andTechnology 1.3 (2010): 127-134.

10. Bernhard S., Neilands JB. Universal chemical assayfor the detection and determination of siderophores.Analytical biochemistry 160.1 (1987): 47-56.

11. Pérez-Miranda S. O-CAS, a fast and universalmethod for siderophore detection.” Journal ofmicrobiological methods 70.1 (2007): 127-131.

12. Machuca A., Milagres AMF. Use of CAS‐agar platemodified to study the effect of different variables onthe siderophore production by Aspergillus.” Lettersin applied microbiology 36.3 (2003): 177-181.

13. Sujatha N., Ammani K. Siderophore production bythe isolates of fluorescent Pseudomonads.International Journal of Current Research andReview 5.20 (2013): 1.

14. Csaky TZ. On the estimation of boundhydroxylamine in biological materials.” ActaChemica Scandinavica 2.5-6 (1948): 450-454.

15. Earle AL. Colorimetric determination of thecomponents of 3, 4-dihydroxyphenylalanine-tyrosinemixtures. Journal of Biological Chemistry 118.2(1937): 531-537.

16. Shenker M. Chemical structure and biologicalactivity of a siderophore produced by Rhizopusarrhizus. Soil Science Society of America Journal59.3 (1995): 837-843.

17. Marmur J. A procedure for the isolation ofdeoxyribonucleic acid from microorganisms, J. Mol.Biol. 3 (1961): 208–218.

18. Altschul SF. Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs.Nucleic acids research 25.17 (1997): 3389-3402.

19. Zhang Z., Schwartz S., Wagner L., Miller W. Agreedy algorithm for aligning DNA sequences. JComput Biol (2000): 7(1-2):203-14.

20. Tamura K., Stecher G., Peterson D., Filipski A.,Kumar S. MEGA6: Molecular EvolutionaryGenetics Analysis version 6.0. Mol Biol Evol. 30.12(2013): 2725-9.

21. Rhoades JD., Chanduvi F. Soil salinity assessment:Methods and interpretation of electrical conductivitymeasurements. Vol. 57. Food & Agriculture Org.,1999.

Table 1. Siderophore production in solid and liquid medium by halophile isolates and quantity of siderophores

22. Karlen DL. Soil quality: a concept, definition, andframework for evaluation (a guest editorial). SoilScience Society of America Journal 61.1 (1997): 4-10.

23. Dave BP., Anshuman K., Hajela P. Siderophores ofhalophilic Archaea and their chemicalcharacterization. Indian journal of experimentalbiology 44.4 (2006): 340.

24. Oren A. Life at high salt concentrations. TheProkaryotes. Springer New York, 2006. 263-282.

25. Santos H., Milton SDC. Compatible solutes oforganisms that live in hot saline environments.Environmental Microbiology 4.9 (2002): 501-509.

26. Gascoyne DJ., Connor JA., Bull AT. Isolation ofbacteria producing siderophores under alkalineconditions. Applied microbiology and biotechnology36.1 (1991): 130-135.

27. Richards AM. Identification and StructuralCharacterization of Siderophores Produced byHalophilic and Alkaliphilic Bacteria. Diss.Washington State University, 2007.

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115


SUBJECT INDEX

In memoriam Prof. Ioan CantacuzinoTHE CAPITAL OF MOLDAVIA AND THE HONOURING OF THE MEMORY OF PROFESSOR IOAN CANTACUZINORichard Constantinescu ............................................................................................................................... 59

MICROBIOLOGY

RETROSPECTIVE STUDY REVEALS THE CIRCULATION OF NOROVIRUS GENOTYPE GII.P21-GII2 IN ROMANIASorin Dinu, Camelia Szmal, Maria Damian, Gabriela Oprişan .................................................................. 5

THE IMPACT OF THE MOLECULAR DIAGNOSIS IN THE SURVEILLANCE OF THE CLOSTRIDIUM DIFFICILE INFECTIONAnda Băicuş, Monica Cîrstoiu, Katia Lambru, Flaviu Plata, Cătălin Florin Cîrstoiu ................................ 12

EXPRESSION SIGNATURE OF SOME IMMMUNITY GENES TRIGGERED IN APIS MELLIFERACARPATICA MODEL BY PSEUDOMONAS ENTOMOPHILA EXPERIMENTAL INFECTIONAlina Neagu, Attila Cristian Raţiu, Mihaela Raluca Mihalache (Radu), Veronica Lazăr, Mariana Carmen Chifiriuc, Alexandru Al. Ecovoiu .................................................................................... 18

RESISTANCE PATTERN OF MULTI-DRUG RESISTANT STRAINS OF MYCOBACTERIUM TUBERCULOSIS AND CHARACTERISTICS OF PATIENTS WITH MULTI-DRUG RESISTANT TUBERCULOSISAdriana Moisoiu, Cristina Iulia Mitran, Mădălina Irina Mitran, Mihaela Roxana Huhu, Octavian Costin Ioghen, Adelina-Silvana Gheorghe, Mircea Tampa, Simona Roxana Georgescu, Mircea Ioan Popa ........................................................................................................................................ 25

EFFECTS OF PSEUDOMONAS AERUGINOSA INFECTION ON THE EXPRESSION PROFILE OF TRANSIENT RECEPTOR POTENTIAL-LIKE (TRPL) GENE FROM DROSOPHILA MELANOGASTERMihaela Raluca Mihalache (Radu), Attila Cristian Raţiu, Alina Neagu, Veronica Lazăr, Mariana Carmen Chifiriuc, Alexandru Al. Ecovoiu .................................................................................... 32

CHARACTERIZATION OF MRSA STRAINS BY PHENOTYPIC AND PCR-BASED METHODSCătălina Luncă, Luminiţa Smaranda Iancu, Teodora Vremeră, Cristina Gabriela Tuchiluş, Olivia Dorneanu .............................................................................................. 37

116


POLYPLOIDIZATION OF SK-N-MC HUMAN NEUROBLASTOMA CELLS INFECTED WITH HERPES SIMPLEX VIRUS 1Zaven Karalyan, Roza Izmailyan, Elena Karalova, Liana Abroyan, Lina Hakobyan, Aida Avetisyan, Zara Semerjyan ................................................................................................................ 44

ISOLATION OF HALOBACILLUS TRUEPERI DS-1 – A SIDEROPHORE PRODUCING HALOPHILIC BACTERIUMLeila Mirfeizi, Mohammad Ali Amoozegar, Ahmad Ali Pourbabaee, Hamid Babavalian, Mehrdad Moosazadeh Moghaddam ........................................................................................................... 109

IMMUNOLOGY

CLINICAL AND LESIONAL DYNAMICS IN AN INFLUENZA A/PR/8/34-H1N1 AEROSOL INDUCED INFECTION IN MICEIoana Sonya Ciulean, Elvira Gagniuc (Gubceac), Manuella Militaru, Crina Stăvaru ................................ 97

THE 46TH ANNUAL IMMUNOLOGY CONFERENCE

Abstracts ..................................................................................................................................................... 63Author Index ............................................................................................................................................... 95

SHORT COMMUNICATION

COULD THE GENEXPERT SYSTEM BE A NEW TOOL FOR POLIOVIRUS DETECTION IN THE SEWAGE WATER?Anda Băicuş ................................................................................................................................................. 52

SUBJECT INDEX ....................................................................................................................................... 115AUTHOR INDEX ....................................................................................................................................... 117


117

AAbroyan Liana 44Amoozegar Mohammad Ali 109Avetisyan Aida 44

BBabavalian Hamid 109 Băicuş Anda 12, 52

CChifiriuc Mariana Carmen 18, 32 Ciulean Ioana Sonya 97Cîrstoiu Cătălin Florin 12 Cîrstoiu Monica 12 Constantinescu Richard 59

DDamian Maria 5Dinu Sorin 5Dorneanu Olivia 37

EEcovoiu Al. Alexandru 18, 32

GGagniuc (Gubceac) Elvira 97Georgescu Simona Roxana 25Gheorghe Adelina-Silvana 25

HHakobyan Lina 44Huhu Mihaela Roxana 25

IIancu Luminiţa Smaranda 37Ioghen Octavian Costin 25Izmailyan Roza 44

KKaralova Elena 44 Karalyan Zaven 44

LLambru Katia 12Lazăr Veronica 18, 32Luncă Cătălina 37

MMihalache (Radu) Mihaela Raluca 18, 32Militaru Manuella 97Mirfeizi Leila 109Mitran Cristina Iulia 25 Mitran Mădălina Irina 25Moghaddam Mehrdad Moosazadeh 109Moisoiu Adriana 25

NNeagu Alina 18, 32

OOprişan Gabriela 5

PPlata Flaviu 12Popa Mircea Ioan 25Pourbabaee Ahmad Ali 109

RRaţiu Attila Cristian 18, 32

SSemerjyan Zara 44Stăvaru Crina 97Szmal Camelia 5

TTampa Mircea 25Tuchiluş Cristina Gabriela 37

VVremeră Teodora 37

AUTHOR INDEX

118

ROMANIAN ARCHIVESOF

MICROBIOLOGYAND

IMMUNOLOGY


in 1928


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Professor Ion Cantacuzino(1863-1934)

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PROFESSOR ION CANTACUZINO - roami.ro · PROFESSOR ION CANTACUZINO in 1928 VOLUME 75 - Issue 3-4 July - December 2016 Published quarterly by Institutul CANTACUZINO Spl. Independenþei