Official Journal of the Romanian Society of Physiological Sciences

CHIEF EDITOR
CO-CHIEF EDITORSASSOCIATE EDITORSEXECUTIVE EDITORSFRANCISC SCHNEIDERIOANA SISKACARMEN TATUMIHAI NECHIFORSORIN RIGA
FLORINA bOjIN
GAbRIELA TANASIEDACIANA NISTORCALIN MUNTEANEDITORIAL BOARD
ARDELEAN AURELBADIU GHEORGHEBĂDĂRĂU ANCA
BENEDEK GYÖRGYBENGA GHEORGHEBUNU CARMEN
COJOCARU MANOLECUPARENCU BARBUCONSTANTIN NICOLAE
HAULICĂ ION
IANCĂU MARIAMIHALAŞ GEORGETA
MUNTEAN DANINAMUREŞAN ADRIANANESTIANU VALERIUOPREA TUDOR(Arad)(Constanţa)(Bucureşti)(Szeged)(Cluj)(Timişoara)(Bucureşti)(Oradea)(Bucureşti)(Iaşi)(Craiova)(Timişoara)(Timişoara)(Cluj)(Craiova)(New Mexico)PĂUNESCU VIRGILPETROIU ANA
POPESCU LAURENŢIU
RÁCZ OLIVERRIGA DANSABĂU MARIUS
SIMIONESCU MAIA
SIMON ZENOSAULEA I. AURELSWYNGHEDAUW BERNARDTANGUAY M. ROBERTTATU FABIAN ROMULUS
VLAD AURELIANVOICU VICTORZĂGREAN LEON

(Timişoara)(Timişoara)(Bucureşti)(Košice)(Bucureşti)(Tg. Mureş)(Bucureşti)(Timişoara)(Chişinău)(Paris)(Canada)(Timişoara)(Timişoara)(Bucureşti)(Bucureşti)AccREDITED By cNcSIS - B+cATEgORy - cODE 240
http://journals.indexcopernicus.com/karta.php?action=masterlist&id=4929
http://www.ebscohost.com/titleLists/a9h-journals.pdf
Publication data: Fiziologia (Physiology) is issued quarterly
Subscription rates: Subscriptions run a full calendar year. Prices
are give per volume, surface postage included.
Personal subscription: Romania - 100 RON, Outside
Romania - 35$ (must be in the name of, billed to, and paid by anindividual. Order must be marked “personal subscription”)
Institutional subscription: 50$ (regular rate)
Single issues and back volumes: Information on availability
and prices can be obtained through the Publisher.
Change of address: Both old and new address should be statedand send to the subscription source.
Bibliographic indices: We hope this journal will be regularly listedin bibliographic services, including “Current Contents”.
Book Reviews: Books are accepted for review by special
agreement.
Advertising: Correspondence and rate requests should be
addressed to the Publisher.

Fiziologia(80)‑ Physiology 2010supplement
2013.24.4
2014.24.1(81)
 Fiziologia- Physiology

1. FOR SUBSCRIPTION ADDRESSHVB Bank TIMISOARA
RO 21 BACX 0000000218508250
TIMISOARA – ROMANIAPENTRU REVISTA„FIZIOLOGIA – PHYSIOLOGY”
2. CORRESPONDENCE SHOULD BE
ADDRESSED TO THE CHIEF EDITOR
PROF. DR. FRANCISC SCHNEIDER
PO BOX 135
300024 – TIMISOARA – ROMANIA
e-mail: carmen.tatu@umft.ro
Editura EUROSTAMPA
Tel./fax: 0256-204816
ISSN 1223 – 2076

11

Official Journal of the Romanian Society of Physiological Sciences

Instructions to Authors
Submission: Only original papers in English areconsidered and should be sent to:Prof. dr. Francisc Schneider
Chief Editor of “Fiziologia”
PO Box 135
300024, TIMISOARA, ROMANIA
Tel./Fax: 40-256/490507
Manuscripts should be submitted in triplicate setsof illustrations (of which one is an original), typewritten
doublespaced on one side of the paper, with a wide margin.
Conditions: All manuscripts are subject to editorial review.
Manuscripts are received with the explicit understanding thatthey are not under simultaneous consideration by any otherpublication. Submission of an article for publication implies thetransfer of the copyright from the author to the publisher upon
acceptance. Accepted papers become the permanent propertyof “Fiziologia” (Physiology) and may not be reproduced by anymeans, in whole or in part, without the written consent of the
publisher. It is the author’s responsibility to obtain permission to
reproduce illustrations, tables, etc. from other publications.
Arrangement:
Title page: The first of each paper should indicate the title(main title underlined), the authors’ names, and the institutewhere the work was conducted. A short title for use as runninghead is also required.
Keywords: for indexing purposes, a list of 3-10 keywords
in English and Romanian is essential.
Abstract: Each paper needs abstract and title in Romanianand English language, fonts size 9, Arial Narrow.
Bady text: fonts size 10, Arial Narrow.
Small type: Paragraphs which can or must be set in smaller
type (case histories, test methods, etc.) should be indicated witha „p” (petit) in the margin on the left-hand side.
Footnotes: Avoid footnotes. When essential, they are
numbered consecutively and typed at the foot of the appropriate
page, fonts size 8, Arial Narrow.
Tables and illustrations: Tables (numbered in Roman
numerals) and illustrations (numbered in Arabic numerals)
should be prepared on separate sheets, fonts size 9, Arial Narrow.
Tables require a heading, and figures a legend, also prepared ona separate sheet. For the reproduction of illustrations, only good
drawings and original photographs can be accepted; negatives
or photocopies cannot be used. When possible, group several
illustrations on one block for reproduction (max. size 140x188mm) or provide crop marks. On the back of each illustration
indicate its number, the author’s name, and article title. Colour

2

illustration are reproduced at the author’s expense.
References: In the text identify references by Arabic
figures, (in brackets), fonts size 9, Arial Narrow. Material
submitted for publication but not yet accepted should be
noted as “unpublished data” and not be included in thereference list. The list of references should include only thosepublications which are cited in the text. The references should
be numbered and arranged alphabetically by the authors’names. The surnames of the authors followed by initials
should be given. There should be no punctuation signs otherthan a comma to separate the authors. When there are morethan 3 authors, the names of the 3 only are used, followedby “et al”. abbreviate journal names according to the Index
Medicus system. (also see International Committee of Medical
Journal Editors: Uniform Requirements for manuscripts
submitted to biomedical journals. Ann Intern Med 1982; 96:766 – 771).

Examples:(a) Papers published in periodicals: Kauffman HF, van der
Heide S, Beaumont F, et al: Class-apecific antibody determination
against Aspergillus fumigatus by mean of the enzyme-linked
immunosorbent assay. III. Comparative study: IgG, IgA, IgM, ELISA
titers, precipitating antibodies and IGE biding after fractionationof the antigen. Int Arch Allergy Appl Immunol 1986; 80: 300 –306.

(b) Monographs; Matthews DE, Farewell VT: Using andUnderstanding Medical Statistics. Basel, Karger, 1985.
(c) Edited books: Hardy WD Jr, Essex M: FeLV-inducted felineacquired immune deficiency syndrome: A model for human
AIDS; in Klein E(ed): Acquired Immunodeficiency Syndrome. Prog Allergy,
Busel, Karger, 1986, vol 37, 353 – 376.
Full address: The exact postal address complete withpostal code of the senior author must be given; if correspondenceis handled by someone else, indicate this accordingly. Add theE-mail address if possible.
Page charges: There is no page charge for papers of
4 or fewer printed pages (including tables, illustrations andreferences).
Galley proofs: unless indicated otherwise, galley proofs
are sent to the first-named author and should be returned withthe least possible delay. Alternations made in galley proofs, otherthan the corrections of printer’s errors, are charged to the author.
No page proofs are supplied.
Reprints: Order forms and a price list are sent with thegalley proofs. Orders submitted after the issue is printed are
subject to considerably higher prices. Allow five weeks fromdate of publication for delivery of reprints.

Fiziologia- Physiology 2014.24.1
2013.24.4(81)(80)
Fiziologia‑ Physiology 2010supplement

STNETNOC
4................................................................................... recnaC fo skramllaH eht gniylrednU erucsbO revewoH ,romuT fo stnatibahnI – stsalborbiF detaicossA-recnaC :weiveR .1
C ucsetianaP ,V ucsenuaP ,G eisanaT ,M acieD ,F nijoB ,L tanreC
31.................................... sitirhtrA decudnI-tnavujdA fo ledoM taR a ni ecnalaB tnadixoitnA-tnadixO eht no sdicA yttaF detarutasnuyloP 3-agemO dna nituR fo stceﬀE ehT .2
A naşeruM ,D dalV ,R navodloM ,D utsopA ,C uicacoS ,A notraM ,CI şihC ,D uratlaB

CONTENTS

81.................................................................. staR ni stceﬀE evitcetorpoidraC trexE dna aixopyH cirabopyH fo stceﬀE eht esreveR nasotihC dna murabraB muicyL ,nitecreuQ .3
P afloB ,G azroB ,D dalV ,R navodloM ,A notraM ,A naşeruM ,CI şihC ,A icivortimuD
32........................................................................................................................................ yhtapolahpecnE citapeH fo ygolohtapoisyhP eht ni lotisonI-oyM fo ecnatropmI ehT .4
IA uărădăB ,AD noI ,R aecocapaP ,C ienroiC ,M aenuB ,M răzaL ,C uaehcS

CONTENTS

62............................................................................................................................................ ksiR ralucsavoidraC fo noitacﬁitartS eht ni ecnacﬁingiS citsongorP :erusserP esluP .5
S șacnaM ,A rimogarD ,G ucsenoI ,D aţâbiâB
13....................................................................................................................... skramdnaL lacimehcotsihonummI dnA lacigolotsiH - amorbﬁoignA laegnyrahposaN elinevuJ .6
D tahroH ,S aeblutoC ,M atumoD ,MR narihG ,G ucsenavoI ,FD aediG

1. Review:
Review:Cancer-Associated
The Analysis of Muscle
Force–Developmentwith Trained
andObscure
Elite Athletes.
..................................................................................................................5
Fibroblasts
Inhabitants of Tumor,
However
Underlying
the Hallmarks of Cancer ...................................................................................4
1.
Review:
Metformin-From
Diabetes
Cancer
Therapy
Milosevic
MB,
Milosevic
MM,toJourkesh
M, BehmC..............................................................................................................................................................4
DG
CernatL, BojinF,Dzoljic
Deica ED,
M, Tanasie
G, Paunescu
V, Panaitescu
Deica M, Sima R, Cernat L, Bojin F, Tatu C, Tanasie G,Panaitescu C, Paunescu V
2. Prevalence of Polyglandular Autoimmune Syndrome Type III in a Group of Children with Thyroid Diseases and Diabetes Mellitus ..........................................12
2. The Eﬀects
and Omega-3 Polyunsaturated Fatty Acids on the Oxidant-Antioxidant Balance in a Rat Model of Adjuvant-Induced Arthritis ....................................13
Gherbon
A of Rutin
2. Adipose-Tissue
Derived Stem Cells: State of the Art Tissue Engineering .................................................................................................................................11
Baltaru D, Chiş IC, Marton A, Socaciu C, Apostu D, Moldovan R, Vlad D, Mureşan A
Hurmuz M, Bojin F, Miu C, Ungurean C, Tatu C, Ionac M, Tatu F
3. In Vitro Toxicity Studies on Breast Cancer Cell Line SK-BR3 and Tumor-Associated Fibroblasts (TAF).........................................................................................17
Deica
M, SimaLycium
R, Cernat
L, BojinandF, Tatu
C, Tanasie
Panaitescu
PaunescuHypoxia
V and Exert Cardioprotective Eﬀects in Rats ..................................................................18
3. Quercetin,
Barbarum
ChitosanReverseG,EﬀectsBehaviorsof C,Hypobaric
3.
Human Recombinant
Inteferon-Alpha
Effects
ontheActivein the Rat Forced
Swimming Test.....................................................................................17
Dumitrovici
A,
Chiş
IC,
MureşanA,
MartonA,
Moldovan
R,
Vlad
D,
Borza
G,
Bolfa
P
Zahiu
D,
Goganau
I,
Braga
RI,
Baca
L,
ZagreanL
4. Lymphocytes Subsets Involved in Alzheimer Disease-Associated Immunosenescence.............................................................................................................22
Groza S, Anghel S, Tatu C, Tanasie G, Panaitescu C, Gavriliuc O, Paunescu V, Bojin F
Importance
Myo-Inositol
in thePhysiopathology........................................................................................................................................23
4. TheCortical
ActivityofandHeart Rate
Changes
as MarkersofofHepatic
NoxiousEncephalopathy
Stimulation Response
during Chloral Hydrate Anesthesia ...................................................22
5.
Methylenetetrahydrofolate
ReductaseandOpportunityof Anticoagulant
Treatment ...................................................................................27
Pavel
B,C,DăneasaCălinM,A,CiorneiRădulescu
Am,GeneRoscaAe,Braga
Ri, Zahiu
L
ScheauLazăr M,A,Bunea
C, Papacocea
R,Mutations
Ion DA,Bădărău
AI D, Zăgrean
Oniceanu FM, Ion I, Adumitresi C, Radulescu N, Farcas C, Stefanescu AC, Chirica R
5.
Arguments
forPrognostic
Using Thrombelastography
in thecation
StudyofofCardiovascularHemostasis Comparedto Other Laboratory Tests .........................................................................26
5. Pulse
Pressure:
Signiﬁcancein the Stratiﬁ
RiskTests............................................................................................................................................26
6.
Correlation
between
Eosinophilic
Cationic
Proteinwith
Other Laboratory
and Atopic Environment in Children with Bronchial Hyperreactivity..............30
Oniceanu
FM,IonI,
Adumitresi
C,Radulescu
N,
FarcasC,
Stefanescu
AC,
ChiricaR
Bâibâţa D, Ionescu
G, Dragomir
A, Mancaș
S S, Mihai CM, Cuzic V
Ungureanu
A, Ileana
I, Adumitresei
C, Chirila
53........................................................................................................................................................................................................... sitilucsaV yranomluP eht otni thgisnI weN .7
MI uracojoC ,DC eibarV ,E usuR ,M uracojoC
83............................................................................................................................................................................. ?gnihT laeR a tI sI ,yhtaporueN citpO detaicossA enoradoimA .8
I goloZ ,M naetnuM ,N ucseluciN

SNIRPUC

4................................................................................................................................................. recnac ni uirotinﬁed lor uc elaromut etnenopmoC – elaromutirep eletsalborbiF .1
C ucsetianaP ,V ucsenuaP ,G eisanaT ,M acieD ,F nijoB ,L tanreC

31............................................... tnavujda ed ăsudni ătirtra uc inaloboş al iţnadixoitna-iţnadixo ieţnalab arpusa 3-agemO iţarutasenilop işarg rolizica la iş iulunituR lutcefE .2
A naşeruM ,D dalV ,R navodloM ,D utsopA ,C uicacoS ,A notraM ,CI şihC ,D uratlaB

81............................ naloboş ed ledom nu-rtnî rotcetorpoidrac snupsăr nu ăticrexe iş erabopih ieixopih eletcefe ăzaeracartnoc lunasotihC iş murabraB muicyL ,lunitecreuQ .3
P afloB ,G azroB ,D dalV ,R navodloM ,A notraM ,A naşeruM ,CI şihC ,A icivortimuD

32............................................................................................................................................................... ecitapeh ieitapolafecne aigolotapoizﬁ nî iululotizoni-oim aţnatropmI .4
IA uărădăB ,AD noI ,R aecocapaP ,C ienroiC ,M aenuB ,M răzaL ,C uaehcS
62................................................................................................................................... ralucsavoidrac iulucsir aeracﬁitarts urtnep acitsongorp aitacﬁinmes :iuluslup aenuiserP .5
S șacnaM ,A rimogarD ,G ucsenoI ,D aţâbiâB
13...................................................................................................................................... ecimihcotsihonumi iş ecigolotsih icitsirecarac – linevuj naignirafosan lumorbﬁoignA .6
D tahroH ,S aeblutoC ,M atumoD ,MR narihG ,G ucsenavoI ,FD aediG
53........................................................................................................................................................................................................................ ăranomlup atilucsav nî iţătilautcA .7
MI uracojoC ,DC eibarV ,E usuR ,M uracojoC

83................................................................................................................................................................ ?etatilaer o etse – anoradoima uc iulutnematart ătaicosa aitaporueN .8
I goloZ ,M naetnuM ,N ucseluciN3ygoloisyhP - aigoloiziF  )77( 1.32.3102

6. BariatricPatient Related
Factors andTherapeutic
Strategy
LarynxSquamous
Cell theCarcinoma
7.
SurgeryPrognostic
in Obese
Patients
IncisionalAnd
Hernia– AinNew
MethodtoLandmarks
ReduceHernia .........................................................................................31
Recurrence Rates...............................................................36
6. Juvenile Nasopharyngeal
Angioﬁbromawith- Histological
Immunohistochemical.......................................................................................................................31
Prodea M,A,Horhat
D,Balica
N,LazarBorugaO, Poenaru M
Dobrescu
Duta
C,
Tarta
C,
F
Gidea DF, Iovanescu G, Ghiran RM, Domuta M, Cotulbea S, Horhat D
7. TheIn VitroCharacterisation
Tumor-AssociatedFibroblastsfromDifferent
Tumors..............................................................................................................35
8.
Use of Perioperative ofIntraperitoneal
Chemotherapy Associated withSolid
Neoadjuvant
Therapy for Advanced Ovarian Cancer as Consolidation Therapy.........40
7. New L,Insightinto
the Pulmonary
Vasculitis...........................................................................................................................................................................................................35
JebereanA, Carabineanu
Ancusa
D,D,Preda
M, Istodor
A, Motoc
A
CernatBojin
F, Deica
M,A,Anastasiu
Tanasie
G, IlinaA,R,Bucur
Paunescu
V, Panaitescu
V
Cojocaru M, Rusu E, Vrabie CD, Cojocaru IM
9.
InducedAdditionin Culture
Media ofEjection
Tumor-AssociatedFibroblasts (TAF).......................................................................................44
8. Changes
Comorbidities
andbyRisk
FactorsofinMetformin
Heart Failure
with Preserved
Fraction...................................................................................................................39
Deica
M,
Sima
R,
Cernat
L,
Bojin
F,
Tatu
C,
TanasieG,
Panaitescu
C,
Paunescu
V
MavreaA, BucurA, Tomescu
8. Amiodarone
AssociatedOpticMNeuropathy, Is It a Real Thing? .............................................................................................................................................................................38
Niculescu N, Muntean M, Zolog I

CUPRINSCUPRINSCUPRINS

1.
Metformin: dela diabet la– terapiaantitumorală
1. Fibroblastele
peritumorale
Componente tumorale.......................................................................................................................................................................4
cu rol deﬁnitoriu in cancer .................................................................................................................................................4
Deica
M,
Sima
R,
CernatL,
Bojin
C,laTanasie
G,Panaitescu
V ............................................................................................................................5
1.
Analizadezvoltarii
forteimuscularesportivii
antrenati
la Paunescu
atletii de elita
Cernat L, Bojin F, Deica M, Tanasie G,F, Tatu
Paunescu
V, Panaitescu
C si C,
Milosevic MB, Dzoljic ED, Milosevic MM, Jourkesh M, Behm DG
2.
CeluleleRutinului
stem derivate
din ţesutuladipos:
actualităţiîningineriatisulară
.................................................................................................................................11
2. Efectulşi al acizilorgraşi polinesaturaţi
Omega-3
asupra balanţei
oxidanţi-antioxidanţi
la şobolani cu artrită indusă de adjuvant ...............................................132.
Prevalența
sindromuluipoliglandular
autoimunIII într-un
HurmuzM, BojinF, Miu C, Ungurean
C, TatuC, Ionac tip
M, Tatu
F grup de copii cu boală tiroidiană și diabet zaharat...................................................................12
Baltaru D,AChiş IC, Marton A, Socaciu C, Apostu D, Moldovan R, Vlad D, Mureşan A
Gherbon
3. Studii
Efecteleinterferonului-alfa
recombinantumanasupracomportamentelor
active la testulînotuluiforţat,la şobolan...............................................................173.
de toxicitate
in vitro efectuate
pe linia
celulara deefectele
cancerhipoxiei
de san SK-BR3
peri-tumorale.
........................................................................17
3. Quercetinul,
LyciumBarbarumşi Chitosanulcontracarează
hipobaresişifibroblastele
exercită un răspuns
cardioprotector
într-un model de şobolan ............................18
Zahiu
D,
Goganau
I,
Braga
RI,
Baca
L,
ZagreanL
Deica M, Sima R, Cernat L, Bojin F, Tatu C, Tanasie G, Panaitescu C, Paunescu V
Dumitrovici A, Chiş IC, Mureşan A, Marton A, Moldovan R, Vlad D, Borza G, Bolfa P
4. Subpopulații
Modificărileactivităţii
corticaleşialeritmuluicardiac
ca markerirăspunsului
la .stimulareanociceptivăîntimpulanestezieicucloralhidrat.............................22
limfocitare
implicateîn imunosenescența
asociatăaibolii
Alzheimer.
...................................................................................................................22
Anghel
S,A,Tatu
Tanasie
G, Panaitescu
C, Gavriliuc
O, Paunescu
V, Bojin LF
4.Groza
Importanţa
mio-inozitoluluiîn ﬁziopatologia
encefalopatiei
Pavel
B,S,DăneasaCălinC, A,Rădulescu
Am, RoscaAe,
Braga hepatice
Ri,
Zahiu...............................................................................................................................................................23D, Zăgrean
Scheau C, Lazăr M, Bunea M, Ciornei C, Papacocea R, Ion DA, Bădărău AI
5.
genei MTHFR si oportunitatea tratamentului anticoagulant.....................................................................................................................................27
5. Mutatiile
Argumentepentruutilizareatrombelastografieiînstudiulhemostazeicomparativ
cu alteteste de laborator .............................................................................26
Oniceanu
FM,
IonI,I,Adumitresi
Adumitresi C,Radulescu
C, Radulescu N,Farcas
FarcasC,C,Stefanescu
StefanescuAC,
AC,Chirica
ChiricaRR
Oniceanu
FM,Ion5. Presiunea pulsului:semniﬁcatia prognosticaN,pentru
stratiﬁcarea riscului
cardiovascular...................................................................................................................................26
Bâibâţa
D,
IonescuG,
DragomirA,
Mancaș
S
6. Corelatiile proteinei cationice eozinofilice cu IgE total, phadiatop infant, numarul total de eozinofile si mediul atopic la copiii cu hiperreactivitate bronsica..........30
6. Factori deA,prognosticlegaţi de pacientşistrategiiterapeutice
Ungureanu
Ileana I, Adumitresei
C, Chirila S, Mihai CM, Cuzic Vîn carcinomullaringian cu celulescuamoase .............................................................................31
Prodea M, Horhat D,Balica N, Boruga O, Poenaru M
6. Angioﬁbromul nasofaringian juvenil – caraceristici histologice şi imunohistochimice ......................................................................................................................................31
7. Chirurgia bariatrica la pacientii obezi cu hernie incizionala – o noua metoda de reducere a ratei de recurenta a herniei.........................................................36
Gidea
DF, Iovanescu
Ghiran
RM, Domuta
M, Cotulbea S, Horhat
7.
Caracterizare
vitro
a C,fibroblastelorperi-tumoraleizolateDdin diferite tumori solide ............................................................................................................35
Dobrescu
A, DutainC,G,
Tarta
Lazar
F
Cernat L, Bojin F, Deica M, Anastasiu D, Tanasie G, Ilina R, Paunescu V, Panaitescu V
7. Actualităţi
vasculita pulmonară
........................................................................................................................................................................................................................35
8.
Utilizarea închimioterapiei
intraperitonealeperi-operative asociata cu terapia neoadjuvanta pentru cancerul ovarian avansat ca terapie de consolidare ........40
JebereanCarabineanu
A,deAncusa
D,IMPreda M, Istodor A,cuBucur
Motoc
A
Cojocaru
M,A,Rusu
E, Vrabie CD,
Cojocaru
8.
Comorbidităţişifactoriiriscîninsuficienţacardiacă
fracţieA,deejecţieprezervată
..............................................................................................................39
Mavrea A, Bucur A, Tomescu M
9.
Modificari asociată
induse deadaugarea cu
metforminului
mediulde cultura
al fibroblastelor peri-tumorale......................................................................................44
8. Neuropatia
tratamentului
amiodarona –inesteo realitate?
................................................................................................................................................................38
Deica
M,
Sima
R,
Cernat
L,
Bojin
F,
Tatu
C,
TanasieG,
Panaitescu
C,
Paunescu
V
Niculescu N, Muntean M, Zolog I
2014.24.1(81) 
Fiziologia - Physiology
2013.24.4
2013.23.1 (80)(77) 
 Fiziologia - Physiology

333

REVIEW
THE ANALYSIS OF MUSCLE FORCE DEVELOPMENT WITHTRAINED AND ELITE ATHLETESMILENKO B. MILOSEVIC1, ELEONORA D. DZOLJIC2, MILOS M. MILOSEVIC3, *MORTEZAJOURKESH4, DAVID G. BEHM5
1Faculty of Management in Sport, Alpha University, Belgrade, Serbia
2
Neurology Clinic, Clinical Center of Serbia, Faculty of Medicine, University in Belgrade3
Department of Psychology, XV Belgrade’s grammar school, Belgrade4
Department of Physical Education and Sports Science, Islamic Azad University, Shabestar Branch, Iran
5
School of Human Kinetics and Recreation, Memorial University of Newfoundland, St. John’s, NL A1C 5S7, Canada

ABSTRACTThe goal of this review was to investigate whether maximal rate of force development (MRFD) represents an excellent indicator for
training planning. The review underlines the influence of the speed of activation, synchronization and optimization of motor units on
MRFD, as well as that MRFD represents an outstanding discrimination factor for achieving optimal sports performance. The analysis focused on research involving force development in athletes who won gold medals in Olympic, world, European and national
competitions as well as non-medal athletes. According to the analyses, MRFD from the beginning of the movement for elite athletes
was produced in time interval ranges from 0.063±0.007s to 0.115±0.016s, using 56% to 89% of maximal force. This data suggests
an emphasis on training to attain MRFD within time intervals of 0.130s. Analyses showed that MRFD developed within this duration
represents an excellent discriminator (P<0.01) between elite athletes competing in different disciplines and between elite athletes
and well-trained individuals. To conclude, MRFD is related to (P<0.01) the conditions and regimes of muscle work, level of force
developed by individual groups of motor units, speed of activation, synchronization and optimization of motor units at intramuscularand inter-muscular levels.
Key words: motor units’ activation speed, motor-unit optimization, maximal force in motor unit groups, model and discrimination value

INTRODUCTION
The time necessary to develop the maximal rate of force isshorter than the time necessary for developing maximal force (2,18, 47, 55, 56, 58, 59, 60). Studies have revealed that a relatively
small number of sport disciplines use an athlete’s maximal capacity for developing force (18, 47, 55, 57, 58, 59, 60). The majorityof sports apply force during intervals ranging from 0.05s to 0.17s(2, 18, 23, 47, 54, 55, 58, 59, 60). Hence, the success of elite
athletes depends more on the maximal rate of force development(MRFD) and factors that influence its level (18, 26, 46, 47, 51, 54,57), than on the maximal force. Consequently, it implies that the
MRFD could be an excellent discrimination measure. Thus MRFD
should be able to discriminate between elite and recreationallytrained athletes. Furthermore, it should be possible to distinguishbetween elite athletes competing in different disciplines basedon the MRFD in time intervals up to 0.13s.
The verification of these hypotheses was performed using data
gathered from the results of elite athletes, winners of national,
European, world championships and Olympic games in basketball, volleyball, soccer, handball, athletics, boxing, weightlifting,karate, and data gathered from testing performed on non-medal
winning professional athletes, using the difference model. Data
was analyzed regarding maximal force and MRFD in different
muscle groups. The literature was also analyzed for the influenceof the following factors on the MRFD: time and type of work of

motor units, types of muscle contraction, muscle length, time(speed) necessary for contracting/extending a muscle, timenecessary for transferring from eccentric to concentric contraction, external conditions and regime of muscle work.
MATERIALS AND METHODS
Subjects
The studies analyzed in this review include force measurements
of professionals athletes that trained daily but did not achievea medal standing (N=300) and medal-winning, elite athletes(N=208) competing in different sports of Olympic, world andEuropean championships (Table I).
Table I. Characteristics of the subjects

Note: All results are mean ± SD

Received September 15th 2013. Accepted October 20th 2013. Address for correspondence: Morteza Jourkesh, Department of Physical Education and Sports Science,
Islamic Azad University, Shabestar branch, IRAN, Phone: +989143124670, e-mail: mjourkesh@iaushab.ac.ir, Jourkesh_2006@yahoo.com

4

Fiziologia - Physiology  2014.24.1 (81)Methodologies for Diagnosing Force-related Measurements
Measurements in studies analyzed in this review were collected using a variety of hardware-software systems (5, 12, 13,30, 53, 54, 55). Measurements included tensiometeric probeswith measurement range of 0-15000 decanewtons (daN) andmeasurement accuracy of 0.0002daN (1 daN = 101N), freeweights with equipment enabling time measurements (s), vertical and horizontal changes in bar position at each 1mm applied
to the entire lifting range, tensiometric platform, electro-opticalsystem for measuring movement parameters during testing (1,2, 4-18, 20, 24, 27-38, 41-43, 49, 50, 52, 53, 56).
The Belt method with a sampling rate of 1000 Hz is used formeasuring isometric force for all muscle groups. The force (Ft)is measured for 1%, 2%... 99% of the maximum force expressedin daN as well as the time of development. The maximum force(Fmax), also expressed in daN, is measured as well as the timeto peak force. The system allows muscle length to be controlled
and varied during the measurement process (the angle of muscle
activity), the time and mode of muscle activity as well as thenumber of muscles – joints included in the measuring process.
Each test is repeated three times and the best result is taken into
account (4, 5, 8-13, 15, 16, 18, 31, 32, 34, 36, 38, 42, 43, 49, 52).
Dynamic measurements of force can be applied during
weight training (6, 35, 36, 37) and using tensiometric platform(7, 10, 12, 18). With the help of tensiometric platforms, muscle
force development during different types of drop reboundjumps, drop jumps or vertical jumps can be measured.
Piezoelectric sensors embedded in the platform register at ahigh speed (500 Hz) the compression forces of jumps allowing
detailed dynamic analysis of all jump phases and a highvalidity for each movement. Apart from standard parameters,
a tensiometric platform allows tracking of the dynamics of theforce development, the speed of executed jumps, the depth ofa drop jump (or depth jump), the effects of stretching, the abilityof rapid replacement of eccentric and concentric contractions
as well as the transfer of elastic energy (7) and a number ofother parameters.
By using weight lifting, the system allows muscle length(angle), time, the mass of free weights, duration and speedof activity, velocity of shortening (concentric contraction) andmuscle elongation (eccentric contraction), the time of transition
from eccentric to concentric contraction (reversible contraction)
to be controlled and varied during the measurement process.
When lifting any weight, the timing of vertical and horizontal
change in the position of the barbells can be measured: verticalchange was measured at 5.8 mm and horizontal was measured at 1 mm, throughout the whole range of muscle activity.
Each test was repeated three times and the best result was
taken into account (6, 8, 17, 28, 30, 35, 37, 49, 50, 53). Those
attempts during which the horizontal change in the weight
position was not in the permitted zone (4-5 cm) were rejected.
On average, 100 points are observed per measurement. The
data on length and time were used to calculate the verticalvelocity and acceleration for each measuring section as thefirst and the second derivative of path by time. The results of2014.24.1 (81)  Fiziologia - Physiology

velocity and acceleration were fitted before the calculation offorce. After that the force was calculated from the fitted data ineach measuring section throughout the entire process of weightlifting according to the formula (6, 35, 36, 37):
F = m (a + 9.81) / 10, where F represents the force expressedin daN, m – the mass of the weight expressed in kg and a –acceleration (m/s2) observed within a specific sample. In bothmodes, the ratio between the force and time was measured
at each 1% of the maximum force as well as the maximum
force and time during which it was developed. From the dataobtained on force and time, the rate of force development iscalculated as their derivatives by time, for all muscle groups
involved in the testing.
Both in the isometric and dynamic assessment mode,throughout the entire force development process, at eachpercentage, the rate of force development were calculatedusing the formula:
RFD=F/t, where RFD represents the rate of force development, expressed in daN/s, F - the appropriate level of force
expressed in daN, and t – the force observation time expressedin seconds (s).
The obtained data on time and force, for both assessment
modes, were used to calculate the change in the rate of force
development, using the formula:
CRFD = (F2-F1) / (t2-t1), where CRFD is a change in the rateof force development expressed in decanewton per second(daN/s) for each percent of the measured force (F) from 1%
to 100% and the time (t) during which it is happening, F1
and F2 - the appropriate level of maximal force expressedin decanewtons (daN), and t1 and t2 - force observation time
expressed in seconds (s).
The data on force and time, obtained during the applicationof the Belt method and weightlifting were used to calculate thespeed of activation of motor units for each Ft using the formula(5, 13, 31, 54, 56):
C = - (1/t) * ln ((1 - Ft/Fmax)), where Ft is a level equaling
to 1%, 2%... 99% of maximum force expressed in daN; Fmax maximum force generated by the engaged muscles, expressedin daN, C – a constant which characterizes the speed ofactivation of motor units expressed in index units (IJ), t - time
during which an adequate level of maximum force is achieved,
expressed in seconds (s).
When using the tensiometric platform for drop jumps, drop
rebound jumps; the speed of activation of motor units is calculated using the following formula (18, 54, 56):
C = Ln (1 – Fexp / A / e B X β exp ) Δ texp, where C is a constant which characterizes the speed of activation of motor
units expressed in index units (IJ); Fexp - the level of force
registered in the dynamic muscle activity over a period during which the muscle strain appears in isometric conditions,expressed in dN, βexp - the angle of the knee joint at which
the angular velocity equals to zero, expressed in degrees;Δtexp - the time interval starting from the contact with thetensiometric platform to the point where the angular velocity of the knee joint equals to zero, expressed in seconds.
5

Fig.1. Speed of change in rate of force development for leg
extensors for two judo champions

Data Analysis
The data were processed by using descriptive statistics, discriminatory analysis, cluster analysis, factor analysis and fittingby using least-squares method. The data on force, time and therate of inclusion of motor units were used to determine, by cluster
analysis, the class of motor units (36, 54, 56) according to the levelof the force development in any given moment of observation ormeasuring section. From the ratio between force and time, the rateof inclusion and force levels of individual motor units throughout theforce development process, the level of synchronization of motor
units (36) was calculated. By fitting the data on the force level ofindividual motor units and time period needed to achieve it throughout the entire force development process for one or more muscles,
the optimization of their activity (36, 38) on the intramuscular or theintermuscular level was calculated.
The Analysis of Results Relating to the Rate of Force Development in Muscles
Maximal Rate of Force Development (MRFD) in Muscles
The results (1, 2, 4, 6, 7, 10, 12, 18, 24, 28, 29, 33, 36, 37,
43, 45, 51, 56, 59) of most studies point to the fact that the RFD
differs according to sports discipline, athlete, muscular group,
regimes, types and speeds of work.
With non-medal winning athletes (BH=1.836±0.05m;
BW=79.92±8.79kg; Age=21±0.65 years), isometric contraction
MRFD is shown in Table II.

In comparison, an elite male judo competitor, winner of Olympic
gold medal, (BH = 1.728m; BW = 73.65kg; Age = 23 years)
achieves much greater RFD with an isometric regime (Table III).
Other elite athletes also achieve much better results when the
RFD is concerned (P<0.01) than non-medal winning athletes,
which is obvious if we take a look at the results of leg extensor
testing at the angle of 1120. Middle and long distance runners
achieve an MRFD of 1382±152.02daN/s in leg extensors.
Basketball, volleyball, handball and soccer players achieve an
MRFD up to 1727.5±103.65daN/s in leg extensors, while athletescompeting in long jump, high jump and triple jump, achieve an
MRFD at 2320±150.8 daN/s. With isometric contractions, forboth non-medal winning athletes and elite medal athletes, thehighest RFD is recorded in hand flexors, while the lowest results
are typical of left (weaker side) hip flexors.
Table III. Descriptive Characteristics and function of development of
MRFD isometric contraction with elite male judo competitor, winner of
Olympic gold medal

With dynamic contractions, the RFD displays even better results; nevertheless these results are different for each individual
and sport. Table IV illustrates the MRFD in leg extensors withan eccentric contraction regime (drop jump – amortization from110cm). The relation between the angle (the length of muscles)
where the amortization ends (X) and the MRFD in leg extensors
is nonlinear (MRFD = 4.674X2 – 965.3X + 53909 for R2 = 1). Table
V illustrates the MRFD in leg extensors with stretch-shortening
cycle (SSC) or reversible type jumps (drop jump-depth jumpfrom the elevation of 80cm).
Table IV. Descriptive Characteristics MRFD eccentric contraction inleg extensors with elite athletes

Table II. Descriptive Characteristics MRFD isometric contraction with
non-medal winning athletes

6

Fiziologia - Physiology  2014.24.1 (81)

If our test sample includes elite athletes competing in different sports (47, 54, 55) and we change the elevation for
performing drop jump-depth jump we will get the following
function, which defines well the relation between the MRFD
in leg extensors and the elevation from which the subjects
jump (X):MRFD = 0.015x3- 3.700x2 + 278.4x – 1E-10 for R2 = 1
In a SSC or reversible activity such as lifting weights(70% of 1RM), a junior medal-winner competing at a
European championship (BH=1.675 m; BW=58.2 kg; Age=21 years) achieved the MRFD in clean phase (shoulder
muscles) at 1984daN/s; 2778daN/s in half squat (legextensors); 1628daN/s in bench press (arm extensors)and 964daN/s in dead lift (back extensors). MRFD in leg
extensors during SSC contractions in standing high jump
starts at 1294±181.16daN/s in non-medal athletes and1582±189.84daN/s in soccer players, to 2000±180.0daN/sin volleyball players and 2432±243.2daN/s in athletes competing in high jump, long jump and triple jump. Consideringblows in martial sports, (30, 56, 57) when executing mae
geri, world karate champion (BH=1.878 m; BW=90.2 kg; Age= 25 years) achieved MRFD in hip flexors at 2132.8daN/s, in
0.050s with the force level at 106.64daN; in knee extensors
at 1035.0daN/s, in 0.050s with the force level at 51.75daN;
in foot extensors at 500.0daN/s, in 0.05s with the force levelat 25.0daN. In an experiment involving a junior medal winner at an European championship (BH=1.675 m; BW=58.2
kg; Age = 21 years), where examiners changed the weight
and the speed of lifting weights from deep squat (Table 6),
it was concluded that the MRFD is achieved when lifting
80% of the maximal weight in one lift (1RM) with maximal
speed of lifting (6, 34, 35, 37).
The analysis of the results enables us to conclude that
the MRFD is much greater in elite champion athletes than in
non-medal winning athletes. With dynamic contractions, theresults may be as much as 12 times greater. With isometriccontractions, differences range between 3-17 times, while
dynamic vs. isometric regimes display results that vary 26
fold (P<0.000). Differences are significant (P<0.01) between
athletes competing in different sports, which imply that the
MRFD is an excellent discrimination factor in both regimes.
Differences in the MRFD for the same muscle groups,
measured during different movements, range up to 31.22
times. The highest RFD is achieved with SSC activities
with high movement dynamics. In an eccentric-concentric
cycle during the phase of the concentric contraction, moreforce is generated over a shorter interval of time (in weightlifting with acceleration exceeding 25 ms-2 one develops
1450.0daN of force in 0.100s with the MRFD at 14500daN/sor during drop jump-depth jump where one develops up to
983.6±113.11daN of force in 0.063±0.007s with the RFD
at 15612.69±1795.46daN/s) (1, 18, 23, 28, 35, 41) thaneccentric or concentric only muscle contraction for several
reasons. First, at the highest point of the cycle, period between the eccentric and concentric contraction, the force is2014.24.1 (81)  Fiziologia - Physiology

being developed in isometric conditions. Secondly, since
the force development starts in the eccentric phase (in drop
jump-depth jump it develops up to 661.89±39.71daN in
0.075±0.004s with the RFD at 8825.08±529.50daN/s) thetime available for developing force in a SSC or reversible
contraction is extended (18, 28, 34). Third, the level of force
is influenced by muscle-tendon elasticity (the accumulationof elastic energy during the eccentric contraction in muscles
and tendons) and fourth, reflex muscle contraction (58, 59,60). All these effects are present when measuring drop jumpdepth jump, weight lifting or other sports movements (1, 18,
23, 28, 35, 41). The muscle length and the level of force, after
making contact with the ground in drop jump-depth jump andafter stopping the first phase of movement in weight liftingor other sports movements, changes abruptly. Muscles areforced to extend and at the same time, their tension rises.
These changes are at the same time, controlled and partiallybalanced by the joint effect of two motor reflexes: myotaticreflex, maintaining an optimal length of muscles, and the Golgi
tendon organ reflex, preventing extremely high and harmful
muscle tension. Myotatic reflex has a positive effect (theincrease in discharge), while Golgi reflex predominantly has
a negative or inhibitory effect influencing the applied force.
The most important thing for learning reversible contraction
during substantial muscle strain is diminishing the activityof the Golgi tendon organ. Nevertheless in drop jumps withamortization or block, in drop jumps-depth jumps from highelevations or when lifting heavy weights from half squat,
bench press, pullover, or during torso rotations with additional
weight, due to the possibility of harming the integrity of one’s
body (1, 3, 18, 28), the CNS reacts by applying facilitation anddisinhibition effects (1, 3, 18, 26, 28, 53) on different levels. In
these cases, the reticular system acts as an amplifier increasing the intensity of efferent signals (58, 59, 60). Furthermore
an inhibition of Renshaw’s inhibitory interneurons as well asother inhibitory motoneurons can also magnify the facilitationof the system. Pre-synaptic inhibition diminishes Golgi tendon
organs inhibition thus minimizing peripheral afferent inhibition
permitting higher levels of MRFD (59). Neural disinhibition,from the abovementioned conditions (1, 3, 18, 22, 26, 28, 41,
44, 60), influences the increase in myotatic reflex (the rateof contraction is significantly (several times) faster and more
powerful than a completely voluntary muscular contraction).
Furthermore, neural disinhibition causes an increase in the
speed of activation of motor units and the level of their synchronization, as well as the reprogramming (by increasing)of the limit of force for all motor units. Therefore, the biggest
influence on the force increase and at the same time, thedecrease in the RFD in reversible contractions, comes fromthe neural component of muscle contraction – most of all, fromthe disinhibition process applied at all levels (1, 3, 18, 22, 25,
26, 28, 41, 47, 49, 51, 54, 55, 57, 59). The biggest challenge
for athletes determined to learn and master reversible (SSC)
activities is to learn and master disinhibition (1, 3, 18, 22, 26,
28, 41, 46, 47, 48, 49, 51, 54, 55, 57, 59).
7

Table V. Descriptive Characteristics MRFD in SSC in leg extensors
with elite athletesMoreover, the analysis of the results enables us to conclude
that a high level of MRFD (isometric) enables elite athletes
to generate in 0.097s, from 56% of maximal force in right hip
rotators to 89% in hip flexors. In contrast, non-medal athletes
in the same time interval achieve from 33% of maximal force
in hip rotators to the right, to 64% in hip flexors. With dynamic
movement (amortization, reversible contractions) due to the high
MRFD, elite athletes achieve much greater values of maximal
force in a much shorter time interval than the values recordedwith isometric contractions (P< 0.000). The relation between
MRFD, time and angle at which the force is developed (lengthof muscles) is nonlinear. The relation between MRFD and theelevation one jumps from is also nonlinear. In the case of elite
athletes, the RFD enables them to develop maximal force inthe amortization period in the time interval ranging from 0.075s
to 0.106s, while the time interval for reversible contractions is0.063s to 0.077s.
In the end, we can conclude that the MRFD in all types ofmuscle contraction (isometric, concentric, eccentric, reversible)
enables elite athletes to develop, in the time interval ranging from
0.063±0.007s. to 0.115±0.016s���������������������������������
, levels of force that are necessary and sufficient to win medals at prestigious competitions.
These results also permit us to recommend the best choice fordeveloping MRFD in leg extensors: reversible contractions during
drop jump-depth jump from 60cm and drop jump-amortizationfrom 120cm (5, 19, 46, 47, 48, 51, 54, 55, 57). For other muscle
groups, we recommend weight lifting with 80% of 1RM andmaximal speed (8, 19, 46, 47, 48, 51, 54, 55, 57). When lifting
weights (80% of 1RM) different muscle groups develop up to
78.5% of maximal force in a time interval ranging from 0.097 to
0.172s (32, 33, 35, 37, 50).
Change in Rate of Force Development (CRFD)
Sometimes, in sport, we need to develop additional, highforces within a certain time interval, with the aim of achieving
an optimal result. Figure 1 clearly shows that the biggest CRFD
in isometric conditions (36), in leg extensors, i.e. the biggest
increase in force, takes place in the interval between 18% and8

32% of maximal level of force. Maximal CRFD in the case of thefirst competitor is 7000daN/s, while in the case of the secondcompetitor it is at 5000daN/s. At that moment, the level of force
was from 94.57daN to 215daN in a time interval ranging from0.059s to 0.086s at the angle of 1250. The level of change isdifferent from athlete to athlete. Non-medal athletes achievemuch lower values, ranging from 1441daN/s (right hand flexors)
to 3947daN/s (leg extensors). The speed of CRFD is different up
to 0.200s and after 0.200 s. The changes are much greater up
to 0.200s than after 0.200s. An experiment performed on elite
judo competitor’s leg extensors (BH=1.91m; BW=97.8kg; Age=25
years) confirms this, i.e. it confirms that the speed of change
in rate of force development (CRFD) in (t) for every % of force
measured (F) from 1% to 100% with isometric contractions at anangle of 1150, can be described up to 0.200s and after 0.200s(R2 = 0.997 and R2 = 0.998) through different formulas (34):
CRFD1(t<0.2) =-179437t2+44792t-428.6.
CRFD2(t>0.2)=254.52t4-1555.2t3+3484t2-3655.4t+2045.4
Table VI. Rate of change for force development and its’ dimensions
with various weight lifts and leg extensors reaction

Note: 50%, 70%, 80%, 90%, 100% - percentage of maximal weight
lifted in one attempt (% of 1RM).

In an experiment that included a junior medal-winner at a European championship (BH=1.675m; BW=58.2kg; Age=21 years),
where scientists changed the weight and speed of weight lifting(Table 6), it was concluded that the biggest CRFD is achieved
with 85% of maximal weight in one attempt (1RM) with maximal
lifting speed (6, 35, 37). In a kettlebell clean, with maximal lifting
speed at 1.68m/s his shoulder muscles achieve CRFD equaling
5832daN/s at 0.150s.
Fiziologia - Physiology  2014.24.1 (81)

His arm extensors in bench press achieve CRFD equaling
4200daN/s at 0.158s with lifting speed at 1.69 m/s. His leg extensors in deep squat, with a lifting speed at 1.74 m/s, achieve CRFD
equaling 7000daN/s at 0.120s. His back extensors in dead liftachieve CRFD equaling 1660daN/s at 0.200s with lifting speed at
1.7 m/s. With reversible or SSC contractions, during drop jumpdepth jump, leg extensors achieve much greater values in RFD
in much shorter time (P<0.01) than with isometric contractions(37, 54, 55). With 85% of maximal weight in one attempt (85% of
1RM) with maximal lifting speed (6, 35, 37) and drop jump-depth
jump from 80cm, we detect the most intensive changes in RFD,
the most intensive processes for producing proteins actin andmyosin (17), but, at the same time, we detect an improvement
in intramuscular coordination.
The research results (1, 2, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 18,
26, 29, 31, 33, 34, 35, 41, 45, 46, 47, 49, 50, 51, 53, 54, 57) tellus that the RFD directly depends on the level of force in certain
groups of motor units, their activation speed, synchronization,
optimization of their work at intermuscular and intramuscularlevels, as well as their ability to sustain high changes in RFD.
Practical Applications
In certain sports activities, the result exclusively depends on
maximal force, which means that the training has to be organizedaccordingly. In most sports and disciplines, the time interval
for developing force is limited to 0.050s - 0.170s. This is why
outstanding results rely on the MRFD and factors that influence
it (maximal force in individual groups of motor units, motor units’
activation speed, synchronization and optimization of motor units’work) more than on the maximal force. The training for developing MRFD should be designed with the aim to achieve maximal
values in the time intervals (0.063s - 0.130s.) as recorded for
elite athletes and medal winners. The best results in developing
MRFD and the factors that influence it, are achieved with maximal
speeds, by using free weight jumps, vaults, drop jumps, drop
jump-depth jumps and sprints with variations in direction. MRFD
is best developed with reversible contractions, in drop jump-depth
jump from 60cm, drop jump-amortization from 120 to 200 cm
and weight lifting at 80% of 1RM. With 85% of maximal weightin one attempt (85% of 1RM) with maximal lifting speed anddrop jump-depth jump from 80cm, we detect the most intensivechanges in RFD, the most intensive processes for producing
proteins actin and myosin (17), but, at the same time, we detect
an improvement in intramuscular coordination. The best training
results are achieved if we apply the abovementioned rules forconstructing training programs for each athlete.
REFERENCES

1. Aagaard P, Simonsen EB, Andersen JL, Magnusson P, HalkjærKristensen J, Dyhre-Poulsen P. Neural inhibition during maximal
eccentric and concentric quadriceps contraction: effects of resistancetraining. J. Appl. Physiol. 2000; 89: 2249-57.
2. Aagaard P, Simonsen EB, Andersen JL, Magnusson P, HalkjærKristensen J, Dyhre-Poulsen P. Increased rate of force development
and neural drive of human skeletal muscle following resistance training.
2014.24.1 (81)  Fiziologia - Physiology

J. Appl. Physiol.2002; 93: 1318-1326.
3. Anohin PK. The theory of functional systems: general questions ofphysiological mechanisms. Moscow: Sciences, 1970.
4. Blagojevic M, Milosevic M, Dopsaj M, Arlov D. Mechanic parametersof trunk flexor and extensor muscle force with well-trained young males.
Exe & Soc. J. of Sports Sci. 1997; 17: 128-129.
5. Blagojevic M, Milosevic M, Dopsaj M. The effect of muscle length
on the change in muscle involvement velocity. Exe & Soc .J. of Sports
Sci. 1997; 21: 213-214.
6. Blagojevic M, Milosevic M, Aleksic V, Papadimitrioiu A, Dopsaj M. The
comparative analysis of force generation velocity and its dimensions at
maximal voluntary contractions in isometric and dynamic muscle work
regime. In: Proceedings of the International Conference on Weightlifting and Strength Training. Editor: Keijo Hakkinen, Lahti, Juvaskyla,
Finland, 1998; 273-274.
7. Bobbert MF, Gerritsen KG, Litjens MC, Van Soest AJ. Why is countermovement jump height greater than squat jump height? Med & Sci
in Sports & Exe. 1996; 28: 1402-1412.
8. Dopsaj M, Milosevic M, Arlov D, Blagojevic M, Stefanovic D. The structure of changes in mechanic contractile characteristics of leg extensor
muscles caused by combined strength training during one-­year motor
learning program in special physical education. In: Proceedings of International Congress on Sports Psychology. Editors: Yannis Theodorakis& Athanasios Papaionnou, Komotini, Greece: Democritos university ofthrace department of physical education& sport science, 1996; 313-318.
9. Dopsaj M, Milosevic M, Matkovic I, Arlov D, Blagojevic M. The relation
between sprint abilities in freestyle swimming and force characteristics
of different muscle group. In: Proceedings of the VIII International Symposium of Biomechanics and Medicine in Swimming. Editors: Keskinen
KL, Komi PV, Hollander AP, Jyvaskyla, Finland, 1998; 58-59.
10. Dopsaj M, Milosevic M, Blagojevi M. Metrological values of selectedmuscle force mechanical characteristics during isometric multi-joint
test. 6th International Congress of Northern Greece Sports Medicine
Association. Thessaloniki, Greece, 1999a.
11. Dopsaj M, Milosevic M, Matkovic I, Arlov D, Blagojevic M. The relation
between sprint ability in freestyle swimming and force characteristics ofdifferent muscle groups. Biomechanics and Medicine in Swimming VIII,
Editors: Keskinen KL, Komi PV, Hollander AP. Department of Biology ofPhysical Activity, University of Jyväskylä, Gummerus Printing, Jyväskylä,
Finland, 1999b; 203-208.
12. Dopsaj M, Milosevic M, Blagojevic M. An analysis of the reliability
and factoral validity of selected muscle force mechanical characteristics
during isometric multi-joint test. In: Proceedings of the XVIII International
Symposium of Biomechanics in Sports. Editors: Youlian H & David PJ,
Hong Kong, 2000; 146-150.
13. Dopsaj M, Milošević M, Blagojević M, Mudric R. A new approach todiscriminating athletes according to their specific fitness status when
considering isometric force. In: Abstract Book of the 3th International
Conference on Strength Training -”Strength Training in Sport and in
Rehabilitation”. Budapest, Hungary, 2002; 15-16.
14. Dopsaj M, Milosević M, Blagojevic M, Mudric, R. Analysis of theeffects that one academic year of Spe has on the characteristics ofmaximal and explosive force of the trunk extensor in policemen. 3thInternational Conference on Strength Training - “Strength Training inSport and in Rehabilitation”. Budapest, Hungary, 2002; 17-18.
15. Dopsaj M, Milosević M, Blagojevic M. The effects of a one-year
swimming course on the swimming skills of police academy students.
Biomechanics and Medicine in Swimming IX, Edited by Jean-Claude C,
Department of Biology and sport medicine, University of Saint-Etienne,
Publications de I’Universite de Saint-Etienne, Saint-Etienne, France,
2003; 445-450.

916. Dopsaj M, Milosevic M, Rajic B, Abella CP, Blagojevic M, Vucković
G. Characteristics of different isometric legs extensors explosive muscle
force parameters at female athletes. Exe & Soc. J. of Sports Sci. 2003;
34: 303-304.
17. Furandžijev V, Abadžijev I. Osnovi na podgotovkata na elitni i podrastvašći sportisti – Adaptacija na kletčno i molekuljarno nivo. Sofija:
Tip-top pres, 2003.
18. Gavrilovic P. Comparative analysis of transitional regime of force
development in athletes competing in different sports with willing contractions and dynamic muscle work. PhD Thesis, Beograd: Faculty ofSport and Physical Education, 1992.
19. Gavrilovic P, Svraka M, Milosevic M. Monitoring Physical Fitness using Biomechanical Methods. Paper presented at Athletics 92. J.of Athl. 92
Belgrade. 1992; 48-57.
20. Grimby L. Firing properties of human single motor units duringlocomotion. J. Physiol. 1984; 346: 195-200.
21. Hannah R, Minshull C, Buckthorpe MW, Folland JP. Explosive
neuromuscular performance of males versus females. Exp .Physiol.
2012; 97: 618-629.
22. Ivancevic T, Jovanovic B, Jovanovic S, Đukic M, Đukic N, Lukman
A. Paradigm shift for future tennis: the art of tennis physiology, biomechanics and psychology. New York: Springer, 2010.
23. Jelen K. Biomechanical estimate of output force of ligamentum patellae
in case of its rupture during jerk. Acta. Uni. Caro. Gym 1991; 2: 71-82.
24. McLellan CP, Lovell DI, Gass GC. The role of rate of force development
on vertical jump performance. J. Strength. Cond. Res 2011; 25: 379-385.
25. Milosevic M, Gavrilovic P. Latent dimensions of strength area in policemen. 13 May. 1985; 2: 79-88.
26. Milosevic M. Determining the structure motor abilities in policemen.
Belgrade: Police College, 1985.
27. Milosevic M, Lazendic O. The relations between the general factor if
specific motor activity of police officers and strength tests. 13 May. 1986;
1: 53-61.
28. Milosevic M, Gavrilovic P, Ivancevic V. Modelling and control of theself-defense system. Belgrade: Scientific Book, 1989.
29. Milosevic M, Dopsaj M, Blagojevic M, Arlov D. Indicator factor
structure of force-­time characteristics of muscle contraction underisometric conditions. XIV International Symposium on Biomehanic,
Portugal, 1996.
30. Milosevic M, Jovanovic S, Arlov D, Blagojevic M, Dopsaj M. The
methodology of assessing the adoption of motoric programs in special
physical education. In: Proceedings of International Congress on Sport
Psychology. Editors: Yannis Theodorakis & Athanasios Papaionnou,
Komotini, Greece, 1996; 297-303.
31. Milosevic M, Laparidis C, Dopsaj M, Arlov D, Blagojevic M. The
analysis of changes of muscle involvement velocity characteristics ofleg extensors by linear and nonlinear methods. Exe & Soc. J. of Sports
Sci. 1997; 17: 167-168.
32. Milosevic M, Takac-Kostic M, Laparidis C, Dopsaj M, Arlov D,
Blagojevic M. The structural change of leg extensor muscle involvement
speed indicators influenced by eight-month strength training. XVII Pan
American & XIII Brazilian Congress of Sports Medicine, Gramado (Porto
Alegro), Rio Grande do Sul, Brazil, 1997,
33. Milosevic M, Blagojevic M, Dopsaj M. Analysing the characteristicof transitory of leg extensor force generation in dynamic strain conditions. Science-Security-Police, J. of Pol. Aca-Belgrade. 1998; 3: 34-40.
34. Milosevic M, Blagojevic M, Dopsaj M. Determining the functions
upon which force generation velocity and its dimensions are changedin leg extensors. In: Proceedings of XVI International Symposium
on Biomehanic in Sports. Editors: Riehle HJ, Vieten MM. Konstanz,
Germany, 1998b: pp. 204-208.

10

35. Milosevic M, Cirkovic Z, Mihajlovic M, Blagojevic M, Dopsaj M. The
analysis of changes in the parameters of velocity, force and its dimensions at lifting different weights from deep squat at different velocities.
In: Proceedings of the International Conference on Weightlifting andStrength Training. Editor: Keijo Hakkinen, Lahti, Jyvaskyla, Finland,
1998; 268-270.
36. Milosevic M, Dopsaj M, Blagojevic M. Comparative analysis of force
generation velocity and its dimensions in leg extensors in top judoists.
Exe & Soc. J. of Sports Sci. 1998; 20: 220-221.
37. Milosevic M, Stefanovic D, Dopsaj M, Blagojevic M. The change
in leg extensor muscle involvement velocity at weightlifting from deep
squat, at different weights and maximal velocity. In: Proceedings of theInternational Conference on Weightlifting and Strength Training. Editor:
Keijo Hakkinen, Lahti, Juvaskyla, Finland, 1998: 271-272.
38. Milosevic M, Takac-Kostic M, Blagojevic M, Cvjetkovic M, Jovanovic
B. Force distribution of motor units of leg extensor muscles. In: Proceedings of the 3rd International Scientific Conference on Prevention of WorkRelated Musculoskeletal Disorders, l3th International Symposium on
Epidemiology in Occupational Health. Helsinki, Finland, 1998; 111-116.
39. Milosevic M, Blagojević M, Tošić B, Pilipović S. Muscular deformations caused by powerful instantaneous force. Science-Security-Police, J.
of Pol. Aca-Belgrade. 1998; 3: 31-41.
40. Milosevic M, Milic Z, Stefanovic D, Cirkovic Z. Methods and tools
for diagnosing and developing speed dimensions for special police
movements. Bezbednost. 1998; 4: 42- 52.
41. Milosevic M, Dopsaj M, Blagojevic M, Papadimitriou K. The analysisof leg extensor muscle involvement velocity at lifting weight from deep
squat in eccentric and concentric contraction phases. Exe & Soc. J. of
Sports Sci. 1999: 21: 130-131.
42. Milosevic M, Blagojevic M, Dopsaj M. Defining the functions that affectthe muscle involvement velocity of leg extensors, back extensors and hand
flexors. 6th International Congress of Northern Greece Sports Medicine
Association. Thessaloniki, Greece, 1999.
43. Milosevic M, Blagojevic M, Dopsaj M. Analysing the characteristicsof transitory of leg extensor force generation in dynamic strain conditions. Science-Security-Police, J. of Pol. Aca-Belgrade. 1999; 3: 34-40.
44. Milosevic M, Blagojevic M, Ciric D, Pilipovic S, Tosic B. Muscles
in isotropic regime. Science-Security-Police, J. of Pol. Aca-Belgrade.
1999; 4: 45-55.
45. Milosevic M, Blagojevic M, Pilipovic S, Tosic B. The muscle contraction and the force production. In: Proceedings of the XVIII International
symposium of biomechanics in sport. Editors: Youlian, H & David, P. J.
Hong Kong, 2000; 183-186.
46. Milosevic M. Physical trainings for top athletes. SQ sports. JBelgrade. 2001; 14: 70-74.
47. Milosevic M. Analysis of the creation of muscular force. SQ sports.
J. Belgrade. 2002; 16: 68-69.
48. Milosevic M. Methods for developing muscle force in top athletes.
SQ sports. J. Belgrade. 2002; 17: 70-71.
49. Milosevic M, Mudric R, Dopsaj M, Blagojevic M. The functions thatdefine maximal values of motor unit involvement velocity in leg extensors
while lifting varied weight at maximal velocity in deep-squat. In: Abstract
Book of the 3th International Conference on Strength Training -”Strength
Training in Sport and in Rehabilitation”. Budapest, Hungary, 2002; 100-101.
50. Milosevic M, Dopsaj M, Blagojevic M, Mudric R. Changes in force andmotor unit involvement velocity (muiv) induced by plyometric training andthe method of incomplete eccentric muscle response. In: Abstract Book of
the 3th International Conference on Strength Training -”Strength Training in Sport and in Rehabilitation”. Budapest, Hungary, 2002; 99-100.
51. Milosevic M. Creation and development of muscle force. SQ sports.
J. Belgrade. 2002; 16: 24-27.

Fiziologia - Physiology  2014.24.1 (81)

52. Milosevic M, Dopsaj M, Blagojevic M, Mudric R. The dynamics ofdeveloping the maximum force in a basketball centre player by applying
modern training technology: One cese study. Exe & Soc. J. of Sports
Sci. 2003; 34: 372-373.
53. Milosevic M, Mudric R, Dopsaj M, Blagojevic M, Papadimitriou E. The
control of force creating in function of the muscle contraction intensity.
In: Book of Abstracts of the 4th International Conference on Strength
Training. Edited by: Kellis E, Amiridis I and Vrabas I. Serres, Greece,
Aristotle University of Thessaloniki, Department of Physical Educationand Sport Science at Serres, 2004; 320-321.
54. Milosevic M. New training technology for top athletes. Belgrade:
Police College, 2005.

55. Milosevic M. Physical preparation of elite athletes: standardizationof management processes. Belgrade: APP, 2010.
56. Milosevic M, Mudric R, Mudric M. The biomechanical analysis of
the karate kick (mae geri) in the function of defining educational training
aims and methods. Sport-sci & pra. 2012; 2: 5-14.
57. Mudrić R, Milošević M, Jovanović S. Attack in karate: education andtraining. Belgrade: Police College, 2004.
58. Thomas R. Physiology of Sports. New York: Taylor & Francis, 2004.
59. Zatsiorsky VM, Kramer WJ. Science and practice of strength training.
Belgrade: Data status, 2009.
60. Wilmore HJ, Cosstill D, Kenney WL. Physiology of Sport and Exercise. London: Human Kinetics, 2007.

ANALIZA DEZVOLTARII FORTEI MUSCULARE LA SPORTIVII ANTRENATISI LA ATLETII DE ELITAREZUMAT

Scopul acestui review a fost de a investiga daca rata maxima de dezvoltare a fortei (MRFD) reprezinta un indicator bun pentru
planificarea antrenamentului. Review-ul subliniaza influenta vitezei de activare, sincronizarea si optimizarea unitatilor motorii la
MRFD, precum si faptul ca MRFD reprezinta un factor de discriminare important pentru obtinerea performantei sportive optime.
Analiza este axata pe cercetarile referitoare la dezvoltarea fortei la sportivii care au castigat medalii de aur la Olimpiada, la competitii
mondiale, europene si competitiile nationale, precum si la sportivii care nu au fost medaliati. Conform acestor analize, MRFD de la
inceputul miscarii in cazul atletilor de elita a fost produsa intr-un interval de timp cuprins intre 0,063±0,007s si 0,115±0,016s, folosind
intre 56% si 89% din forta maxima. Aceste date indica o crestere a antrenamentului pentru a tinge MRFD intr-un interval de timp de
0.130s. Analizele au aratat ca MRFD dezvoltata in acest interval de timp reprezinta un factor de discriminare semnificativ (P<0.01)
intre atletii de elita din diferite discipline si intre atletii de elita si indivizii bine antrenati. In concluzie, MRFD este corelata (P<0.01)
cu conditiile si regimuirle de travaliu muscular, cu nivelul fortei dezvoltate de grupuri individuale de unitati motorii, viteza de activare,sincronizarea si optimizarea unitatilor motorii la nivel intra- si inter-muscular.
Cuvinte cheie: viteza de activare a unitatilor motorii, optimizarea unitatilor motorii, forta maxima in grupurile de unitati motorii,
valoare model si de discriminare

2014.24.1 (81)  Fiziologia - Physiology

11

PREVALENCE OF POLYGLANDULAR AUTOIMMUNE SYNDROME
TYPE III IN A GROUP OF CHILDREN WITH THYROID DISEASESAND DIABETES MELLITUS
ADRIANA GHERBON
Department of Functional Sciences, Physiology, “Victor Babeş” University of Medicine and Pharmacy Timişoara

ABSTRACT

Background&Aims: Polyglandular autoimmune syndromes (PAS) (otherwise known as polyglandular failure syndromes) are a constellations of multiple endocrine gland insufficiencies.Polyglandular autoimmune syndrome type III (PAS III) comprises autoimmune
thyroiditis, immune-mediated diabetes mellitus, celiac disease, hypogonadism, myasthenia gravis, sarcoidosis, Sjogren syndrome,rheumatoid arthritis, gastric neoplasia, and malabsorption. The purpose of this study is to determine the prevalence of PAS typeIIIin a group of children with thyroid diseases and diabetes mellitus (DM) type 1. Methods: The studied group was of 83 children with
DM type 1 (71 girls and 12 boys), aged between 7 and 17 years.The methods of investigation were represented by clinical, imaging,biochemical, hormonal and immunological parameters.Results: The prevalence of PAS III in the study group was 59.03 % (85.71%
girls and 14.29% boys, p < 0.001, X2 = 50). Also, the subtype was III a (autoimune thyroiditis + DM type 1), and prevailed in girls.
In 1.2% cases were associated non endocrine disease as vitiligo and decalvant pelada.Conclusions: In the children group we have
PAS III a, which prevailed in girls. Because the frequent association was between Autoimmune thyroiditis and DM type 1, if we havea child with an autoimunne disease is better to investigate him for another autoimmune disease, because these can coexist and beasymtomatic.
Keywords: diabetes mellitus, autoimmune thyroid disease, polyglandular autoimmune syndromes type III, children.

INTRODUCTION
Polyglandular autoimmune syndromes (PAS) (otherwise
known as polyglandular failure syndromes) are a constellationsof multiple endocrine gland insufficiencies.
Essentially, 2 types of PAS exist, type I and the more common
type II, also known as Schmidt syndrome. A third type (typeIII), which occurs in adults, has been described. Type III does
not involve the adrenal cortex, but it includes 2 of the following:
thyroid deficiency, pernicious anemia, type 1A diabetes mellitus,
vitiligo, and alopecia. Other disorders also have been described inassociation with the PAS syndromes; pulmonary hypertension inassociation with PAS syndrome type II (PAS-II) is one example (1).
In 1980, Neufeld and colleagues distinguished two major PAS
that contained Addison’s disease (PAS I and PAS II) and one
PAS that was like PAS II but without the involvement of Addison’s
disease, which was classified as PAS III (2, 6). In retrospect, thelatter two PAS are sufficiently related that PAS II a with Addison’s
disease and PAS II b without Addison’s disease would seem
more appropriate and have been used herein.
PAS 2 a/2 b appeared biased to female patients with components expressed in successive generations, suggesting adominant mode of transmission.
The evidence supporting the autoimmune nature of the component diseases of the PAS is compelling: (1) affected organs
demonstrate a chronic inflammatory infiltrate composed mainly of
lymphocytes, sometimes aggregating into follicle formation; (2) someof the component diseases are associated with immune-response
genes encoded by class-II loci of the HLA complex and more recently,
the cytotoxic T lymphocyte antigen-4 (CTLA-4) locus(2); and (3) the
syndromes are replete with auto antibodies reacting to targetedtissue-specific antigens, which often are targeted organ-specificenzymes, secretor products of the cells or their receptors.
PAS III (or PAS II b) is characterized by the presence ofautoimmune thyroid disease in association with one of the otherorgan-specific autoimmune diseases such as atrophic gastritis/
pernicious anemia, vitiligo, primary hypogonadism (female>male)
and/or DM type 1, but in the absence of Addison’s disease (3).
Frequent associations common to PAS II b as centered aroundDM type 1, myasthenia gravis (5), and vitiligo (7) have been well
documented. Graves’ disease and Hashimoto’s thyroiditis are both
frequent in PAS II, as are vitiligo and pernicious anemia. The authors
recommend routine thyroid autoantibody screening of all DM type1 diabetes patients and full endocrine autoantibody testing in thosefound to be positive. However, physicians should routinely elicit
historical and physical features relevant to the diagnostic triad in
all patients with DM type 1 and/or autoimmune thyroiditis. A family
history of poly-glandular failure is often present in past generations
that can serve as a flag for those patients who need extra monitoring.
Close to half of cases are familial, though the patterns of heritability
are variable. (4) The presence of non-endocrine autoimmune
disease, such as alopecia or vitiligo, is less common than in PAS I.
When such manifestations are present, however, they are important

Received January 5th, 2014.Accepted February 20th, 2014. Address for correspondence:Adriana Gherbon, MD, PhD, Department of Physiology, “Victor Babes” Universityof Medicine and Pharmacy Timisoara, Romania, Eftimie Murgu Square No. 2A, 300041, Timisoara, phone/fax: +40256220479; e-mail: adrianagherbon@yahoo.com

12

Fiziologia - Physiology  2014.24.1 (81)clinical indicators, especially if they are profound. The mortality risk ofuntreated adrenocortical failure in the 2% of patients with myasthenia
gravis who develop associated endocrinopathies requires that
all these patients under 40 years should be assessed closely forendocrinological disorders during their initial investigations.
So, polyglandular autoimmune syndrome type III (PAS - III)(9) is associated with the following diseases: celiac disease,hypogonadism, myasthenia gravis, sarcoidosis, Sjogren’s
syndrome, rheumatoid arthritis, gastric cancer, malabsorption
due to the pancreatic exocrine deficiency, and can be classified
in three subcategories:̶ PAS III a - autoimmune thyroiditis with DM type 1
̶ PAS III b - autoimmune thyroiditis with pernicious anemia
̶ PAS III c - autoimmune thyroiditis with vitiligo and / or alopecia and / or other autoimmune diseases
PAS III exact prevalence is unknown, but it is more common
in women than in men and it is typically observed in middle-aged
women, but can occur in people of any age. PAS III mortality
and morbidityare determined by the individual components ofthe syndrome.
MATERIAL AND METHOD
Investigated population
The group of children was represented by 83 subjects aged
between 7-17 years.All children from the study group had Type1 diabetes.In the studied group, the gender distribution of thechildren was 5.9/1, represented by 71 girls (85.54%) and 12
boys (14.45%).
The methods of investigation were represented by clinicaldata - case history, current status, imagistic- thyroid ultrasound,biochemical - for glycemic balance: fasting blood glucose, glycosylated hemoglobin, investigation of the thyroid gland: TSH,
FT4, FT3, thyroid antibodies, investigation of the adrenal gland:cortisol, 21-hydroxylase antibodies, gonadotropins: FSH, LH andappropriate sex hormones (testosterone, estradiol), investigation of celiac disease: anti-tissue-transglutaminase antibodies,
investigation of pernicious anemia: complete blood count withmean cell volume and vitamin B12 levels.
Determination of plasma glucose was performed byenzyme technique with glucosooxidasis. Normal values weretaken between 70 - 110 mg%; diabetes mellitus - values equal
or over 126 mg%, impaired glucose tolerance - values between
110 - 125 mg% and the OGTT at2 h between 140 - 200 mg%
and impaired fasting glucose - values between 110 - 125 mg%
and OGTT at 2 h under 140 mg%.
Determination of HbA1c was achieved through the DiaStat
for measuring HbA1c reported to the total HbA.
To determine the TSH level in plasma, the free fraction of triiodotironin (FT3), and the plasma free fractionof thyroxin (FT 4)were performed a quantitative method
ARCHITECT; witch is an immunological method,Chemilum
nescentMicroparticle Immunoassay (CMIA). Normal values
were following: TSH = 0.465 - 4.68 Miu/ml, FT3 = 3.69 -10.4
pmol/l, FT 4 = 10 - 28.2 pmol/l.
2014.24.1 (81)  Fiziologia - Physiology

To obtain the level of cortisol was performed the technique
IMMULITE / IMMULITE 1000, an immunometric method, in solid
phase, competitive, of chemiluminescent, Immuno-ChemiluminoEnzymometric assay (ICEM). It was considered normal: a.m.
5 - 25 microgram/dl.
FSH level was measured quantitatively by the ARCHITECT
method; a ChemilumnescentMicroparticle Immunoassay. Reference values: determined with ARCHITECT test (Table I).
Table I. The reference values for FSH

Population field
Women:- Follicular phase- Ovulating phase- Luteal phase- Postmenopausal
MenmIU/ml3.35 – 21.63
4.97 – 20.82
1.11 – 13.99
2.58 – 150.53
1.37 – 13.58

LH level was measured quantitatively by the ARCHITECT method;a ChemilumnescentMicroparticle Immunoassay. Reference values:
determined with ARCHITECT test (Table II).
Table II. The reference values for LH

Population field
Women:- Follicular phase- Ovulating phase- Luteal phase- Postmenopausal
MenmIU/ml1.26 – 10.05
2.57 – 26.53
18.06 – 90.23
0.67 – 23.75
1.09 – 92.45

Testosterone was determinate by ELISA method. The references values are depending by age and gender:
Adults:- men: 0.019 - 0.145 nmol/L;- women in fertile period: < 0.014 nmol/L;- pills: 0.001 - 0.0069 nmol/L;- postmenopausal: 0.0003 - 0.0058 nmol/L.
Estradiolwas determinate by immunochemical with electrochemiluminiscent detection method (ECLIA). The references
values are depending by age and gender, and at women alsowith the menstrual cycle period and pregnancy (Table III).
Table III.The reference values for estradiol

Age and gender

Adults – Women • Follicular phase                           • Ovulating phase                           • Luteal phase               • Postmenopausal– Men
Pregnancy (first quarter)
Children (1-10 years) • girls
                          • boysReferences values(pmol/L)
46.0 - 607
315 - 1828
161 - 774
<18.4 - 201
28.0 - 156
789 – 15781
22.0 - 99.1
<18.4 - 99.1
13

The immunological parameters were represented by autoimmune thyroid markers - antibodies(antiTPO and antiTg antibodies).
To determine serum levels of antiTPO antibodies it wasused the kit AxSYMantiTPO, an immunological method (Microparticle Enzyme Immunoassay) (MEIA). Normal values: antiTPO
antibodies <35 IU/ml.
To determine serum levels of antiTg antibodies it was used
the kit AxSYMantiTg, a MEIA method as well (Microparticle Enzyme Immunoassay). Normal values: antiTg antibodies <55 IU/ml.
To determine 21-hydroxylase (anti 21-OH antibodies) antibodies level it was used the radioimunodetermination method
combined with a technique of imunoprecipitation, based on human 21-OH marked with I 125 reacting with the antibodies anti
21-OH from the samples test and forming immune complexes
that precipitated with the solid-phase of protein A. Normal range:<1 IU/ml
Antitissuetransglutaminase antibodies were determinate
by ELISA method.
Referencesvalues: IgA, IgG :<10 U/mL: negative; ≥10 U/
mL: positive.
Vitamin B12 levels were determinate by immunochemical withelectrochemiluminiscent detection method (ECLIA). References
values: 191 - 663pmol/L (for European population).
Determination of complete blood count was achieved with
automatic method: electric impedance method. Normal values(for children): erythrocytes = 4-5.5 mil/mm3, leukocytes = 450011000 mil/mm3, plateletes = 150000-450 000/mm3, hematocrit(Ht): 32-44 %, hemoglobin (Hb): 9.5-15.5 g/dl.
Constantsandred cellindicesare calculatedautomatically, depending on the values of Hb, Ht and red blood cells (RBC) count.
Normal values: mean corpuscular volume(MCV) = 80 - 100 fl,mean corpuscular hemoglobin concentration (MCHC) = 32-36
g Hb/100 ml erythrocytes, mean corpuscular hemoglobin(MCH)= 27-32 pg.
Thyroid ultrasound was performed in all cases and allowed
us to measure thyroid volume, thyroid study and the changes
in parenchyma’s density.
An increased density, uniform, characterizes normal thyroid
parenchyma easily distinguished from the neck muscles thatare hypo dens.
Inflammatory processes and autoimmune pathology appearshypo dens. The scale was assessed as being discreet +,moderate ++ and marked +++.
In the autoimmune thyroid disease the parenchyma of thegland appears hypo dens.
Chronic autoimmune thyroid disorder appears with a hypoecogenity of the parenchyma and normal or increased thyroid volume.
STATISTICAL ANALYSIS
For statistical analysis we used Microsoft Excel and POP Tools
from Microsoft Office 2003 and EPI 2000 program. To measure
the quantitative variables were determined average (A) andstandard deviation (SD), and to assess the gender differences
we used the unpaired t test and ANOVA test, considering statistically significant a p < 0.05.
14

RESULTS AND DISCUSSION
The prevalence of PAS III in the study group was 59.03 %(85.71% girls and 14.29% boys, p < 0.001, X2 = 50).
The main endocrine autoimmune association was represented
by autoimmune chronic thyroiditis and DM type 1. In 4.82%
cases was associated Graves’ disease to DM type 1. Other nonendocrine disease association was represented by vitiligo anddecalvandpelada. (Table IV).
Table IV. Prevalence of endocrine and non – endocrine disorders inthe studied group

Associations

Subjects group
No%
DM type 1
83
DM type 1 + ACT
47
56.63%
DM type 1 + ACT + vitiligo
1
1.2 %DM type 1 + ACT + decalvantpelada
1
1.2%
DM type 1 + Graves – Basedow disease 4
4.82%
From the 83 cases of children and adolescents, in 47 cases
were associated with two autoimmune endocrine disorders, andin 2 cases three disorders. Type 1 diabetes was present in all
53 cases. In 49 cases with type 1 diabetes was associated ACT,
and in 4 cases Graves’ disease.
The most frequently detected PAS was PAS III containing after
Blizzard’s and Neufeld classification, association of autoimmune
thyroid disease (Hashimoto’s chronic thyroiditis, idiopathic myxedema, asymptomatic autoimmune thyroiditis, Graves’ disease,endocrine ophthalmopathy) with:- one or more autoimmune diseases - diabetes type 1 - type III a- association with gastric atrophy, pernicious anemia - type III b- association with vitiligo, alopecia, myasthenia gravis - type III c
Another classification of autoimmune polyendocrinopathies
and of associated non endocrine systemic imunopathies was
proposed by Volpe (Table V):
Table V. Autoimmune endocrino pathies and system icimunopathies
commonly associated (R. Volpe, 1985 modify.) (10)Autoimmuneendocrinopathies Non endocrine disorders
GravesBasedowdisease

Perniciousanemia

Hashimoto’s thyroiditis

Vitiligo

IdiopathicAddison’s disease

Myastheniagravis

Insulin dependent diabetes Sjogrensyndrome
mellitus
Autoimmunegonadalinsuffi- Rheumatoid arthritis
ciencyAutoimmune hypoparathyroid- Thrombocytopenicidiopathism
icpurpura
Autoimmune pituitary disease

Chronic activehepatitis

Autoimmune sterility(through Primary biliary cirrhosis
antisperm antibodies)
In the case of PAS III, its worldwide prevalence is not known.
Fiziologia - Physiology  2014.24.1 (81)

It turned out that occurs more frequently in women than in men
and at middle age but can occur at any age. The most common PAS III is the type 3a (DM type 1 association with chronic
autoimmune thyroiditis). DM type 1 can be consecutive or may
precede thyroiditis (10).
In the children and adolescents group of, in all cases of diabetes type 1/ACT association, the first immunopathy was DM type1, followed by ACT appearance at a variable period of time (10cases after 10-15 years, 34 cases after 6 – 10years and 5 cases
after 1-5 years of development of DM type 1) (Figure 1). In thecase of association DM type 1/Graves-Basedow disease, thelatter appeared before type 1 diabetes with 1 year in all 4 cases.
The medium interval between onset age of type 1 diabetes
and the occurrence of ACT was 8.59 ± 3.24 years.
No case presented the first endocrine immunopathy ACT,
followed by type 1 diabetes.

Fig. 1. Appearence interval of ACT after DM type 1

In the study group was a net predominance of females (85.71%
girls and 14.29% boys, p < 0.001, X2 = 50).

It occurred before the onset of endocrine immunopathies. Decalvantpelada was met in 1 case and also precede the onset of
autoimmune endocrinopathies.
In literature, a case of PAS III c in 12-year-old boy with generalized vitiligo, alopecia universalis, and Hashimoto thyroiditis wasrecently reported from Turkey, and the patient was the youngest
of previously reported cases (8).
In 4 cases we found the initial presence of Graves’ disease,followed by an interval of 1 year by the occurrence of DM type 1.
So, in our study, the prevalence of PAS type III was 59.03 %(85.71% girls and 14.29% boys, p < 0.001, X2 = 50). The subtype
was III a (autoimmune thyroiditis + DM type 1), and prevailed ingirls. In 1.2% were associated non endocrine disease as vitiligo
and decalvantpelada, and in 4.82% cases we have association
between DM type 1 and Graves’ disease.
CONCLUSION
In the children group we have PAS III a, which prevailed in girls.
Because the frequent association was between autoimmune
thyroiditis and DM type 1, if we have a child with an autoimmune
disease is better to investigate him for another autoimmune
disease, because these can coexist and be asymptomatic.
Many disorders involved in PAS present a long prodromalphase, characterized by the presence of characteristics antibodies for each disorder in part, before the clinical manifestations.
Because the second disease appears after 6 – 10 years after
the first and in this interval can be present, but asymptomatic,
and because the first was DM type 1 follow by ACT after a variable period, and this can evolve with hypothyroidism it is better
if we have an autoimmune disease to investigate it for anotherautoimmune disease.
Because the frequent association was between DM type 1
and ACT, it is better to determinate in the case of a patient with
DM type 1 TSH and specific antibodies, and in the case of ACT
fasting glucose annual.
The PAS (and mostly PAS III) classification is not final. Thismay change over time, with the onset of new endocrine disorders
or associations with new autoimmune determination.
REFERENCES

Fig. 2. Gender distribution of PAS III in the study group

Two endocrine immunopathies may be associated with a variable incidence no endocrineorgan-specific systemic diseases.In
our studied group, the patients presented no endocrineorganspecific autoimmune diseases. They were represented by vitiligo
met at 1.2% cases and by the decalvantpelada found in 1.2% ofcases. In 1 case was associated vitiligo, characterized by skinpigmentation due to autoimmune destruction of melanocytes.
2014.24.1 (81)  Fiziologia - Physiology

1. Aung K, Salmon M. Polyglandular Autoimmune Syndrome, Type III,In Endocrinology (electronic book), 2006, 1-18.
2. Maclaren N. Autoimmune polyglandular syndromes. Endocrinology,
Fifth Edition, 2009, Chapter 149.
3. Maclaren NK, Riley WJ. Thyroid, gastric, and adrenal autoimmunities
associated with insulin-dependent diabetes mellitus. Diabetes Care,
1985, 1:34.
4. Neufeld M,Maclaren NK, BlizzardRM. Two types of autoimmune
Addison’s disease associated with different polyglandular autoimmune(PGA) syndromes. Medicine (Baltimore), 1981; 60:355.
5. Osserman KE. Muscels (myasthenia gravis). Grune and Stratton,
New York,1969.
6. Song YH, Connor EL, Muir A, She JX, Zorovich B, Derovanesian D,
Maclaren N. Autoantibody epitope mapping of the 21-hydroxylase antigen in autoimmune Addison’s disease. J Clin Endocrinol Metab,1994;
78:1108.

15

7. Song YH, Connor E, LiY, ZorovichB, BalducciP, Maclaren N.The roleof tyrosinase in autoimmune vitiligo. Lancet, 1994; 344:1049.
8. Turkoglu Z, Kavala M, Kolcak O, Zindanci I, Can B. Autoimmune polyglandular syndrome-3C in a child. Dermatol Online J,
2010;16(3):8.

9. Valerio G, Maiuri L, Troncone R. Severe clinical onset of diabetes
and increased prevalence of other autoimmune diseases in children
with coeliac disease diagnosed before diabetes mellitus. Diabetologia,
2002; 45: 1719-22.
10. Zosin I. Curs de endocrinologieclinică, Lito UMF, 1997; 59-107.

PREVALENŢA SINDROMULUI POLIGLANDULAR AUTOIMUN TIP III ÎNTR-UN
GRUP DE COPII CU BOALĂ TIROIDIANĂ ŞI DIABET ZAHARATREZUMAT

Introducere: Sindromul poliglandular autoimun (SPA) (cunoscut şi ca sindromul insuficienţelor poliglandulare) reprezintă o asociere amai multor insuficienţe a glandelor endocrine. Sindromul poliglandular autoimun tip III (SPA III) cuprinde tiroidita autoimună, diabetul
zaharat mediat imun, boala celiacă, hipogonadismul, miastenia gravis, sarcoidoza, sindromul Sjogren, artrita reumatoidă, neoplazia
gatrică, şi malabsorbţia. Scopul acestui studiu este de a determina prevalenţa PAS III într-un grup de copii cu boală tiroidiană şi
diabet zaharat (DZ) tip 1. Metode: Grupul de copii a cuprins 83 de copii cu DZ tip 1 (71 fete şi 12 băieţi), cu vârsta între 7 şi 17 ani.
Metodele de investigaţie au fost reprezentate de determinarea unor parametrii clinici, imagistici, biochimici, hormonali şi imunologici.
Rezultate: Prevalenţa SPA III în grupul de studiu a fost de 59,03% (85,71% fete şi 14,29% băieţi, p < 0.001, X2 = 50). De asemenea,
subtipul SPA a fost tip III a (tiroidită cronică autoimună + DZ tip 1) şi a predominat la fete. În 1,2 % cazuri s-au asociat şi afecţiuni
neendocrine ca vitiligo şi pelada decalvantă. Concluzie: În grupul de copii s-a întâlnit SPA III a, care a predominat la fete. Deoarececea mai frecventă asociere a fost cea dintre tiroidita cronică autoimună şi DZ tip 1, în cazul în care avem un copil cu o boală autoimună
este bine să-l investigăm şi pentru alte boli autoimune, deoarece acestea pot coexista şi pot fi asimptomatice.
Cuvinte cheie: diabet zaharat, boală autoimună tiroidiană, sindrom poliglandular autoimun tip III, copii

16

Fiziologia - Physiology  2014.24.1 (81)IN VITRO TOXICITY STUDIES ON BREAST CANCER CELL
LINE SK-BR3 AND TUMOR-ASSOCIATED FIBROBLASTS (TAF)
MEDA DEICA, RALUCA SIMA, LAURA CERNAT, FLORINA BOJIN, CARMEN TATU, GABRIELATANASIE, CARMEN PANAITESCU, VIRGIL PAUNESCU
Department of Functional Sciences, ”Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACT

Solid tumors are formed of tumor cells and stroma. Within the tumor stroma, one of the most important component are the tumorassociated fibroblasts, which provide support for tumor cells development, growth, and metastasis. The purpose of this study was
to investigate the effects of metformin, a well-known anti-diabetic drug, on breast cancer cell line (SK-BR3) and tumor-associatedfibroblasts (TAFs) and to reveal the possible role of this drug as anti-tumor adjuvant treatment. We used two comparative methods,
Alamar Blue assay which is an indicator of cellular metabolic activity, and xCELLigence system, which quantifies proliferation, viability
and adherence of tested cells. We concluded that metformin induced a blockage of TAFs proliferation ability, but did not influence
the viability of these cells.
Key words: Alamar Blue, tumor-associated fibroblasts, xCELLigence system, breast cancer

INTRODUCTION
Tumors are formed of two compartments: tumor cells and avariety of untransformed cells within a specialized extracellularmatrix – stroma. Auxiliary cells found within the tumor stroma
can be: fibroblasts, myofibroblasts, leukocytes, endothelial cells,
and bone marrow-originating cells.
Recent studies showed that tumorigenesis depends uponsignals which are received by tumor cells from peri-tumoral
stroma. Due to diverse pathophysiological processes, the stroma
is remodeling tumor progression. In normal tissues it has abarrier role against tumorigenesis, by blocking proliferation oftumor cells. However, during the tumorigenesis process it wasproven that stroma helps tumor growth as a response to signals
received from tumor cells.
Tumor development and progression towards advanced
stages of the disease require co-evolution of both tumor andstromal cells. In order to facilitate this process, diverse changes
take place at stromal level. Stromal fibroblasts are an importantcomponent of stroma, which suffer genetic and epigenetic transformations, thus modulating tumor progression. These cells arealso known as myofibroblasts, tumor-associated fibroblasts orreactive fibroblasts.
The first hypothesis about inflammation role in tumorigenesis
was elaborated by Virchow, who noticed that inflammationinduced by certain irritant factors can enhance cellular proliferation (1). Several studies showed that fibroblasts are endowedwith important role in immune response due to chemokinesand cytokines production (2). It was supposed that chronic
inflammation is strongly connected with abnormal fibroblasts
behavior, which is not interrupting the inflammatory program

and lead to to unsuitable survival signals and retention of leukocytes within inflamed tissue (3). From the clinical point of view,
chronic inflammation and cancer are strongly related, cancers
being considered as a wound that never heals. Tumor cells are
secreting pro-inflammatory cytokines, while tumor-associatedfibroblasts (TAFs) attract immune cells towards tumor tissue.
Thus, macrophages, neutrophils, and lymphocytes are recruitedby tumor stroma by diverse factors secreted by fibroblasts.
Macrophages, once reaching tumor stroma, begin to differentiate into tumor-associated macrophages and help developmentof tumor and metastasis. By secretion of different factors, suchas VEGF, HGF, MMP2, and IL-8, they can influence behavior
of endothelial cells, which begin to be actively involved into theangiogenesis process (4).
Tumor invasion or metastasis is a complex process in whichtumor cells of primary tumor tissue reach and colonize othertissues and organs. For a long time it was considered that metastasis is strictly influenced by genetic and epigenetic changesof tumor cells, but latest studies showed that interaction between
tumor cells and stroma facilitate tumor invasion.
Different studies demonstrated that presence of a large population of myofibroblasts within the stroma of different tumor types
is associated with an increased metastasis risk and poor prognosis. Cross-signaling between tumor cells and tumor-associatedfibroblasts contribute to changes of extracellular matrix andbasal membrane. It is know that basal membrane breakage isthe first step towards intravasation of tumor cells within systemic
circulation. On the other hand, extracellular matrix remodelingcan alter expression of genes which are essential in cytoskeleton
organization (5). Actually, tumor-associated fibroblasts have an

Received 5th of January 2014.Accepted 25th of January 2014. Address for correspondence: MedaDeica, MD, PhD student, Department of Functional Sciences, Physiology, “Victor Babes” University of Medicine and Pharmacy Timisoara, EftimieMurgu Square No. 2A, RO-300041, Timisoara, Romania, Phone/fax: + 40 256490507;
email: bravameda@yahoo.com

2014.24.1 (81)  Fiziologia - Physiology

17invasive potential, demonstrated by in vivo studies. Using transgenic GFP (green fluorescent protein) mice, under the VEGF
promoter control, Fukumura discovered that tumor-associatedfibroblasts positive for GFP invade tumor sites (6). Another roleof the fibroblasts is to direct tumor invasion towards a certain site.
The results of in vitro experiments on co-cultures showed thatcells guiding tumor invasion are of stromal origin and facilitate
tumor cells migration due to degradation of extracellular matrix
and basal membrane, thus inducing structural and proteolyticchanges (7).
The purpose of this study was to investigate the effects of metformin, a well-known anti-diabetic drug, on breast cancer cell line
(SK-BR3) and tumor-associated fibroblasts (TAFs) and to reveal
the possible role of this drug as anti-tumor adjuvant treatment.
MATERIALS AND METHODSxCELLigence system
We used RTCA DP Analyzer (ACEA Biosciences.Inc, San
Diego, USA) for assessment of proliferation rate of breast cancer
cell line SK-BR3 and tumor-associated fibroblasts (TAF). The
cells were detached from the culture plate using Trypsin, andcellular suspension was replated in specific 16-well culture plate(E-plate 16), in humid atmosphere, with 5% CO2, at 37o C. For
each well we added 5000 cells in 150 µl of specific media. 24hours after cell addition, we added metformin (Sigma-AldrichCompany, Germany) in 2 concentrations, of 5 mM and 10 mM.
The results were interpreted before metformin addition, and 48
hours after addition of the chemical substance. The experimentlasted for 144 hours, and results interpretation was performed
using RTCA Software (ACEA Biosciences.Inc).
xCELLigence system is based on using gold biosensors for
detecting impedance change as a result of cellular interaction
and the adherent surface of each well. Each sample is analyzed
at least as duplicate. On the base of each well a microelectrodeis placed to detect changes in cellular number, cellular morphology, cell adhesion and cell membrane integrity for specified time
duration.
Alamar Blue assay
The proliferation rate was tested in parallel using AlamarBlue® (Invitrogen). AlamarBlue® is a non-toxic reagent which isused for evaluation of metabolic activity of live cells, which will
reduce resazurin (dark blue colored compound) with intrinsic
fluorescence, to resorufin, a pink-colored compound, highly
fluorescent (extinction 579 nm, emission 584 nm). Maximum
absorbance of resazurin is at 605 nm, while for resorufin it is573 nm.
For AlamarBlue® assay the cells were cultured in 96-wellplates, at a cellular density of 7000 cells/well, in quadruplicates.
24 hours after incubation, metformin was added in concentrations of 5 and 10 mM in specific media, while AlamarBlue®
was added after 48 hours. The values of sample extinctions
were read at 570 and 600 nm using Bio Rad Microplate Reader
spectrophotometer (Benchmark). Interpretation of the Ala18

marBlue® test results uses the following calculation formula,which provides the level of reagent reduction due to cellular
metabolic activity:

Where:- AB%N-percentage value of Alamar Blue reagent reduction
compared to negative control (culture media without cells)- O1- molar extinction coefficient (E) for oxidized Alamar Blueat 570 nm- O2- E for Alamar Blue oxidized at 600 nm- R1- E for Alamar Blue reduced at 570 nm- R2- E for Alamar Blue reduced at 600 nm- A1- absorbance of tested cells at 570 nm- A2- absorbance of tested cells at 600 nm- N1- absorbance of negative control at 570 nm- N2- absorbance of negative control at 600 nm
Molar extinction coefficients for Alamar Blue are:Wave length

Reduced form (R)Oxidized form (O)570 nm

15567780586

600 nm14652
117216
Increased percent of reduced Alamar Blue is indicative ofintense cellular metabolic activity, and high viability and cellular proliferation, because proliferating viable cells induce
a chemical reduction of Alamar Blue reagent, while deadcells or non-proliferating cells maintain an oxidized form of
the reagent.
RESULTS
xCelligence System assays
Analysis of cellular proliferation rate was performed inthree stages: before metformin addition, 48 hours after
metformin addition, and the following 48 hours. Considering the positive control proliferation rate (cells with specificmedium), we noticed that 72 hours after plating (3 days) the
proliferation rate begins to decrease. This meant that without
any intervention, cellular viability is impaired. So, this was
the reason why we considered the 48 hours time interval
after addition of metformin (72 hours from cellular plating)as the relevant time interval for analysis of metformin effecton cellular proliferation.
Proliferation graph given by xCelligence system suggest that
SK-BR3 cells proliferation increases with increase of metformin
concentration (Figure 1). The graphs depicting time and proliferation velocity (doubling time and slope) support previous results.
Three stages analysis showed that 48 hours after metformin
addition in culture medium, proliferation velocity of SK-BR3 cells
is more increased for 5 mM metformin concentration than for 10
mM metformin concentration (Figures 2 and 3).
Fiziologia - Physiology  2014.24.1 (81)Fig. 1. Proliferation rate expressed by Cell index for SK-BR3 breast
cancer cells during the entire experiment (from 0 to 144 hours).

Proliferation diagram given by xCelligence system suggestthat TAFs proliferation decreases when cells are treated with
metformin (Figure 4). The graphs representing doubling timeand proliferation velocity (slope) are also supporting this result.
Three stages analysis showed that 48 hours after metformin addition in culture medium, proliferation velocity of TAFs decreased
proportional with metformin concentration, being lower for 10 mM
than 5 mM metfomin concentration (Figures 5 and 6).

Fig. 4. Proliferation rate expressed by Cell index for TAFs during theentire experiment (from 0 to 144 hours).

Fig. 2. Upper panel – Doubling time; lower panel – Slope (proliferation
velocity) for SK-BR3 cells during the entire experiment (0-144 hours)

Fig. 5. Upper panel – Doubling time; lower panel – Slope (proliferation velocity) for TAFs during the entire experiment (0-144 hours)

Fig. 3. Proliferation velocity (slope) for SK-BR3 cells. Upper panel– before metformin addition; middle panel – first 48 hours after
metformin addition; lower panel – after addition of 5 mM and 10 mM
metformin
2014.24.1 (81)  Fiziologia - Physiology

19Fig. 6. Proliferation velocity (slope) for TAFs. Upper panel – before
metformin addition; middle panel – first 48 hours after metformin addition; lower panel – after addition of 5 mM and 10 mM metformin

Alamar Blue assay results
Alamar Blue assay was performed during three days, anddaily we tested the metabolic activity of SK-BR3 cells andTAFs, untreated, and treated with metformin in 5 mM and 10
mM concentration.
Within the analysis we monitored time-dependent and metformin concentration-dependent metabolic activity of the cells.
For SK-BR3 we noticed a decrease in metabolic activity in thefirst day, followed by an increase in the following two days (day2 and 3). We can notice a higher decrease of metabolic activity
for cells treated with lower metfromin concentration (Figure 7).
For TAFs, we were not able to detect any change in metabolic
activity, for any of metformin concentrations. Moreover, related totime, metabolic activity remains constant (Figure 8). The results
suggest that 5 mM metformin concentration negatively influences
metabolic activity of SK-BR3 cells, more than higher metformin
concentration (10 mM). Cellular metabolic activity of TAFs is not
influenced by metformin.

Fig. 8. Alamar Blue assay results for TAFs during 3 days of experiment after addition of 5 mM and 10 mM, comparative to positive
control cells (untreated) and negative control cells (DMSO-treated).

Fig. 9. Comparative analysis of metabolic activity for TAFs and SKBR3 when treated with 5 mM and 10 mM metformin, during 3 days ofexperiment.

Fig. 7. Alamar Blue assay results for SK-BR3 during 3 days of experiment after addition of 5 mM and 10 mM, comparative to positive
control cells (untreated) and negative control cells (DMSO-treated).

20

The results of in vitro toxicity studies performed with xCelligence system showed that SK-BR3 proliferation rare increases
when metformin is added into culture media. Although proliferation velocity is increased in presence of metformin, cellular
metabolic activity of SK-BR3 cells is negatively influenced.
Toxicity studies employing Alamar Blue reagent suggest that 5
mM metformin concentration induced more important changes
in metabolic activity than 10 mM concentration. Interestingly, following the diagram of proliferation velocity within 48 hours after
metformin addition, we noticed that proliferation velocity (slope)
increases for 5 mM metformin canocentration, more than for 10
mM metformin concentration, with no statistic significance. So,
we may conclude that metformin effect on cellular proliferationand metabolic activity is augmented for 5 mM compared to 10
Fiziologia - Physiology  2014.24.1 (81)mM. Metformin does not stop SK-BR3 cells proliferation, but
stimulates it, inducing in the same time changes in cellular metabolism. In order to establish the connection between increased
proliferation velocity and decreased cellular metabolism, manyelaborated cellular metabolism tests are required.
In contrast with results obtained for SK-BR3 cells, metformin
decreases TAFs proliferation, without affecting cellular metabolism. Proliferation diagram obtained using the xCelligence systemdemonstrates that TAFs proliferation decreases when metformin
is added into the culture medium, anti-proliferative effect being
stronger for 5 mM metformin concentration. According to Alamar Blue tests, metabolic activity of TAFs is not influenced by
metformin. So, we may conclude that metformin decrease theproliferation rate, without affecting the cellular viability results.
Metformin is not inducing cellular death, but probably blocks thecells in a certain phase of cellular cycle.
DISCUSSION AND CONCLUSION
There are numerous studies related to metformin action onbreast cancer cells. Most studies are performed on MCF-7 cell line,
and only few studies included SK-BR3 cells. Vasquez-Martin et al.
studied the influence of metformin on Her2 expression in SK-BR3
cells (8). In this study, SK-BR3 were treated with diverse metformin
concentrations and they noticed a decreased cellular metabolic
activity and viability, directly proportional with concentration increase. Our results also showed that metabolic activity decreases
with metformin addition, but we were not able to demonstrate
the dose-effect relationship (lower concentration induced a moredecreased viability compared to higher metformin concentration).
Metformin action was tested on different tumor cells, but there
was no study published regarding metformin effects on tumorassociated fibroblasts. There are many studies supporting theimportance of fibroblasts for tumor growth and development.
Considering all the above, we investigated metformin effects
on tumor-associated fibroblasts. As a result of our experiments,
we concluded that metformin induces a decrease of TAFs proliferation capacity, but is not influencing cellular metabolism andviability. Thus, we concluded that further analyses are required,including cell cycle assays, to investigate whether metforminblocks the cell cycle, and to show in which of its phase. There
are studies showing that metformin blocks cell cycle in pancreatictumor cells (9), and triple negative breast cancer cells (10), while

metformin blocks prostate cancer cells in G(0)/G(1) phase (11).
As a conclusion, we may say that metformin can perform its
anti-tumor action on tumor-associated fibroblasts and can beconsidered as adjuvant treatment to anti-tumor chemotherapy.
Acknowledgements
This work was supported by the Sectorial Operational Programme for Human Resources Development, financed from theEuropean Social Fund, FSE POSDRU 107/1.5/S/78702 and by
UEFISCDI, PNII-Idei, Project No. 318/2011.
REFERENCES

1. Balkwill F, Mantovani A. Inflammation and cancer: back to Virchow?
The Lancet, 2001; 357(9255): 539-45.
2. Buckley C, Filer A, Haworth O, Parsonage G, Salmon M. Defining
a role for fibroblasts in the persistence of chronic inflammatory joint
disease. Ann Rheum Dis, 2004; 63: ii92-ii95.
3. Buckley CD, Pilling D, Lord JM, Akbar AN, Scheel-Toellner D, Salmon
M. Fibroblasts regulate the switch from acute resolving to chronic persistent inflammation. Trends Immunol., 2001; 22(4): 199-204.
4. Leek RD, Harris Al. Tumor-associated macrophages in breast cancer.
J Mammary Gland Biol Neoplasia, 2002; 7(2): 177-189.
5. Lukashev ME, Werb Z. ECM signalling: orchestrating cell behaviour
and misbehaviour. Trends Cell Biol., 1998; 8(11): 437-441.
6. Fukumura D, Xavier R, Sugiura T, Chen Y, Park EC, et al. Tumor
induction of VEGF promoter activity in stromal cells. Cell, 1998; 94(6):715-25.
7. Gaggioli C, Hooper S, Hidalgo-Carcedo C, Grosse R, Marshall JF,
Harrington K, Sahai E. Fibroblast-led collective invasion of carcinoma
cells with differing roles for Rho-GTPases in leading and following cells.
Nat. Cell Biol., 2007; 9(12): 1392-1400.
8. Vazquez-Martin A, Oliveras-Ferraros C, Menendez JA. The antidiabetic drug metformin suppresses HER2 (erbB-2) oncoprotein
overexpression via inhibition of the mTOR effector p70S6K1 in humanbreast carcinoma cells. Cell Cycle, 2009; 8(1): 88-96.
9. Wang LW, Li ZS, Zou DW, Jin ZD, Gao J, Xu GM. Metformin induces
apoptosis of pancreatic cancer cells. World J. Gastroenterol., 2008;
14(47): 7192-98.
10. Liu B, Fan Z, Edgerton SM, Deng XS, Alimova IN, Lind SE, Thor
AD. Metformin induces unique biological and molecular responses in
triple negative breast cancer cells. Cell Cycle, 2009; 8(13): 2031-2040.
11. Sahra IB, Laurent K, Loubat A, Giorgetti-Peraldi S, Colosetti P, etal. The antidiabetic drug metformin exerts an antitumoral effect in vitro
and in vivo through a decrease of cyclin D1 level. Oncogene, 2008;27(25): 3576-86.

STUDII DE TOXICITATE IN VITRO EFECTUATE PE LINIA CELULARA DE
CANCER DE SAN SK-BR3 SI FIBROBLASTELE PERI-TUMORALE (TAF)
REZUMAT

Tumorile solide sunt formate din celule tumorale si stroma. La nivelul stromei tumorale, una dintre cele mai importante componentesunt fibroblastele peri-tumorale, care asigura suport pentru dezvoltarea tumorala, crestere si metastazare. Scopul acestui studiu afost investigarea efectelor metforminului, un anti-diabetic oral frecvent utilizat, asupra celulelor liniei de cancer mamar (SK-BR3) si
la nivelul fibroblastelor peri-tumorale (TAFs), pentru a releva un posibil rol al acestui medicament in terapia adjuvanta anti-tumorala.
Am utilizat doua metode comparative, testul Alamar Blue, care este un indicator al activitatii metabolice celulare si sistemul xCELLigence, care poate oferi informatii referitoare la proliferarea, viabilitatea si aderenta celulara. In urma analizei rezultatelor obtinuteputem concluziona ca metforminul induce blocarea capacitatii de proliferare a TAFs, dar nu influenteaza viabilitatea acestor celule.
Cuvinte cheie: Alamar Blue, fibroblaste peri-tumorale, sistem xCELLigence, celule tumorale mamare
2014.24.1 (81)  Fiziologia - Physiology

21

LYMPHOCYTES SUBSETS INVOLVED IN ALZHEIMER DISEASEASSOCIATED IMMUNOSENESCENCEGROZA SABINE, ANGHEL SIMONA, CARMEN TATU, GABRIELA TANASIE, CARMEN PANAITESCU,OANA GAVRILIUC, VIRGIL PAUNESCU, FLORINA BOJIN
Department of Functional Sciences, “Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACT

Immunosenescence is defined as progressive alteration of morphological and functional characteristics, at the level of immune
system, during the aging process. The purpose of this study was to define the immunosenescence status, to characterize proliferative potential and apoptosis intensity in T lymphocyte subpopulations, as well as to define the Alzheimer disease (AD)-associatedimmunosenescence in clinically diagnosed dementia, as well as prodromal stages (pre-dementia). The expected impact of thisstudy is related to significant development of state of the art in the field, related to mechanisms and clinical symptoms occurring in
immunosenescence and AD-related immunosenescence.
Key words: immunosenescence, Alzheimer disease, pre-dementia

INTRODUCTION
Alzheimer’s disease (AD) is one of the most common forms
of dementing disorder, characterized by deterioration of functional and cognitive ability. From the neurological point of view,
Alzheimer disease (AD) patients present loss of synaptic andneuronal capacity, and extracellular plaques, mainly consistingof amyloid-β (aβ), and intracellular neurofibrillary tangles of the
microtubule-associated protein Tau. The amyloid hypothesis isbased on increased production of aβ, cytoskeletal changes, andneuronal loss. These aβ changes are associated with diseasecausing mutations in genes related to the generation of aβ.
The pathology typically starts in the structures of the medial
temporal lobe, and extends to the other cortical areas in a hierarchical manner (1,2), leading to vascular alterations withdeposition of aβ in the vessel walls, especially in carriers of theapolipoprotein (apoE) ε4 allele (3), as well as various inflammatory reactions (4).
The possible influence of viral infections on AD development
has been investigated (5), such as herpes simplex virus andhuman cytomegalovirus (CMV). These viruses cause chronicallypersistent infection that in the immunocompetent adult rarely escapes immune surveillance, but can causesevere disease in patients with suppressed immune function (6,7). The CMV hasbeen
shown to inflict a deep imprint in the host T-cell compartmentthat is characterized by an age-related oligoclonal expansionof differentiated CD8 (CD27-CD28-) cellsand a corresponding
decrease inproportion of naïve cells (8-11). Also, the degreeof differentiation in the CD4compartments has been shown tocorrelate with levels of CMVIgG(12). Alterations in systemic
immunity have been shown to occurin the elderly, and the term
immunosenescence is used to describe the age-related decline
in capacity and regulatorybalance of both innate and adaptive

immune responses (13,14). Deregulation of immunoactive cells
could also explainthe progression of baseline systemic inflammation called inflammaging, which is considered a risk factor
for several age-relateddiseases and where the role of CMV has
beeninvestigated (15). Clinically important immunosenescencesigns include an impaired vaccine response and an increased
incidence of severe bacterial and viralinfections in the elderly.
In Swedish octogenarian andnonagenarian cohorts (16,17) a
defined immune risk profile(IRP) consisting of a shift in CD4/CD8
ratio was associatedwith increased morbidity and mortality (18).
Alzheimer’s disease has previously been associated withshifts
in CD4 as well as CD8 T-cell subsets (19-21).
The aim of this study was to establish the correlation between
AD-associated immunosenescence and T cell subpopulation
characteristic markers, in different stages of AD, comparatively
with control, healthy patients.
MATERIALS AND METHODSIn order to establish the study groups for patients with AD in
different stages, we evaluated 24 patients, age between 57-84
years old. After signing the Informed Consent form (elaborated
based on a well-established protocol, approved by the Ethics
Committee of “Victor Babes” University of Medicine andPharmacy Timisoara), two tests were applied to these subjects:
Standardized Mini-Mental State Examination and Clock drawing
test.
As a result of this evaluation, correlated with other clinicalsymptoms, the subjects were divided in two groups: early stage
dementia (10 patients) and severe dementia (14 patients).
As control group, we investigated 20 elderly patients without
dementia, with similar age range.

Received 10th of January 2014. Accepted 25th of March 2014. Address for correspondence: Sabine Groza, MD, PhD student, Department of Functional Sciences, Immunology, “Victor Babes” University of Medicine and Pharmacy Timisoara, EftimieMurgu Square No. 2A, RO-300041, Timisoara, Romania, Phone/fax: + 40 256490507;
email: sabine_groza@yahoo.com

22

Fiziologia - Physiology  2014.24.1 (81)Blood harvesting and cell processing
10 ml peripheral blood was harvested on anticoagulant(Heparin) from both study and control groups. Blood was diluted
with Dulbecco’s Phosphate Buffered Saline (PBS; Sigma-AldrichCompany, St. Louis, USA) with a dilution ratio of 1:1, and thenwe used gradient centrifugation (Ficoll-Hipaque, GE Healthcare
Bio-Sciences AB) for 25 minutes at 2500 rpm for peripheral bloodmononuclear cells (PBMCs) separation. All PBMCs samples
were cryopreserved until further use.
Cell cycle analysis
PBMCs were submitted to cell cycle analysis, performed using
CycleTest™ Plus, DNA Reagent Kit (Becton-Dickinson, San
Jose, CA, USA). This method involves dissolving cell membrane
lipids, eliminating cellular cytoskeleton and nuclear proteins,digestion of cell RNA, and stabilization of nuclear chromatin,
followed by propidium iodide (PI) binding to the clean, isolated
nuclei. All procedures were according to the manufacturer
protocols and included a first step of addition of 250 μL of Solution
A (trypsin buffer) which was left to interact for 10 minutes at room
temperature. A second step of 200 μL of Solution B (trypsin
inhibitor and RNase buffer) addition and 10 minutes resting atroom temperature was followed by the last step of 200 μL of cold
(2° to 8°C) Solution C (propidium iodide stain solution) additionin the same tube, gentle mixture and incubation for 10 minutes
in the dark in the refrigerator.
Annexin V/PI viability assay
Annexin V-FITC (MiltenyiBiotec, Gladbach, Germany) wasused in cell death flowcytometric studies (apoptosis) combinedwith Propidium Iodide Staining Solution (BD Biosciences,
San Jose, CA, USA) following the manufacturer protocol.
Shortly, 106 cells were washed in 1 x Annexin V Binding Buffer(BD Pharmigen) and centrifuged at 300 x g for 10 minutes,
resuspended in the same solution and incubated with 10 μl ofAnnexin V-FITC for 15 minutes in the dark. After washing thecells with 1 ml specific binding buffer and centrifugation, thecell pellet was resuspended in 500 μl binding buffer and 1 μg/
ml of PI solution was added immediately prior to analysis byflow cytometry.
Data acquisition for Annexin V/PI assay and Cell cycle
flowcytometric procedures was performed on a four-color
capable FACSCalibur (Becton-Dickinson) flow cytometer,while data analysis employed CellQuest Pro software (BectonDickinson).
Immunophenotypical analysis of PBMC
For flowcytometric analysis of surface and intracellularmarkers, PBMCs were washed twice in PBS and resuspended
in 100 μL PBS for a concentration of 105 cells/mL. Cells wereincubated at room temperature, in the dark for 30 minuteswith mouse anti-human antibodies, conjugated with specificfluorochromes, at a dilution specified by the producer. Cells werethen washed twice with 1 mL of cell wash solution (Cell Wash
Solution - BD Biosciences, San Jose, CA, USA) and resuspended
2014.24.1 (81)  Fiziologia - Physiology

in 500 μL of the same solution for further acquisition and analysis
with FACSCalibur flowcytometer (Becton-Dickinson), which
can detect 4 colors simultaneously. PBMCs were analyzed
for presence of the following markers: CD3 (FITC)/CD16+56(PE)/CD45 (PerCP)/CD19 (APC); CD3 (FITC)/CD8 (PE)/
CD45 (PerCP)/CD4 (APC); CD45RA (FITC)/CD45RO (PE)/
CD3 (PerCP)/CD4 (APC); CD25 (FITC)/CD154 (PE); CD28
(FITC)/CD184 (PE); Bcl2 (FITC)/TCR γδ (PE). Conjugatedantibodies used were purchased from BD Pharmingen™.
Sample acquisition and analysis was performed using CellQuest
Pro software (Becton-Dickinson), and Flowing Software 2.5,
respectively.
RESULTS AND DISCUSSION
Statistical analysis of PBMCs in different phases of cell cycle
showed that AD patients present a decrease of cells in DNA
synthesis and replication phase (S) or mitosis preparation
phase (G2/M), which demonstrates that proliferation rate ofthese cells is much decreased. Similar results can be observedin case of patients with pre-Alzheimer, where there is an increase of cells in growth and DNA synthesis preparation phase(G0/G1), but this increase ratio is not found in other phases ofcell cycle, demonstrating blockage of cell proliferation (Figures
1, 2 and Table I).

Fig. 1. Graphic results for cell cycle analysis in young subjects/older
subjects groups

Fig.2. Graphic results for cell cycle analysis in pre-Alzheimer/Alzheimer patients group23

Table I. Statistic analysis of PBMC in different phases of cell cycle

Sample Sub-G0 (%)

G0/G1 (%) S (%)

G2/M (%)

Control

88.12±2.24

0.32±0.05

8.9±1.03

1.53±0.24

Pre-Al- 91.56±1.85
zheimer

1.13±0.08

6.09±0.89 0.63±0.12

Al94.23±1.79 0.49±0.04 4.37±0.76 0.39±0.13zheimer
Statistical analysis of apoptosis propensity in different study
groups (Figure 3) shows that, compared to control subjects,
intensity of apoptotic processes in pre-Alzheimer patients andAD patients, respectively, is significantly increased compared tocontrol group (Table II). There are no statistical significant differences between pre-Alzheimer and AD patients regarding thetotal apoptotic processes. Moreover, cell death is similar between
different study groups, significant differences being in case ofearly apoptosis (36% compared to 5%) and late apoptosis (8%compared to 2%), respectively (data not shown).

Table II.Statistical analysis of PBMC in different apoptosis phases incontrol, pre-Alzheimer, and Alzheimer subjects

SampleUn-functional cells(dead cells, late/early apoptotic
cells)

Viable cells

Control

15.13±1.2%84.87±1.3%

Pre-Alzheimer

62.09±0.98%

37.91±1.02%

Alzheimer
67.7±0.95%
32.3±1.04%
Flowcytometric analysis of T cells subpopulations showed normal distributions of cytotoxic T cells (CD4+), with significant proportion of activated cells (CD25+), less naïve T cells (CD45RA+),and increased number of memory T cells (CD45RO+). The ratio
between Th/CTL is maintained at 2/1.
For intracellular staining of Bcl2 we used monoclonal antibody
mouse anti-human Bcl2 oncoprotein/FITC (DakoCytomation)
and DakoCytomation Intrastain – intracellular staining kit, in
accordance with producer recommendation. Bcl2oncoprotein
is associated to mitochondria, smooth endoplasmic reticulum,and peri-nuclear membrane, being endowed with central role inapoptosis inhibition (programmed cell death). Expression of thisprotein in T cells from the control group was evaluated at 10%
from total T lymphocytes (Figure 4).

Fig. 4.Flowcytometric analysis of T cells subpopulations for subjects
in control group. Dotplot graphs for markers representative to studied
lymphocytic subpopulations.

Fig. 3. Graphic aspects of Annexin V/PI viability assay in control/preAlzheimer/Alzheimer patients groups

24

In case of patients from the ��������������������������������
pre-Alzheimer and AD groups, respectively, we used the same panel for acquisition of analysis
of T cells subpopulation. We revealed a change in Th/CTL ratio
from 2/1 to less than 1, with increased proliferation of cytotoxic
CD8+ T cells. However, CTLs are naïve cells (CD8+CD45RA+),
wich are not activated at global level (CD25-). Bcl2 oncoprotein
expression level was evaluated at 8% from total T lymphocytes,
Fiziologia - Physiology  2014.24.1 (81)

significantly decreased compared to control groups, both young
and elders (Figure 5).

Fig. 5. Flowcytometric analysis of T cells subpopulations for subjects
in pre-Alzheimer and AD groups. Dotplot graphs for markers representative to studied lymphocytic subpopulations.

proportion of highly differentiated T cells, which exhibits surface
phenotypic markers and present specific functional characteristics (22,24). Additionally, alteration in cytokines production or inrecognition of these cytokines by the cells of immune system, areresponsible of changes observed during the immunosenescenceprocess, especially associated with AD (25).
Expression of co-stimulatory molecules CD27 and CD28
was used for study of differentiation phenotype of antigen-specificcells, during chronic phase of persistent infection (5). According
to T lymphocytes differentiation pattern, CD27+CD28+ T cells
represent population of naïve cells or cells in early stages of differentiation, which progresses towards CD27-CD28- phenotype,which represent finally differentiated T cells (26). Due to association of CD27 and CD28 expression with different differentiation
stages of both CD4+ and CD8+ T cells (5), this classification
is used for comparing the subsets of CD4+ T cells in young
persons, elders, and neurodegenerative disorders patients(AD). For studying differentiation status of CD4+ T cells, someother markers may be used, such as CD45RA, CD11a, CCR7,
perforines production, and telomere length.
Although cytokines production is controversial in elders,
it is well known that senescence is associated with increased
global production of INFγ (22). INFγproduction by CD8+ T cells
was investigated and correlated with accumulation of effector
T cells CD28-, terminally differentiated, while their number increases dramatically in neurodegenerative disorders. However,there are few data related to cytokines profile of CD4+ T cells
subpopulations, correlated with senescence process, especiallyin AD-associated immunosenescence. Another unsolved aspectis the correlation between cytokines production related to T cells
shift, from naïve CD4+ cells to effector, memory cells. CD4+ T cells
are essential for induction of immunity as a result of vaccination.
Due to decreased vaccination protection in elders, further directions of research would be to monitor and compare proliferation
capacity of CD4+ T cells in healthy youngers and elders, to AD
patients in different disease stages.
Acknowledgements
This work was supported by UEFISCDI, PNII-Idei, Project No.
259/2011.
REFERENCES

Fig. 6. Flowcytometric analysis of T cells subpopulations, comparatively for all studied groups. Histogram graphs for markers representative to studied lymphocytic subpopulations (analysis was performed
using Flowing Software 2.5)

As the individuals are growing older, there is an important
decrease in immune response. This leads in most of the cases tooccurrence of severe infections and decreased protective effectof vaccination (22). Thymus involution and decreased productionof T lymphocytes are the most frequent changes which occur inelders (23). This is the reason why in elders there is an increase
2014.24.1 (81)  Fiziologia - Physiology

1. 1Hyman BT, Van Hoesen GW, Damasio AR, Barnes CL. Alzheimer’s
disease: cell-specific pathology isolates the hippocampal formation.
Science, 1984; 225: 1168-1170.
2. Braak H, Braak E. Neuropathologicalstageing of Alzheimer-relatedchanges. ActaNeuropathol, 1991; 82: 239-259.
3. Premkumar DR, Cohen DL, Hedera P, Friedland RP, Kalaria RN.
Apolipoprotein E-epsilon4 alleles in cerebral amyloid angiopathy andcerebrovascular pathology associated with Alzheimer’s disease. Am J
Pathol, 1996; 148: 2083-2095.
4. Nilsson L, Rogers J, Potter H. The essential role of inflammationand induced gene expression in the pathogenic pathway of Alzheimer’s
disease. Front Biosci, 1998; 3: d436-d446.
5. Marques AR, Straus SE, Fahle G, Weir S, Csako G, et al.Lack ofassociation between HSV-1 DNA in the brain, Alzheimer’s disease and25apolipoprotein E4. J Neurovirol, 2001; 7: 82-83.
6. Boeckh M, Nichols WG. The impact of cytomegalovirus serostatusof donor and recipient before hematopoietic stem cell transplantationin the era of antiviral prophylaxis and preemptive therapy. Blood, 2004;
103: 2003-2008.
7. Sund F, Lidehäll AK, Claesson K, Foss A, Tötterman TH, et al. CMVspecific T-cell immunity, viral load, and clinical outcome in seropositive
renal transplant recipients: a pilot study. Clin Transplant, 2010; 24:
401-409.
8. Khan N, Shariff N, Cobbold M, Bruton R, Ainsworth JA, et al. Cytomegalovirus seropositivity drives the CD8 T cell repertoire toward greaterclonality in healthy elderly individuals. J Immunol, 2002; 169: 1984-1992.
9. Vescovini R, Biasini C, Fagnoni FF, Telera AR, Zanlari L, et al.Massive
load of functional effector CD4+ and CD8+ T cells against cytomegalovirus in very old subjects. J Immunol, 2007; 179: 4283-4291.
10. Vescovini R, Telera A, Fagnoni FF, Biasini C, Medici MC, etal.Different contribution of EBV and CMV infections in very long-term
carriers to age-related alterations of CD8+ T cells. ExpGerontol, 2004;
39: 1233-1243.
11. Derhovanessian E, Maier AB, Hähnel K, Beck R, de Craen AJ, et al.
Infection with cytomegalovirus but not herpes simplex virus induces theaccumulation of late-differentiated CD4+ and CD8+ T-cells in humans.
J Gen Virol, 2011; 92: 2746-2756.
12. Alonso Arias R, Moro-García MA, Echeverría A, Solano-Jaurrieta
JJ, Suárez-García FM, et al. ������������������������������������������
Intensity of the humoral response to cytomegalovirus is associated with the phenotypic and functional status of
the immune system. J Virol, 2013; 87: 4486-4495.
13. Solana R, TarazonaR, Aiello AE, Akbar AN, Appay V, et al. CMV and
Immunosenescence: from basics to clinics. Immun Ageing, 2012; 9: 23.
14. Herndler-Brandstetter D, Landgraf K, Tzankov A, Jenewein B,
Brunauer R, et al. The impact of aging on memory T cell phenotype andfunction in the human bone marrow. J LeukocBiol, 2012; 91: 197-205.
15. Bartlett DB, Firth CM, Phillips AC, Moss P, Baylis D, et al. The age31: 685-90.

related increase in low-grade systemic inflammation (Inflammaging) is
not driven by cytomegalovirus infection. Aging Cell, 2012; 11: 912-915.
16. Wikby A, Johansson B, Olsson J, Löfgren S, Nilsson BO, et al.
Expansions of peripheral blood CD8 T-lymphocyte subpopulations
andan association with cytomegalovirus seropositivity in the elderly:
theSwedish NONA immune study. ExpGerontol, 2002; 37: 445-453.
17. Wikby A, Maxson P, Olsson J, Johansson B, Ferguson FG. Changes
in CD8 and CD4 lymphocyte subsets, T cell proliferationresponses andnon-survival in the very old: the Swedish longitudinalOCTO-immune
study. Mech Ageing Dev, 1998; 102: 187-198.
18. Colonna-Romano G, Akbar AN, Aquino A, Bulati M, Candore G, etal. Impact of CMV and EBV seropositivity on CD8 T lymphocytes inan
old population from West-Sicily. ExpGerontol, 2007; 42: 995-1002.
19. Pellicanò M, Larbi A, Goldeck D, Colonna-Romano G, Buffa S,et al.Immune profiling of Alzheimer patients. J Neuroimmunol, 2012;
242:52-59.
20. Larbi A, Pawelec G, Witkowski JM, Schipper HM, DerhovanessianE, et al. Dramatic shifts in circulating CD4 but not CD8 T cell subsetsin
mild Alzheimer’s disease. J Alzheimers Dis, 2009; 17: 91-103.
21. Pirttilä T, Mattinen S, Frey H. The decrease of CD8-positivelymphocytes in Alzheimer’s disease.J NeurolSci, 1992; 107: 160-165.
22. Gruebeck-Loebenstein B, Wick G. The aging of the immune system.
Adv. Immunol., 2002; 80:243.
23. Gardner EM, Murasko DM. Age-related changes in type 1 and type2 cytokine production in humans. Biogerontology, 2002; 3:271.
24. Banderes E, Merino J, Vazquez B, et al. The increase INF-gamma
production through aging correlates with the expanded CD8(+high)CD28(-)CD57(+) subpopulation. Clin.Immunol., 2000; 96:230.
25. Fulop T, Larbi A, Pawelec G. Human T cell aging and the impact of
persistent viral infections. Front Immunol, 2013; 4:27
26. Derhovanessian E, Theeten H, Hahnel K, et al. Cytomegalovirus
associated accumulation of late-differentiated CD4 T cells correlateswith poor humoral response to influenza vaccination. Vaccine, 2013;SUBPOPULAŢII LIMFOCITARE IMPLICATE ÎN IMUNOSENESCENŢA
ASOCIATĂ BOLII ALZHEIMERREZUMAT

Imunosenescenta este definita ca alterarea progresiva a aspectului morphologic si functional, care apare la nivelul sistemului imunin timpul procesului de imbatranire. Scopul acestui studiu a fost definirea statusului de imunosenescenta, caracterizarea potentialului
proliferativ si a gradului de apoptoza a subpopulatiei de limfocite T si definirea imunosenescentei asociate bolii Alzheimer (AD) in
dementa clinic diagnosticata si in stadiile prodromale (predementa). Impactul asteptat al acestui studiu este legat de dezvoltarea
semnificativa a nivelului cunoasterii in acest domeniu, in ceea ce priveste mecanismele si manifestarile imunosenescentei si imunosenescentei asociata bolii Alzheimer.
Cuvinte cheie: imunosenescenta, boala Alzheimer, predementa

26

Fiziologia - Physiology  2014.24.1 (81)METHYLENETETRAHYDROFOLATE REDUCTASE GENE
MUTATIONS AND OPPORTUNITY OF ANTICOAGULANTTREATMENTFLORINA-MADALINA ONICEANU, ILEANA ION, CECILIA ADUMITRESI, NINELA RADULESCU,CRISTINA FARCAS, ANABELLA-CRISTIANA STEFANESCU, CHIRICA R
Faculty of Medicine, “Ovidius” University Constanta

ABSTRACT

Considering that hypercoagulable status means accurate diagnostic, starting from clinical signs, most of them unspecific, alongwith expensive lab work, we considered useful studying thrombotic events in patients with modified molecular genetic analysis andestablishing a connection between the existence of these mutations and the risk for thrombotic events. Also, we considered the opportunity of anticoagulant treatment to decrease this pathology. Existing studies and guidelines concluded that testing for hereditary
thrombophilia is not recommended in first degree relatives of the patients who had at least once in their life a thrombotic manifestation. Prophylaxis against thrombosis in patients with gene mutations with a predictable trigger factor for this pathology is indicated.
Keywords: thrombophilia, molecular genetic analysis, anticoagulant.

INTRODUCTION
Methylenetetrahydrofolate reductase (MTHFR) is an essential
enzyme in folate metabolism. MTHFR reduces 5,10 methylenetetrahydrofolate to 5-methyltetrahydrofolate, which representsa cofactor for homocysteine remethylation to methionine. The
involvement of MTHFR in pathology was first described in apatient presenting homocystinuria, with a severe defeciency inthe enzyme activity (1).
In 1988, a ‘thermolabile’ enzyme with reduced activity wasdescribed, associated with high risk of coronary disease. After
1994, MTHFR analysis became more intense and mild or severe
deficiencies of this enzyme were identified (2).
The thermolabile variant was associated with mutation in
nucleotide 677, where cytosine (C) is substituted with thymine(T), with high risk of coronary disease and elevated level of homocysteine in the blood in patients with folate deficiency. The
mechanism based on which MTHFR mutations are involved in
vascular pathology it is not well established. One hypothesis
stands for associated high levels of homocysteine in the blood,
which may determine vascular endothelial injury with secondary
venous thrombembolism (3).
MTHFR gene mutations were connected to various pathology,
including colon cancer, acute leukemia, vascular diseases, depression, schizophrenia, migraine, glaucoma, Down syndrome,neural tube defects.
The most frequent MTHFR mutations are C677T and A1298C.
The first one makes MTHFR 20% less active in metabolizing
homocysteine. Studies report an incidence of 11% in Caucasian
population for the homozygous state of this mutation, associatedwith risk of elevated levels for homocysteine in the blood andseveral pregnancy complications – chromosomal anomalies,

congenital malformations, pregnancy loss, pathology of placenta
and preeclampsia, including thrombotic events late in pregnancy
and also in the postpartum period (4).
Heterozygous patients for this mutation have normal levels ofhomocysteine in the blood and low risk for thrombosis.
A1298C mutation is not associated with hyperhomocysteinemia, no matter the hetero- or homozygous state (5).
The combined heterozygous state for both MTHFR mutations
can generate clinical manifestations similar to the homozygous
state for C677T; this association is recognized as a risk factor
for neural tube defects (6).
MATERIALS AND METHOD
The present study was performed on 16 patients admitted
in the Onco-Hemathology Department, Paediatric Clinic of
the County Clinical Hospital of Constanta from January 2012
to January 2013 for thrombotic events or representing first
degree relatives of children with thrombotic events. They all
have modified genetic test already known to increase the risk
for thrombosis.
Following data was charted for these patients:− age;− gender;− basic coagulation tests, like PT, APTT, INR, number of platelets;− clinical manifestations at the onset of the acute event;− thrombophilia profile: C protein, S protein, antithrombin, Leiden
factor mutation, lupic anticoagulant;− mutation for prothrombin gene;− anticardiolipin antibodies;− homcysteine in blood and urine;− MTHFR gene mutations.

Received 5th of January 2014.Accepted 25th of January 2014. Address for correspondence: Dr. Florina-Madalina Oniceanu MD, Phdd, Faculty of Medicine, Physiology
dept, “Ovidius” University Constanta, No. 1 Al. Universitatii, Constanta, Romania; phone: 0722648830; e-mail: oniceanuflorinamadalina@yahoo.com

2014.24.1 (81)  Fiziologia - Physiology

27

The data obtained were analyzed and presented as graphics
and tables.
RESULTS
From the total of 16 patients, 25% of them are adults and
75% children under 18 years of age. The results show that thehypercoagulable state is more frequently seen at early age. 62%of the persons in the study are female.

Fig. 1. Age distribution in the study group

Fig.2. Gender distribution of the patients in the study group

For all the patients with MTHFR gene mutations, the incidence
for thrombotic events was 62% in the studied group. The mostfrequent affected areas were represented by cerebral pathologystrokes, followed by thrombotic events regarding pregnancy.
Also, from the 62% with clinical manifestations, none is homozygous for MTHFR gene mutations.
In the study group, 25% presented C677Thomozygous
state, 37% are C677T heterozygous, 25% have heterozygous
A1298C mutation and only 13% are double heterozygous forboth mutations.

Fig.3. Distribution of MTHFR gene mutations in the study group28

The levels for homocysteine in the blood were normal for all
patients. We can think that homocysteine did not represent a
trigger or even a predictable factor for thrombosis.
Table I. Homocysteine levels in the bloodNo.

Homocysteine (µmol/l)No.

Homocysteine(µmol/l)

1.

4.29

9.

6.1

2.

2.25

10.

7.05

3.

6.79

11.

6.98

4.

5.25

12.

5.8

5.

6.22

13.

5.95

6.

7.1

14.

5.7

7.

6.09

15.

7.01

8.
5.1
16.
6.29
Patients with homozygous mutations in the study group haven’t
had any clinical thrombotic event, at least not yet.
DISCUSSION
Based on existing literature, we can say that not the genemutations represented the trigger factor for thrombosis in thisstudy; not even the homocysteine level, but other causes must
be considered in the presence of a thrombotic manifestation.
The authors highlight a particular case report of a nine year old
boy who was admitted to hospital for stroke. The trigger factors
considered for the thrombotic event appears to be a trip in themountains, standing still for several hours and also a certain
degree of dehydration. All these factors could represent trigger
factors in a patient with later discovered MTHFR gene mutations,heterozygous state.
After molecular analyses, the authors noticed that the boy’s
father and sister carry together homozygous MTHFR mutations
and they haven’t had thrombotic events until the moment of determinations. This idea stands for the fact that genetic mutations
are not enough for thrombotic pathology and we must consider
the trigger factors which should be well known and avoided inthese patients.
Thrombosis with normal levels for homocysteine enhances
the idea that its elevation must be just a secondary effect after
the already produced vascular damage.
We considered the opportunity of guidelines in testing hereditary thrombophilia. Many international studies for hypercoagulable state mention the utility of guidelines in testing hereditary
thrombophilia; they also offer information for prophylaxis ofthrombotic events.
CONCLUSION
Primary hypercoagulable state remains a diagnostic based onvery sensitive lab work, with high specialization for the people
involved in this work, including high costs.
The MEGA study concludes that it is not justified testing for
genetic defects in first degree relatives of the patients with
Fiziologia - Physiology  2014.24.1 (81)thromboembolic events in early childhood, even if hereditary
thrombophilia is proved – “testing does not reduce the recurrence of thrombosis”.
We have to take into consideration the factors that could
become trigger for thrombosis: large varices, oncologic andhemathologic pathology, with high viscosity, severe infections,neprotic syndrome, preeclampsia, congestive heart failure,important hemorrhage, dehydration.
Prophylaxis against thrombosis must be initiated knowing all
the possible trigger factors and, of course, we should take into
account the risk for adverse reactions.
Homocysteine levels weren’t relevant in monitoring the risk
for thromboembolic events, probably because this determination
was always made after the acute event; homocysteine remains

a parameter for secondary hypercoagulable state.
REFERENCES

1. Montgomery RR, Scott JP. Hemoragic and thrombotic disease. In
Nelson textbook of Pediatrics, ed. 17, 2004.
2. Rey E, Kahn SR, David M, Shrier I. Thrombophilic disorders and fetal
loss: a meta-analysis. Lancet 2003; 361: 901-908.
3. Serban M, Schramm W. Hemostazeologie clinica, Ed. Brumar,
Timisoara, 2001.
4. Steegers-Theunissen RP, Van Iersel CA, Peer PG, et al. Hyperhomocysteinemia, pregnancy complications, and the timing of investigation.
Obstet Gynecol 2004; 104: 336-343.
5. http://www.synevo.ro/profil-trombofilie-2/
http://www.synevo.ro/gena-mthfr-mutatii-c677t-a1298c-risc-trombofilie/

MUTATIILE GENEI MTHFR SI OPORTUNITATEA TRATAMENTULUIANTICOAGULANTREZUMAT

Tinand cont de faptul ca statusul hipercoagulabil are la baza un diagnostic elaborat, incepand de la manifestarile clinice, multe dintre
ele nespecifice, impreuna cu analize de laborator cu cost crescut, am considerat util studiul manifestarilor trombotice la pacientii cu
analize modificate de genetica moleculara si stabilirea unei legaturi intre existenta acestor mutatii si riscul de evenimente trombotice.
De asemenea, am luat in discutie oportunitatea tratamentului anticoagulant pentru scaderea incidentei acestei patologiii. Toate
studiile si ghidurile pana in acest moment au concluzionat ca testarea pentru trombofilia ereditara nu este recomandata la rudele
de gradul I ale pacientilor care au suferit cel putin o data in viata lor o manifestare trombotica. Profilaxia trombozei la pacientii cu
mutatii genetice cu un factor declansator predicitibil pentru aceasta patologie este indicata.
Cuvinte cheie: trombofilie, analize de genetic moleculara, anticoagulant.

2014.24.1 (81)  Fiziologia - Physiology

29

CORRELATION BETWEEN EOSINOPHILIC CATIONIC PROTEIN
WITH OTHER LABORATORY TESTS AND ATOPIC ENVIRONMENT
IN CHILDREN WITH BRONCHIAL HYPERREACTIVITYADINA UNGUREANU², ION ILEANA¹,², CECILIA ADUMITRESEI², SERGIU CHIRILA², CRISTINAMARIA MIHAI¹,², VIVIANA CUZIC¹,²
1County Emergency Clinical Hospital of Constanţa, Romania
2
Faculty of Medicine, “Ovidius University “of Constanţa, Romania

ABSTRACT

Asthma is one of the most common chronic diseases in children, which causes a significant number of emergency hospitalizations
and has a great impact on the lives of children and their family. In most children with asthma, airway hyperreactivity, manifested as
an exaggerated bronchoconstrictor response, is interrelated with bronchial inflammation and responsible for the clinical manifestations.The aim of the study is to determine the value of new methods of non-invasive investigations of bronchial inflammation. It
represents a prospective study conducted on a sample of 199 patients with obstructive respiratory disease. The sample lot of 199
patients was observed for a period of 4 years or until the diagnosis of asthma was established. By the end of the period of the study,
we identified a number of 126 cases of children with asthma and 73 cases without asthma. We determined the serum eosinophil
cationic protein (ECP) levesl and assessed the relationship between the values of eosinophilic cationic protein and IgE values whichshows an important positive correlation, statistically significant between the values of the two tests (p < 0.001). Comparison of thevalues of eosinophilic cationic protein with the result of the Phadiatop Infant test showed no statistically significant difference, thesame result being obtained in the case of comparing the values of eosinophilic cationic protein with the evaluation of the presenceof atopic environment.
Keywords: asthma, Ig E, eosinophilic cationic protein

INTRODUCTION
In recent years the focus is increasingly higher on non-invasive
explorations of bronchial inflammation, thus, in 2005 a new field
called “inflammamometry” was considered at European Respiratory Society congress in 2005, highlighting the importanceof pathophysiologic substrate in the diagnosis, treatment andfollow-up of patients with obstructive pulmonary diseases.
Asthma is one of the most common chronic diseases inchildren and causes a significant number of hospitalizations
and emergencies. It has a significant impact on the daily lifeof the child, family and entourage where the child with asthma
lives (4,5).
Asthma is a chronic disease, and for many patients has
unpredictable evolution or progression, which may affectphysical activity, social activities and quality of life. Conventional
measurements as spirometry or bronchial hyperreactivity bring
important information about airways clinical conditions, but do notoffer information about local bronchial inflammation. Lately, thefocus is increasingly higher on exploring non-invasive bronchialinflammation (6).
High serum ECP level may be a predictor and a risk factor forasthma exacerbation and may be used for guiding treatment (7).
OBJECTIVESThis study aims to determine the value of the new methods of

non-invasive investigation of inflammatory bronchial eosinophilic
cationic protein, the correlation that exists with the IgE levels,
eosinophils level and the differences in patients with a positive
Phadiatop Infant test and those with a negative Phadiatop
Infant test.
MATERIAL AND METHODS
The study is a prospective study, conducted on a sample of
199 cases of respiratory disease cases, which were followed fora maximum period of 4 years, from 2009 to 2013. The patientswere consulted yearly for a reevaluation of their medical status,and once it was possible, spirometry was conducted in orderto certify the diagnosis of bronchial obstruction. Patients were
followed until the diagnosis of asthma was established, or until
the end of the study period.
The inclusion criteria for the study were the presence ofwheezing in children with ages between 2 and 5 years old. Exclusion criteria are represented by previous diagnosis of chronic
obstructive pulmonary disease.
We measured the Eosinophilic Cationic Protein (ECP), IgE
values, eosinophils values and we performed Phadiatop Infant
testing at admittance, using standard procedures recommended
by the laboratory.
Eosinophilic Cationic Protein (ECP) was determined using immunoenzymatic with detection by fluorescence (FEIA)

Received January 15th, 2014. Accepted February 20th, 2014. Address for correspondence: Adina Ungureanu, MD, PhD, Pediatric Department, Faculty of Medicine,“Ovidius University “of Constanţa, Romania; phone: +40723499317; e-mail: adinaungureanu2007@yahoo.com

30

Fiziologia - Physiology  2014.24.1 (81)

method. IgE was determined with a COBAS 6000 device
produced by Hitachi using immunochemistry with detection byelectrochemiluminescence (ECLIA) method. Phadiatop Infant
was determined with UNICAP 250 device using imunoenzymatic with detection by fluorescence (FEIA) method. Bloodeosinophils were determined with SYSMEX XT 1800i device
using fluorescent flow cytometry, using semiconductor lasers
and focusing hydrodynamic technologies (8). The presenceof atopic environment was assessed during the initial consult
using a questionnaire.
For the analysis of the data we used descriptive statistics.
Shapiro-Wilk test was used to determine the normality of the
distribution of the values, and according to the result obtained
we used parametric or nonparametric methods. For parametric methods of testing the null hypothesis we used t test andPearson correlation, while for nonparametric testing we used
Mann-Whitney U test and Spearman rho in case of correlations.
The p value to consider the result as being statistically significant
is less than 0.05.
From the total of 199 patients, by the end of the study, 126
patients were diagnosed with asthma (63.3%) and 73 cases
without asthma, which remained assigned to the group calledwheezing (36.7%) (Figure 1)

Fig.1. Distribution of cases by respiratory disease

At the beginning of the study,the patients from the study
sample were distributed equally according to their age(Figure 2).

The distribution according to sex for the whole study groupreveals a higher proportion of males, 118 cases (59.3%) compared to females, 81 cases (40.7%) (Figure 3).

Fig.3. Distribution of cases included in the study according to gender

RESULTS AND DISCUSSION
Our study investigated ECP due to eosinophils important rolein the pathophysiology of asthma. ECP is stored in the granulesof eosinophil granulocytes together with the major basic protein(MBP), eosinophil peroxidase (EPO) and eosinophil - derived
neurotoxin / eosinophil protein X (EDN/EPX) \ which havebeen demonstrated to cause airway damage (10,11). During
eosinophil’s degranulation ECP is released in the tissues upon
exposures to surfaces coated with immunoglobulins and complement with the escape of some ECP to circulation (12). SerumECP can also be used to assess the exposure to environmental
allergens (13) and as a measure of allergen provocation causing
increased eosinophil activity (14).
Recently, serum ECP is studied to conclude if it represents
a valuable marker for the diagnosis and prognosis of bronchialasthma (9).
We measured serum eosinophil cationic protein and compared
the results with those of the tests exploring bronchial hyperactivity. We evaluated the relationship between the values of
eosinophilic cationic protein and IgE values in all patients in the
study, patients that have bronchial hyperreactivity. The meanvalue measured for eosinophil cationic protein is 21.228 mg/dl,
with standard deviation of 12.37 mg/dl. The mean value for IgE is648.89 IU / ml, with standard deviation of 918.68 IU/ml (Table I)
Table I. Descriptive analysis of eosinophilic cationic protein and IgEvalues in all patients with bronchial hyperreactivity
Eosinophil
Cationic
Protein

IgE

Valid

199

196

Missing

0

3

Mean

21.228648.8995

Median17.400315.0000

Mode

32.0

45.00

Std. Deviation

12.3776918.68976

Variance153.204

843990.872

Skewness

1.034

3.553

N

Fig. 2. Distribution of the patients included in the study according toage
2014.24.1 (81)  Fiziologia - Physiology

31

Std. Error of Skewness

.172

.174

Kurtosis.773

18.135

Std. Error of Kurtosis

.343

.346

Range

66.9

7354.50

Minimum

3.1

3.50

Maximum

70.0

7358.00

tive correlation, statistically significant between the values of thetwo tests (r = 0.542, p <0.001). Therefore, we conclude that in
the case of patients from the wheezing group considered for thisstudy, there is an important relationship between the values of
the ECP and the values of the IgE.

The result of statistical analysis (Figure 4) shows a major positive correlation, statistically significant between the values of thetwo tests (r = 0.537, p <0.001). Therefore, we conclude that in
the case of patients with bronchial reactivity considered for thisstudy, there is an important relationship between the values of
the ECP and the values of the IgE. This data is in accordance
to other studies (15,16).

Fig. 5. Correlation between eosinophil cationic protein and IgE values for the wheezing group

The descriptive analysis for the patients that were diagnosed with asthma during the period of the study, is available
in Table III.
Table III. Descriptive analysis of eosinophilic cationic protein and IgEvalues in patients diagnosed with asthma

Eosinophil
Cationic Protein

IgE

N

73

71

Range

66.9

5115.50

Minimum

3.1

3.50

Maximum

70.0

5119.00

Mean

17.148413.2283

Std. Deviation

11.6145743.66841

Variance

134.897553042.711

Skewness
Std. Error of Kurtosis

1.989

4.278

.281

.285

KurtosisStd. Error of Kurtosis

5.442

23.449.555.563

Fig.4. Correlation between eosinophil cationic protein and IgE values
in all patients with bronchial hyperreactivity
Table II. Descriptive analysis of eosinophilic cationic protein and IgEvalues in patients in the wheezing group

Eosinophil
Cationic
Protein

IgE

N

73

71

Range

66.9

5115.50

Minimum

3.1

3.50

Maximum

70.0

5119.00

Mean

17.148413.2283

Std. Deviation

11.6145743.66841

Variance

134.897553042.711

Skewness
Std. Error of Kurtosis

1.989

4.278

.281

.285

KurtosisStd. Error of Kurtosis

5.442

23.449.555.563
The result of statistical analysis (Figure 5) show a major posi-

32

The result of statistical analysis (Figure 6) shows a moderate
positive correlation, statistically significant between the valuesof the two tests (r = 0.465, p <0.001). Therefore, we conclude
that in the case of patients from the asthma group considered forthis study, there is a moderate relationship between the valuesof the ECP and the values of the IgE.
Fiziologia - Physiology  2014.24.1 (81)no statistically significant difference between the two groups(U= 3125, z=1.554, p=0.120)
Exposure to atopic environment particularly in early life
may be an important factor in the pathogenesis of atopic
and asthmatic symptoms (17). The most important inhaledallergen frequently associated with asthma is house dust miteand maybe prevention is needed to minimization the potential
allergens and pollutants from the house (17). In our study,
63.3% of the patients were not exposed to an atopic environment, while the other 73 were considered to live in an atopic
environment (Figure 7).

Fig.6. Correlation between eosinophil cationic protein and IgE values
for the asthma group

A comparison was also followed with eosinophilic cationicprotein test values and Phadiatop Infant. From the total of 199
cases, this testing was conducted in 185 of them. From these, 57
had a negative result, while the other 128 had a positive result.
Table IV. Phadiatop Infant distribution of the patients according to theresult

FrePerquency cent
Valid

CumuValid
lativePercent Percent

Negative 57

28.6

30.8

30.8

Pozitive

128

64.3

69.2

100.0

Total

185

93.0

100.0

14

7.0

Missing System

Total
199
100.0
Mean eosinophilic cationic protein value in patients with negative Phadiatop infant test is 18.684, with standard deviation of
9.85. For those with Phadiatop infant positive test result, mean
eosinophil cationic protein is 22.623 with standard deviation of
13.39 (Table V).
Table V. Comparisson of eosinophilic cationic protein values between
groups with positive and negative result in Phadiatop Infant test

Phadiatop
InfantN

Negative

57

Pozitive

128

Mean

Std. Deviation

Median18.6849.855917.40022.62313.3950

18.250Total
18521.41012.519317.600
Following statistical analysis, using Mann-Whitney U
test, as the distribution of the values differs significantly
from a normal distribution (Shapiro-Wilk test rejects the null
hypothesis with a p value less than 0.001 for cases withpositive Phadiatop Infant test and with p=0.001 for caseswith Phadiatop Infant negative test), we observe that there is
2014.24.1 (81)  Fiziologia - Physiology

Fig. 7. Distribution of all patients according to the exposure to atopic
environment

Eosinophilic cationic protein assessment accordingly to environmental presence of atopic factors show that the averagevalues of eosinophilic cationic protein in patients not exposed to
atopic environment is 19.881 with standard deviation of 10.49.
Patients exposed to an atopic environment have average valueof 23.553 for ECP with a standard deviation of 14.88. The data
is available in Table VI.
Table VI. Evaluation of eosinophil cationic protein according to thepresence of atopic environment

Exposure toatopic environment

N

Mean

Std. Deviation

Median73

23.55314.8866

19.300No

12619.881

10.4900

16.650Total

199

21.22812.377617.400Yes

We observed in patients exposed to atopic environment wider
range of values for eosinophilic cationic protein (Figure 8). Test
for normality reveals that distributions of eosinophilic cationicprotein values are significantly different from a normal distribution
in both groups (p <0.001 respectively p = 0.004).
Despite these differences, the statistical testing, doneusing Mann-Whitney U test shows no statistically significant difference between the two groups (U=4150, z=1.147,
p=0.251).
33

Fig.8. Evaluation of eosinophil cationic protein according to the presence of atopic environment

Eosinophils are major inflammatory cells present in bronchialasthma patient’s respiratory tract mucosa and submucosa (18). A
subpopulation of low density eosinophils with increased cytotoxic
activity and increased numbers of activated blood T cells represent part of the pathophysiological processes of asthma (19,20).
We considered the correlation between eosinophilic cationic protein and blood eosinophilia but it has shown a negative
weak correlation (r=-0.197), which is not statistically significant(p=0.081) (Table VII).
Table VII. Correlation between eosinophilic cationic protein and blood
eosinophilia
Correlations

Spearman’s
rho

ECP

Eosinophils

ECP

Eozinophils

Correlation Coefficient

1.000

-.197

Sig. (2-tailed)

.

.081

N

199

79

Correlation Coefficient

-.197

1.000

Sig. (2-tailed)

.081

.

N

79

79

CONCLUSION
In our study, ECP serum concentration, blood eosinophil number and total IgE in serum are significantly higher in asthmaticchildren demonstrating the role of eosinophilic inflammation in
the pathogenesis of asthma.
Evaluation of the relationship between the values of eosinophil cationic protein and IgE values in patients with bronchialabnormal reactivity show a positive correlation major statisticallysignificant between the two analysis values (r = 0.537, p <0.001).
For the population in our study, there was no statistically34

significant difference between patients with a positive Phadiatop
Infant test and the ones with a negative one (U= 3125, z=1.554,
p=0.120), even though the values observed in the group with anegative test have a lower average value compared to the values
observed in the group with a positive result.
There is no statistically significant difference for the valuesof the ECP levels between patients that come from an atopic
environment and the ones that live in an atopic-free environment(U=4150, z=1.147, p=0.251).
The values of the ECP do not correlate with the values of
eosinophils number. (r=-0.197, p=0.081).
ECP levels in serum reflect better than IgE allergic inflammatory process, ie the activation of eosinophils, correlate wellwith disease activity, so the severity of atopy. This laboratory
parameter is useful for objectification and surveillance therapy
in asthma and atopic dermatitis.
Direct measurement of airways inflammation using biologicalmarkers like ECP, could potentially enhance asthma management. This explains the interest in measuring the levels of
eosinophil granule proteins especially in asthma.
REFERENCES

1. Sterk PJ, Fabbri LM, Quanjer P, et al. Airway responsiveness: standardized challenge testing with pharmacological, physical and sensitizing
stimuli in adults. Eur Respir J, 1993; 6(16): 53-83.
2. Bierman CW, Shapiro GG. Clinical expression of bronchial hyperreactivity in children. Clin. Rev Allergy, 1989; 7(3): 301-20.
3. Francis JG, Warren L. The burden of paediatric asthma: economic
and familiar. European Monograph, 2012; 56: 71.
4. Juniper EF, Guyatt GH, Feeny DH, et al. Measuring quality of life in
the parents of children with asthma, Quality of Life Research, 1996;
5: 27-34.
5. Ion I, Badiu G. Fiziologie-curs, Ed Ex Ponto, Constanţa, 2000
6. Restrepo RD, Peters J. Near-Fatal Asthma: Recognition and Management. Curr Opin Pulm Med, 2008; 14(1): 13-23.
7. Oddera S, Silvestri M, Penna R, et al. Airway eosinophilic inflammation and bronchial hyperresponsiveness after allergen inhalation
challenge in asthma. Lung, 1998; 176: 237-247.
8. Laborator Synevo. Referintele specifice tehnologiei de lucru utilizate
2010. Ref Type: Catalog.
9. Koh GCH, Shek LPC, Goh DYT, et al. Eosinophilic cationic protein:is it useful in asthma? A systemic review. Respiratory Medicine, 2007;
101: 696-705.
10. Byström J, Garcia RC, Håkansson L, et al. Eosinophil cationicprotein is stored in, but not produced by peripheral blood neutrophils.
Clin Exp Allergy, 2002; 32:1082-91.
11. Bousquet, Chanez P, Lacoste Y, et al. Eosinophilic inflammation inasthma. New Eng J Med, 1990; 323: 1033-9.
12. Lee BioSolutions, Inc. http://www.leebio.com/eosinophil-cationicprotein-human-P359.html
13. Nieto A. What role does ECP have in the evaluation of asthma
severity. Allergol Immunopathol, 2000; 28:119-124.
14. Oddera S, Silvestri M, Penna R, et al. Airway eosinophilic inflammation and BHR after allergen inhalation challenge in asthma. Lung,
1998; 176: 237-247.
15. Esengül M, Hamza Y, Arzu G. To evaluate serum eosinophil cationic protein and total IgE concomitantly may predict the persistenceof wheezing in young children. ISRN Pediatrics International Scholarly

Fiziologia - Physiology  2014.24.1 (81)Research Network, 2010.
16. Mohammad RK, Majid M, Mojtaba S, et al. Is serum or sputum
eosinophil cationic protein level adequate for diagnosis of mild asthma?
Iran J Allergy Asthma Immunology, 2009; 8(3):155-160.
17. Sporik R, Chapman MD, Platts-Mills TA. House dust mite exposure
as a cause of asthma. Clin Exp Allergy, 1992; 22: 897-906.
18. Koh Y, Kang H, Kim CK. Ratio of serum eosinophil cationic protein/blood eosinophil counts in children with asthma: comparison between
acute exacerbation and clinical remission. Allergy Asthma Proc, 2003;
24(4): 269-274.
19. Koller DY, Herouy Y, Götz M, et al. Clinical value of monitoring
eosinophil activity in asthma. Arch Dis Child 1995; 73:413-417.
20. Seroogy CM, Gern J. The role of T regulatory cells in asthma. J
Allergy Clin Immunol, 2005; 116:996-9.

CORELATIILE PROTEINEI CATIONICE EOZINOFILICE CU IG E TOTAL,PHADIATOP INFANT, NUMARUL TOTAL DE EOZINOFILE SI MEDIULATOPIC LA COPIII CU HIPERREACTIVITATE BRONSICAREZUMAT

Astmul este una dintre cele mai comune boli cronice la copii, care provoacă un număr semnificativ de spitalizări de urgență și are un
impact mare asupra vieții copiilor si familiilor lor. Scopul acestui studiu este de a determina valoarea unor noi metode de investigație
non-invazive a inflamației bronșice. Studiul este un studiu prospectiv realizat pe un eșantion de 199 de pacienți cu boli respiratorii
obstructive. Am determinat nivelul seric al proteinei cationice eozinofilice (ECP). Studiul a cuprins un număr de 199 de pacienți,urmăriți o perioadă de 4 ani sau până ce diagnosticul de astm a fost stabilit. Până la sfârșitul perioadei de studiu, am identificat unnumăr de 126 de cazuri de copii cu astm bronsic și 73 de cazuri fără astm. Evaluarea relației dintre valorile ECP și valorile IgE arată
o corelație pozitivă importantă, semnificativă statistic între valorile celor două teste (r=0,537 p <0,001). Compararea valorilor ECP
în funcție de rezultatul testului Phadiatop Infant a arătat lipsa unor diferențe semnificativ statistic între cei cu test pozitiv și cei cu
test negativ, același rezultat fiind obținut în cazul comparării valorilor ECP bazate pe evaluarea prezenței mediului de viață atopic.
Cuvinte cheie: proteina cationică eozinofilica, IgE, astm bronşic.

2014.24.1 (81)  Fiziologia - Physiology

35

BARIATRIC SURGERY IN OBESE PATIENTS WITH INCISIONALHERNIA – A NEW METHOD TO REDUCE THE HERNIA RECURRENCERATESDOBRESCU A, DUTA C, TARTA C, LAZAR F
Surgical II Department, University of Medicine and Pharmacy “Victor Babes”Timisoara, Romania

ABSTRACT

Post-incisional hernia occurs in approximately 10% of cases of abdominal surgery and it is the most common long-term complication, one of the main risks for developing a post-incisional hernia is obesity. Laparoscopic incisional ventral hernia repair (LIVHR)
became increasingly popular in the last years due to faster recovery and lower rate of wound infection. We used laparoscopic greater
curvature plication (LGCP) of the stomach for treating obese patients. The present study was a prospective case series that aimed toevaluate the short and the long term outcomes of combined LIVHR and LGCP of the obese patients with incisional hernia operated
in 2012 in Surgical 2 Clinic of County Emergency Hospital Timisoara. 6 patients were enrolled, all females. 4 patients had 24 months
follow-up, the others two had 20 and 16 months follow-up. Median age was 42, median wall defect was 56 cm2, and median hospital
stay was 5 days, complications rates were 83%. Complications were nausea in 5 patients, vomiting in 2 cases, seroma formation inone. Median body mass index dropped from 36.7 kg/m2 to 29.85 kg/m2. Excess weight loss was 52.6% at two years, no recurrenceof the hernia occurs during follow-up. LIVHR repair is a safe method in obese patients, if LGCP is added in the same operative time
the rate of short term minor complication is increased, but long term recurrence is decreased due to weight loss. Larger studies are
required to drown a final conclusion upon our combined method.
Keywords: bariatric surgery, LIVHR, gastric plication

INTRODUCTION
Post-incisional hernia occurs in approximately 10% of casesof abdominal surgery and it is the most common long-term
complication (1), one of the main risks for developing a postincisional hernia is obesity (2). Laparoscopic incisional ventral
hernia repair (LIVHR) became increasingly popular in the lastyears due to faster recovery and lower rate of wound infection.
Also for obese patients bariatric surgery seems to have thehighest rate of success in reducing the excess weight. One of
the methods used for treating obese patients is laparoscopic
greater curvature plication (LGCP) of the stomach; some of theadvantages of this method are the low rate of post-operativecomplications, lower cost and the digestive tube is not opened.
We decide to use a combined operation for the obese patientswith incisional abdominal hernia – LGCP and LIVHR in the same
operating time.
METHODS
The present study was a prospective case series that aimed
to evaluate the short and the long term outcomes of combined
LIVHR and LGCP. The study begun in 2012, morbidly obese
subjects referred to our surgery clinic with incisional ventral
hernia (IVH) were offered simultaneous LIVHR and LGCP. During 2012 the patients were enrolled, follow-up was planned at
1, 3, 6, 12 and 24 months.
Inclusion criteria were the presence of an IVH, obesity and theeligibility of the patient to undergo laparoscopic surgery.
The maximum size of the IVH was 10 cm diameter; only
patients with reducible hernias and undergoing elective surgery
were included in the study.
Patients required a body mass index (BMI) between 30-40
kg/m2 and an absence ofpsychological conditions that might
influence postoperative evaluations and recommendations.All
recruited patients underwent a pre-surgicalevaluation, including
consultations with a cardiologist, psychologist, and nutritionist
plus routine laboratory test. They must have a strong motivation
to lose weight and the potential for continuous diet and exercise
after operation.
Patients with multiple abdominal surgeries, irreducible hernias
and with no will to lose weight were not offered the combined
procedure.
During the surgical procedure first step was to dissect andreduce the content of incisional hernia. Pneumoperitoneum wascreated inserting the Veress’ needle at the Palmer’s point, left
mid clavicular line below the last rib either using the open Hasson technique for the insertion of the first trocar on the left flank.
Another two trocars are inserted in the left flank. The adhesions
in the abdomen were lysed with or without the use of an energy
source. This choice was dictated by the proximity of the bowel
and the surgeon preference. No energy was applied adjacent toany structure that might result in an injury. Missed enterotomy
is one of the most feared laparoscopic complications of hernia

Received January 7th, 2014. Accepted February 25th, 2014. Address for correspondence: Amadeus Dobrescu, MD, PhD student, Surgical II Department, “Victor Babes”
University of Medicine and Pharmacy Timisoara, Romania, Eftimie Murgu Square No. 2A, 300041, Timisoara, phone/fax: +40256220479; e-mail: lazarfulger@yahoo.com

36

Fiziologia - Physiology  2014.24.1 (81)

repair. Once the hernia defect or defects were revealed we determined their dimensions. In two cases additional three trocars
were inserted in the right flank for proper hernia dissection.
Then the patient was placed 30° reverse Trendelenburg position, with the operating surgeon standing between patient legs.
Dissection started at the greater curvature of the stomach fromthe middle of the antrum and continued to the left diaphragmaticcrus, and downwards to the level of the pyllor. Communicating
vessels were ligated by LigaSure (LigaSureTM Covidien, USA),
Ultracision (Harmonic® Ultrasonic, Ethicon, USA) or clips.
Continuous suturing from 2 cm below the cardia through theantrum, 4 cm distance above the pyllorus, was performed inthis stage, making one and then two layers of plicated stomach
from the anterior wall of the stomach to the posterior wall. For
suturing 2-0 prolene (2-0 PROLENE™ Polypropylene Suture,
Ethicon, USA) was used, and the bulk of each stitch was 1 cm
with a 1-cm interval. For gastric tube calibration we used a 36
Fr tube. Sutures were extramucosal, preventing absorption by
gastric acid (3).
Next step was to place the mesh in order to cover the parietal
defect. After we determined the defect size we used a mesh atleast 8 cm larger which provided an overlap over the fascialedges of the hernia of at least 4 cm on both sides. Then weplaced four transfascial sutures using fascia closure. Then themesh final fixation was done using titanium either absorbabletacks. The use of a single row of tacks either the double-crowntechnique was left at the surgeon preference.
Drainage was used for 24 hours in order to see if there wasany bleeding.
Post-operative we administrated to all the patients proton
pump inhibitors 2x40 mg/day, anti-inflammatory and anti-nausea/
vomiting drugs. Low molecular heparins were administrated for
up to three weeks to prevent deep vein thrombosis and superior
mesenteric vein thrombosis.
Postoperative diet was resumed 24 hours after surgery andconsisted only in fluids up to the 9th POD. Next three weeks all
the food will be semi-liquid.
The demographics and the comorbidities of the patients wereassessed. The peri-operative data gathered regarded operatingtime, blood loss, parietal defect size, wound infection, seroma
formation, nausea and vomiting, re-admission, hospital stay,first bowel movement, morbidity and mortality in the first 30
post-operative days.
Follow-up regarded recurrence of the hernia, percentage ofthe excess weight loss, BMI, comorbidities resolution, mortality.
RESULTS
6 patients were enrolled, all females. 4 patients had 24
months follow-up, the others two had 20 and 16 months
follow-up. The primary surgery was open cholecystectomyin three cases: one perforated appendicitis with generalized
peritonitis, and two hysterectomies for benign lesion of theuterus. Demographics of the patients are showed in Table I.
None of the patients had diabetes mellitus. Peri-operative data
are shown in Table II.
2014.24.1 (81)  Fiziologia - Physiology

Table I. Demographics of the patientsMedian age

42Median BMI

36.70 kg/m2

Comorbidities

83%

ASA I

2

ASA II

4

BMI – body mass index

Table II. Peri-operative data of the patients

OR time (median)

105 min (75;135)

Blood loss (median)

90 ml (70;130)

Defect size (median)

56 cm2 (24;82)

First bowel movement

POD 1

Hospital stay (median)

5 days (3;10)Complications

83%

POD – post-operative day

All the surgery were started and finished laparoscopically. No
perforation of the stomach either of the bowel happened. For the
first three days IV fluids were administrated. The patients wererecommended to walk in the same day with the surgery and oral
diet was resumed first POD.
Complications were nausea in 5 cases and vomiting in two
out of these five. Seroma was search with the ultrasound and all
the patients developed seroma, but only one needed punctureaspiration in order to evacuate the fluid. Mortality was null.
Follow-up data (Table III) – no recurrence of the hernia or mortality was recorded. No bulging at the hernia site was observed.
No regain happened in the follow-up period.
Table III. BMI Follow-up

preoperative

1 month

3 months

BMI

36.7

33.82

30.1

EWL %

-

23.61

52.59

6 months

12 months

24 months

29.51

28.9

29.85

54.5

55.1

52.6

BMI
EWL %

EWL - excess weight loss; BMI - Body mass index

CONCLUSION
LIVHR repair is a safe method in obese patients, if LGCP isadded in the same operative time the rate of short term minor
complication is increased, but long term recurrence is decreased
due to weight loss. Larger studies are required to drown a final
conclusion upon our combined method.
DISCUSSION
LIVHR is becoming increasingly popular; one of the reasonsis the spread of the laparoscopic technique and the consecutive
raise in laparoscopic skills of the surgeons, the other is the lower
37

complications rates of the minimally invasive method. A metaanalysis done by Goodney et al. (4) identified eight studies fora total of 712 patients which have two arms comparing LIVHR
and open incisional hernia repairs. Patients undergoing a LIVHR
in these studies were 58% less likely to develop a perioperativecomplication compared to those undergoing an open repair.
The new technologies advance – dual mesh, biologique mesh,absorbable tackers, glue – make LIVHR looking more attractive
than ever. Another benefit of the laparoscopic approach is identifying small fascial defects, known as ‘‘Swiss cheese’’ defects,
which may be missed during an open repair. These small fascial
defects are a source of incisional hernia recurrence; therefore
identification is important for a successful hernia repair (5).
The laparoscopic approach of incisional hernia is ideal in
the obese patient, due to the smaller wounds and theoretically,
decreased wound complications (6). Sugermanet al. (7) reported
that obesity grade II or more (BMI >35 kg/m2) was a greater risk
factor for incisional hernia and recurrence than chronic steroid
use. Same group has shown that morbid obese patients, andespecially those with central obesity, have increased basal intraabdominal pressure (8). This higher pressure creates more strain
on the mesh placed for the hernia repair and reduces the bloodflow in the abdominal wall, which are probably responsible forthe higher rate of recurrence following repair.
Adding a restrictive procedure to the hernia repair we try to
decrease one of the most important risk factor for the recurrence - obesity. In our pilot study of six patients we didn’t had
any recurrence in the follow-up period of two years, one of the
explanation might be the use of laparoscopy, and the bariatricprocedure that allowed our patients to lose more than 50% of
the excess body weight. We choose to follow-up our patients for
two years because most of the recurrences occur in this period;
approximately 50% of all incisional hernias develop within thefirst 2 years after surgery (9).
We chose to associate LGCP to LIVHR because of the uniquefeature of this procedure among bariatric procedure – the digestive tube stays closed during procedure, there is no digestive
spillage in contact with the mesh lowering the risk of mesh infection. Also LGCP has a low rate of gastric fistula and complications
as Talebpour et al reported in the largest series of LGCP that
8 patients out of 800 cases (1%) required reoperation due tocomplications like: micro perforation, obstruction and vomiting
following adhesion of His angle (10).
The results of LGCP are satisfying with obese persons with a
BMI lower than 40 kg/m2 and a strong motivation to lose weight(10). Long-term preservation of weight reduction is a challengein patients with greater BMI. There is only one report regarding
long-term outcome of LGP that reported weight regain of 15 %(11 of 75), 30 % (10 of 35), and 50 % (5 of10) after 3, 7, and 10
years, respectively (11).
There is no bulging at the hernia repair site; although wethought that reducing the volume of the abdomen will lead tobulging. Seroma near to mesh implant was present, but in only
one case required drainage by repeated puncture.
LGCP provide a good loss of the excess body weight, 41% to38

62% at 24 months, but the overall excess weight loss was lower
at 24 months than at 12 months, these patients are watchedcarefully and other restrictive bariatric surgery like laparoscopic
gastric by-pass or duodenal switch might be more effective on
the long term.
5 patients had nausea after surgery, and 2 out five vomited up
to 7 days post-operatively. In order to prevent dehydration weadministrated IV fluids up to 4 liters/day until the vomiting stops.
Great care was taken to prevent electrolyte imbalance. Atlas etal had 79.5% nausea and vomiting in their 44 patients’ series up
to ten days (12), and generally it’s considered that nausea andvomiting appears because of the gastric wall edema.
Our study had only six patients due to difficulty to enrolled morepatients, most of the obese patients grade I and II with abdominal
incisional hernia did not want a gastric restrictiveprocedure same
operative time with the LIVHR. But our results are encouraging
us to propose our combined method to new patients.
Aknowledgements: The research of Dr. Dobrescu Amadeus
was supported by PhD fellowship POSDRU 107/1.5/S/78702.
Disclosure: The authors have nothing to disclosure.
REFERENCES

1. Santora TA, Roslyn JJ. Incisional hernia. Surg Clin North Am, 1993;
73: 557-570.
2. Yahchouchy-Chouillard E, Aura T, Picone O, Etienne JC, Fingerhut
A. (2003) Incisional hernias and related risk factors. Digest Surg, 2003;
20: 3-9.
3. Copaescu C. Plicaturarea marii curburi gastrice pe cale laparoscopicã
(pentru tratamentul obezitãtii morbide). Chirurgia, 2011; 106: 91-97.
4. Goodney PP, Birkmeyer CM, Birkmeyer JD. Short-term outcomesof laparoscopic and open ventral hernia repair: a meta-analysis. ArchSurg, 2002; 137:1161-65.
5. Rudmik L R, Schieman C, Dixon E, Debru E. Laparoscopic incisional
hernia repair: a review of the literature. Hernia, 2006; 10: 110-119.
6. Ramshaw BJ, Esartia P, Schwab J, et al. Comparison of laparoscopic
and open ventral herniorrhaphy. Am Surg, 1999; 65(9): 827-31.
7. Sugerman HJ, Kellum JM Jr, Reines HD, et al. Greater risk ofincisional hernia with morbidly obese than steroid-dependent patientsand low recurrence with prefascial polypropylene mesh. Am J Surg,
1996; 171(1):80-4.
8. Sugerman H, Windsor A, Bessos M, et al. Effects of surgically induced
weight loss on urinary bladder pressure, sagittal abdominal diameter andobesity co-morbidity. Int J Obes Relat Metab Disord, 1998; 22(3): 230-5.
9. Anthony T, Bergen PC, Kim LT, et al Factors affecting recurrence
following incisional herniorrhaphy. World J Surg, 2000; 24: 95-100.
10. Talebpour M, Motamedi SM, Talebpour A, Vahidi H. Twelve year
experience of laparoscopic gastric plication in morbid obesity: development of the technique and patient outcomes. Ann Surg Innov Res,
2012; 22; 6(1): 7.
11. Talebpour M, Vahidi H, Talebpour A. Eleven years experience
about the new technique “laparoscopic vertical gastric plication” inmorbid obesity introduced the first time in the world. In: Proceedingof the XVI Congress of the International Federation for the Surgery
of Obesity and Metabolic Disorders (IFSO); Hamburg, Germany,
2011: 1017.
12. Atlas H, Yazbek T, Garneau PY, Safa N, Denis R. Is there a future
for laparoscopic gastric greater curvature plication (LGGCP)? A reviewof 44 patients. Obes Surg, 2013; 23(9):1397-403.

Fiziologia - Physiology  2014.24.1 (81)

CHIRURGIA BARIATRICA LA PACIENTII OBEZI CU HERNIE INCIZIONALA– O NOUA METODA DE REDUCERE A RATEI DE RECURENTA A HERNIEIREZUMAT

Hernia post-incizionala apare in aproximativ 10% dintre cazurile de interventii chirurgicale abdominale si este cea mai comuna complicatie pe termen lung, unul dintre riscurile majore pentru aparitia herniei post-incizionale fiind obezitatea. Repararea laparoscopica
a herniei ventrale incizionale (LIVHR) a devenit o metoda foarte populara in ultimii ani datorita recuperarii mai rapide si a ratei scazutede infectie. Am utilizat plicaturarea laparoscopica a marii curburi (LGCP) a stomacului pentru tratarea pacientilor obezi. Studiul de fata
este un studiu prospectiv al unei serii de cazuri clinice, care si-a propus evaluarea rezultatelor pe termen lung si scurt ale combinatiei
LIVHR si LGCP pentru pacientii obezi cu hernie incizionala operati in anul 2012 in Clinica de Chirurgie 2 a Spitalului Judetean deUrgente Timisoara. Au fost inrolati in acest studiu 6 pacienti de sex feminin. 4 dintre pacienti au fost monitorizati 24 de luni, ceilalti
2 fiind monitorizati pe o perioada de 20 si respectiv 16 luni. Varsta medie a pacientilor a fost de 42 de ani, iar dimensiunea medie a
defectului parietal a fost de 56 cm2, durata medie de spitalizare a fost de 5 zile, iar rata complicatiilor de 83%. Complicatiile intalnite
au fost: greturi la 5 dintre pacienti, sindrom vomitiv la 2 dintre pacienti si formarea seromului la unul dintre pacienti. Indicele de masa
corporala a scazut de la 36,7 kg/m2 la 29,85 kg/m2. Pierderea de greutate excesiva a fost de 52,6% la doi ani, fara recurenta aherniei in acest interval de timp. Repararea prin LIVHR este o metoda sigura pentru pacientii obezi, iar daca este adaugata si LGCP
in aceasi sedinta chirurgicala, rata complicatiilor minore pe termen scurt este crescuta, dar pe termen lung recurenta este scazuta
datorita scaderii in greutate. Sunt necesare studii mai ample pentru a avea o concluzie finala asupra metodei noastre combinate.
Cuvinte cheie: chirurgie bariatrica, LIVHR, plicaturare gastrica

2014.24.1 (81)  Fiziologia - Physiology

39

THE USE OF PERIOPERATIVE INTRAPERITONEALCHEMOTHERAPY ASSOCIATED WITH NEOADJUVANTTHERAPY FOR ADVANCED OVARIAN CANCER AS
CONSOLIDATION THERAPYJEBEREAN A1,2, CARABINEANU A1,2, ANCUSA D1,2, PREDA M1,2, ISTODOR A2, BUCUR A1,
MOTOC A1,2
1
University of Medicine and Pharmacy “Victor Babes” Timisoara
2
Clinical Emergency County Hospital Timisoara

ABSTRACT

Objectives: To determine the effectiveness of perioperative intraperitoneal chemotherapy and the safety of this method for patientswith ovarian neoplasm who have high residual volume after primary surgery. Methods: A retrospective clinical study in which 24
female patients with ovarian neoplasm, stage FIGO IIIC, operated in the Surgical Oncology Clinic of the Municipal Hospital Timisoara
over a period ofsix years (2003-2009), were evaluated. Results: The female patients studied were diagnosed after surgical staging+/- suboptimal tumor cytoreduction, had voluminous residual tumor after surgery, have been given cisplatin and perioperatively
endoxan, followed by six cycles of neoadjuvant systemic chemotherapy. Among the side effects of the chemotherapy administration
into the peritoneal cavity, pain was present in 18 patients, ileus in 5 patients, leukopenia in 2 patients and renal toxicity in 2 patients.
The treatment response evaluated after a further surgery in 20 patients and by computed tomography in 4 patients was complete in
9 cases (37.5%), partial in 10 patients (41.66%) and negative in 5 patients (20 83%), the disease being stagnant or in evolution. The
median survival of patients was 30.9 months, with a median of 24.3 months. Conclusions: Intraperitoneal chemotherapy used as
consolidation treatment in patients with high residual volume after primary surgery is devoid of major complications and in combination with systemic chemotherapy and cytoreductive surgery may improve long-term prognosis.
Keywords: intraperitoneal chemotherapy, ovarian cancer, cytoreductive surgery.

INTRODUCTION
The epithelial ovarian cancer is an important cause of deathamong gynecological cancers in women, with an incidence in theEuropean Union by 18/100.000 women/year and a mortality of
12/100.000 women/year (1). The gloomy prognosis is becauseof the fact that approximately 75% are diagnosed in advanced
stages (FIGO III-IV), when there are usually about peritoneal,
hematogenous or lymphatic disseminated metastasis (2,3).
The primary route of dissemination of ovarian cancer is theperitoneal by implanting intraperitoneal cancer cells in different
locations, following the peritoneal fluid hydrodynamicsthroughthe spaces parieto-colice to diaphragm and back to pelvis (4,5).
This hypothesis has captured the attention of studies in recent
decades, directing therapeutic protocols toward intraperitoneal
administration of cytostatic agents.
For advanced ovarian neoplasm the basic principle of treatment is the combination of primary cytoreductive surgery withthe remnants of tumor volume as low as possible and adjuvant
chemotherapy, six cycle of platinum-based regimens (cisplatin/
carboplatin) and taxanes (6,7,8).For female patients with unresectable tumors is recommended between 3 and 6 cycles of neoadjuvant chemotherapy with the same platinum-based regimens andtaxanes followed by “surgery interval” with cytoreductive purposes.

Multiple studies have reported benefits in terms of survival
and disease-free period for intraperitoneal administration ofcytotoxic agents (9,10). The probable mechanisms by which
the use of cytostatics directly into the peritoneal cavity bring thisbenefit are: getting an increased levels of cytotoxic substancein contact with the peritoneal serous, having a low clearance
from the systemic circulation; stimulation of local immunologicalmechanisms of the peritoneal cavity with increasing cytotoxicity
antineoplastic agents; conversion from “chemoresistant” tumors
to “chemosensitive” tumors; direct penetrating of chemotherapy
in tumor tissue (11).
Although current indications of intraperitoneal cytotoxic drugs inovarian cancer are the small residual volume after primary cytoreductive surgery or recurrent disease, in our study we aimed to highlightthe possible advantages by using this method also to the female
patients that present high residual volume after primary surgery.
METHODS
The study was conducted on a group of 24 patients diagnosed
with advanced ovarian cancer who were hospitalized in the General Surgery II and Surgical Oncology Clinic of the Emergency
Municipal Hospital Timisoara, between 2003-2009. Patients wereselected from a total of 236 sick subjects, diagnosed and treated

Received 15th of January 2014.Accepted 25th of February 2014. Address for correspondence: Dr. Adrian Jebereanu, MD, Clinical Emergency County Hospital
Timisoara, University of Medicine and Pharmacy “Victor Babes” Timisoara, Eftimie Murgu Square No. 2A, RO-300041, Timisoara; phone/fax: +40256220497; e-mail:jeberean_adi@yahoo.com

40

Fiziologia - Physiology  2014.24.1 (81)in our clinic during that time.
The inclusion criteria of the patients in the study were represented by: histopathologic diagnosis of epithelial ovarian cancer,
locally advanced disease, residual tumor volume increased afterstaging laparotomy or cytoreductive surgery performed as thefirst therapeutic act, patients who had performance index increased, following postoperatively intraperitoneal chemotherapy.
Were excluded from the study patients who had significant
comorbidities (other neoplastic diseases associated with heart,kidney or liver failure), who showed a lower performance index.
All patients were diagnosed by staging laparotomy +/- tumor
cytoreduction, which was the first therapeutic approach.Only
5 patients underwent cytoreductive tumor, but did not reach
optimal, they presented postoperatively increased residual volume (> 2 cm) and in the remaining 19 patients, the surgery was
limited only to the exploratory laparotomy, considering abdominal
tumors as unresectable. Postoperatively, all the patients received
intraperitoneally chemotherapy by normothermia treatment.
Subsequently, patients who survived the postoperative period
received adjuvant chemotherapy or neoadjuvant systemic including regimens platinum agents (cisplatin/carboplatin) according
to the treatment guidelines, 20 patients were subjected to a newsurgery with cyto-reductive purposes.
Intraperitoneal chemotherapy technique: intra-operatively acatheter is placed pelvic which externalizes through counterincision in the right flank for administration and a drainage tube also
placed pelvic for disposal. During the first 3-4 hours after surgery
is administered50 mg Cisplatin and 1000 mg Endoxan added to1000 ml of normal saline heated to 37 ° for one hour and evacuated after 12 hours. The procedure repeats itself in the next twodays with only 50 mg Cisplatin diluted in 1000 ml saline under thesame conditions. Throughout the treatment is being maintained adiuresis of 100 ml/h and a maximum creatinine 2 mg/dl.
Postoperatively, the following parameters were monitored:
blood counts, urea, creatinine, transaminases, coagulation times,
diuresis, resumption of intestinal transit time, postoperativecomplications. After the completion of systemic chemotherapy
sessions it was appreciated the response to treatment with a
new cyto-reductive surgery in 20 patients, and the remaining 4
patients by computed tomography.
The study is based on retrospective analysis of survey sheets,
discharge registers, records of outpatient consultation, operators protocols, histopathological bulletins and oncology records.
RESULTS
The study followed 24 patients hospitalized and treated in the
Surgical Oncology Clinic, with an average age of 58.49 years
with a minimum value of 30 and maximum of 77 years. The peak
incidence was recorded in the age group 50-65 years was 62.5%.
After the staging laparatomy, 24 patients have been in thestage FIGO IIIC, 22 patients presented voluminous neoplastic
ascites and all the patients presented generalized peritoneal
carcinomatosis.
Regarding histopathology (Figure 1), papillary adenocarcinoma was the most common result (83.33%); histopathological2014.24.1 (81)  Fiziologia - Physiology

diagnosis showed mucinous adenocarcinoma in two cases(8.33%); in only a case was present adenocarcinoma with clear
cell (4.16%), and in other case was present undifferentiated carcinoma (4.16%). The G2/G3 tumor grading report ratio was 16/8.

Fig.1. Statistical histopathological results regarding the incidence ofdifferent carcinoma types

Postoperatively patients received intraperitoneal chemotherapy by normothermia treatment and subsequent neoadjuvant systemic chemotherapy. The side effects related to theintraperitoneal administration of anti-neoplastic agents were:18 patients presented abdominal pain during administration, 2patients experienced leukopenia (WBC < 4,000/mm3), 5 patients
had prolonged postoperative ileus (over 4 days), 2 patients had
renal toxicity (creatinine > 3 mg/dl), and 2 patients had postoperative wound infections.
Of the 24 patients only 20 patients were subjected to a new
purpose cytoreductive surgery, 4 patients being diagnosed with
progressive disease, therefore surgery was considered inadvisable. After the second surgery 9 patients were in complete remission, showing parts of sterile resection; 10 patients had partial
remission, in 6 patients we could perform optimal cytoreductive(residual tumor <1 cm) and in 4 patients we could perform suboptimal cytoreduction to a residue tumor postoperatively between
1 and 2 cm; in one patient surgery was limited to exploratory
laparotomy, presenting voluminous tumor masses un-resectable.
The short-term result in terms of response to neoadjuvant
chemotherapy and systemic intraperitoneal postoperatively,
after surgery evaluation by clinical examination and by clinicaland imaging examination (Figure 2) was favorable to 79.16%,
the remaining 20.83% showing chemoresistance.

Fig.2. Statistical representation of treatment outcomes in cancer
patients

41

The median survival of patients was 30.9 months, with a
median of 24.3 months. For the patients who had a favorable
response after neoadjuvant therapy there was a median survivalof 35.62 months, with a median of 29.16 month. The survival
rate for the same group of patients who received neoadjuvant
therapy and cyto-reductive surgery was 12 months to 95.85%,
24 months to 50%, 36 months to 29.16% and 5 years to 16.66%.
DISCUSSION
Ovarian neoplasm is an important cause of mortality and morbidity in the malignancy. Optimal tumor cytoreduction performed
both as a first therapeutic act as well as after neoadjuvant chemotherapy is considered a favorable prognostic factor in terms ofsurvival for patients with advanced ovarian neoplesm (12,13).
Residual tumor volume after primary surgery is closely related tosurvival, such a study on 3,126 patients where du Bois et al. (14)found an average survival of only 29.6 months in patients who
had residual disease after primary surgery > 1 cm. Considering
75% (2) of ovarian neoplasm cases are diagnosed in advanced
stages, being almost impossible an oncologic surgery, the analysis and evaluation of adjuvant or neoadjuvant opportunities thatwould make possible the application of a radical onco-surgicalprotocol requires searching for new methods for these cases.
One such method is the intraperitoneal chemotherapy applied
according to numerous studies (GOG-114, GOG-158, GOG-172)
at patients that could get a small residual tumor after primary
surgery, the survival rate and disease-free period being significantly elevated for patients with intraperitoneal chemotherapy.
The problem of this method occurs in patients whose primarytumor cytoreduction cannot achieve the optimal threshold. In
our study we analyzed the data and the results of applying thismethod to patients without optimal cytoreduction who undergo
neoadjuvant systemic chemotherapy.
Intraperitoneal administration of cytostatic agents is associated with greater toxicity than intravenous administration. Of theside effects related to intraperitoneal administration of cytostatic
agents remember abdominal pain during the treatment, marrow
toxicity, renal toxicity, infectious complications, neuropathy andeven intestinal fistulas. In the present study we observed that theabdominal pain was the most frequently encountered side effect,
followed by delaying intestinal transit, the remaining reactions
being negligible due to the relatively low percentage.
There are some studies in the literature showing the effectiveness and safety of intraperitoneal chemotherapy in patients who
have voluminous disease after staging surgery (16, 17). Thus S.
Nagao et al. in a study of 44 patients after a combination therapy
with intraperitoneal carboplatin and paclitaxel intravenously
had a favorable answer to ~ 80% of patients, 16 patients werein complete remission, 19 had a partial response and 9 hadstagnant or progressive disease and the median survival was
31 months. In another study conducted by Zylberberg B et al.
on 13 patients with ovarian cancer stage IIIC and who had highintra-abdominal tumor volume received a chemotherapy regimen
based on cisplatin and immunotherapy between 4 and 10 cycles,
6 patients had complete remission (18). U Beller et al, using a42system of consolidation intraperitoneal chemotherapy with cisplatin and cyclophosphamide intravenously achieved complete
remission in 32 patients (65%) with advanced ovarian cancerfrom a total of 49 patients, but the median survival for patients
who had residual volume increased after primary surgery was
only 24 months (19).
Analyzing the results of our study and comparing them withthose in the literature we can notice the fact that there are insufficient data in order to demonstrate the superiority for the useof cytotoxic agents in patients with voluminous residual disease
after primary surgery. In our study, the results obtained are in areasonable cited in the literature.
CONCLUSIONSThe perioperative administration of cytostatic agents into theperitoneal cavity is not associated with major complications, butpatients require additional follow up.
The use of perioperative intraperitoneal chemotherapy as
consolidation treatment in neoadjuvant systemic chemotherapy
made possible the tumor cytoreduction to an increased numberof patients.
Patients with advanced ovarian cancer, who cannot make
optimal primary cytoreductive surgery, the prognosis can be
improved by using peritoneal and systemic cytotoxic agents incombination with secondary cytoreductive surgery.
REFERENCES

1. Aebi S, Castiglione M. On Behalf of the ESMO Guidelines Working
Group. Newly and relapsed epithelial ovarian carcinoma: Esmo Clinical
Recommendations for diagnosis, treatment and follow-up. Annals of
Oncology, 2009; 20(4): iv21-iv23.
2. Ozols RF. Update on gynecologic Oncology Group (GOG) trials in
ovarian cancer. Cancer Invest. 2004; 22(2):11-20.
3. Thigpen T. First-line therapy for ovarian carcinoma: what’s next?
Cancer Invest. 2004; 22(2):21-28.
4. Markman M. Intraperitoneal chemotherapy in the management ofovarian cancer: rationale and results. In Abstract book of 20th International Congress on Anticancer Treatment. Paris, February 3-6, 2009:
140-142.
5. Vincent T, DeVita Jr, Hellman S, Steven A, Rosenberg. Cancer:
Principles and Practice of Oncology, 7th ed., 2005.
6. Ledermann JA, Raja FA, Fotopoulou C, Gonzalez-Martin A, Colombo
N, Sessa C. Newly diagnosed and relapsed epithelial ovarian carcinoma:
ESMO Clinical Practice Guidelines for diagnosis, treatment and followup. Annals of Oncology, 2013; 24(6): vi24-vi32.
7. Hoskins.WJ. Surgical staging and cytoreductive surgery of epithelial
ovarian cancer. Cancer, 1993; 71:1534-40.
8. Vergote I, Tropé CG, Amant F, Kristensen GB, Ehlen T, Johnson N,
Verheijen RHM, et al. European Organization for Research andTreatment of Cancer–Gynaecological Cancer Group and the
NCIC Clinical Trials Group-a Gynecologic Cancer Intergroup
Collaboration, Neoadjuvant Chemotherapy or Primary Surgery inStage IIIC or IV Ovarian Cancer. N Engl J Med, 2010; 363: 943-953.
9. Markman M, Bundy BN, Alberts DS, et al. Phase III trial of standarddose intravenous cisplatin plus paclitaxel versus moderately high-dosecarboplatin followed by intravenous paclitaxel and intraperitoneal
cisplatin in small-volume stage III ovarian carcinoma: an intergroup

Fiziologia - Physiology  2014.24.1 (81)

study of the Gunecologic Oncology Group, Southwestern Oncology Group, and Eastern Cooperative Oncology Group. J Clin Oncol,
2001;19(4):1001-1007.
10. Armstrong DK, Bundy B, Wenzel L, et al. Intraperitoneal cisplatin
and paclitaxel in ovarian cancer. N Engl J Med, 2006; 354(1):34-43.
11. Helm CW, Edwards RP. Intraperitoneal Cancer Therapy; Ed.
Humana Press Totowa, New Jersey 2007.
12. Hoskins WJ, McGuire WP, Brady MF, Homesley HD, Creasman WT,
Berman M, et al. The effect of diameter of largest residual disease on
survival after primary cytoreductive surgery in patients with suboptimal
residual epithelial ovarian carcinoma. Am J Obstet Gynecol, 1994;
170:974-80.
13. Bristow RE, Tomacruz RS, Armstrong DK, Trimble EL, Montz FL.
Survival effect of maximal cytoreductive surgery for advanced ovariancarcinoma during the platinum era: A meta-analysis. J Clin Oncol,
2002; 20: 1248-59.
14. du Bois A, Reuss A, Pujade-Lauraine E, et al. Role of surgicaloutcome as prognostic factor in advanced epithelial ovarian cancer:
a combined exploratory analysis of prospectively randomized phase
3 multicenter trials: by the Arbeitsgemeinschaft Gynaekologische
Onkologie Studiengruppe Ovarialkarzinom (AGO-OVAR) and thegrouped’ Investigateurs nationaux our Ies Etudes des cancers de
I’Ovaire (GINECO). Cancer. 2009; 115(6):1234-1244.

15. Markman M, Reichman B, Hakes T, Rubi S, Lewis JL Jr, Jones W,
Barakat R, Curtin J, Almadrones L, Hoskins W. Evidence supporting
the superiority of intraperitoneal cisplatin compared to intraperitoneal
carboplatin for salvage therapy of small volume residual ovarian cancer.
Gynecol Oncol. 1993: 50(1): 100-104.
16. Nagao S, Fujiwara K, Ohishi R, Nakanishi Y, Iwasa N, Shimizu M,
Goto T, Shimoya K. Combination chemotherapy of intraperitoneal carboplatin and intravenous paclitaxel in suboptimally debulked epithelial
ovarian cancer. Int J Gynecol Cancer.2008; 18: 1210-1214.
17. Fujiwara K, Nagao S, Kigawa J, Noma J, Akamatsu N, Miyagi Y,
Numa F, Okada M, Aotani E. Phase II study of intraperitoneal carboplatinwith intravenous paclitaxel in patient with suboptimal residual epithelial
ovarian or primary peritoneal cancer: a Sankai Gyecologic cancer groupstudy. Int J Gynecol Cancer. 2009; 19: 834-837.
18. Zylberberg B, Dormont D, Janklewicz S, Darai E, Bretel JJ, Poncelet
C, Guillet JL, Madelenat P. Response to neo-adjuvant intraperitoneal
and intravenous immunochemotherapy followed by interval secondary cytoreduction in stage IIIc ovarian cancer. Eur J Gynaecol Oncol.
2001; 22(1):40-5.
19. Beller U, Speyer J, Colombo N, Sorich J, Wernz J, Hochster H,
Zeleniuch-Jacquotte A, Porges R, Beckman EM. Consolidation with
Intraperitonealcisplatin in first-line therapy of advanced ovarian cancer.
J Clin Oncol. 1991; 9(5): 809-17.

UTILIZAREA CHIMIOTERAPIEI INTRAPERITONEALE PERI-OPERATIVEASOCIATA CU TERAPIA NEOADJUVANTA PENTRU CANCERUL OVARIAN
AVANSAT CA TERAPIE DE CONSOLIDAREREZUMAT

Obiective: Determinarea eficacitatii chimioterapiei peri-operative intraperitoneale si a sigurantei acestei metode la pacientii cu cancer
ovarian care prezinta volum rezidual crescut dupa chirurgia primara. Metode: Studiu clinic retrospectiv in care au fost incluse 24 depaciente cu cancer ovarian, stadiul FIGO IIIC, care au suferit intervetie chirurgicala in Clinica de Oncologie a Spitalului Municipal
Timisoara pe o perioada de 6 ani (2003-2009). Rezultate: Pacientele cuprinse in studiu au fost diagnosticate dupa stadializarea
chirurgicala +/- citoreductia tumorala suboptimala, prezentand tumora reziduala voluminoasa dupa interventia chirurgicala; a fostadministrat cisplatin si endoxan perioperator, urmat de 6 cicluri de chimioterapie sistemica neoadjuvanta. Printre efectele adverse
ale administrarii chimioterapiei la nivelul cavitatii peritoneale s-au numarat: durere la 18 paciente, ileus la 5 paciente, leucopenie
la 2 paciente si toxicitate renala la 2 paciente. Raspunsul la tratament a fost evaluat dupa interventia chirurgicala ulterioara la 20
paciente prin tomografie computerizata; la 9 paciente raspunsul a fost complet (37,5%), partial la 10 paciente (41,66%) si negativ la
5 paciente (20,83%), afectiunea fiind stagnanta sau in evolutie. Rata de supravietuire a pacientelor a fost de 30,9 luni, cu o medie
de 24,3 luni. Concluzie: Chimioterapia intraperitoneala folosita ca tratament de consolidare la pacientii cu volum rezidual crescut
dupa chirurgia primara este lipsita de complicatii majore, iar in combinatie cu chimioterapia sistemica si chirurgia citoreductiva poate
imbunatatii prognosticul pe termen lung.
Cuvinte cheie: chimioterapie intraperitoneala, cancer ovarian, chirurgie citoreductiva

2014.24.1 (81)  Fiziologia - Physiology

43

CHANGES INDUCED BY ADDITION OF METFORMIN IN CULTURE
MEDIA OF TUMOR-ASSOCIATED FIBROBLASTS (TAF)
MEDA DEICA, RALUCA SIMA, LAURA CERNAT, FLORINA BOJIN, CARMEN TATU, GABRIELATANASIE, CARMEN PANAITESCU, VIRGIL PAUNESCU
Department of Functional Sciences, ”Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACT

Considering the important role played by tumor-associated fibroblasts (TAFs) in tumor development and progression, we tried toinvestigate in this study the influence of metformin on TAFs’ phenotype. We isolated TAFs from surgical resection pieces obtained
from patients with diagnosis of infiltrative ductal mammary carcinoma. Cultured cells were treated with metformin in two concentrations (5 mM and 10 mM) for 72 hours, and then the cells were flowcytometricaly analyzed for expression of surface markers CD44
and TGFβ-RII. Additional flowcytometric procedures included viability assay using Annexin-V/PI. The results showed that metformintreated TAFs present an increased expression of CD44 and a decrease in TGFβ-RII expression on cellular surface. We concluded
that metformin can induce changes in TAFs phenotype, which could switch their behavior towards a less aggressive phenotype,which will not provide support for tumor cells.
Key words: tumor-associated fibroblasts (TAF), metformin, surface markers, apoptosis

INTRODUCTION
Origin of tumor-associated fibroblasts (TAFs)
Within tumor tissues, myofibroblasts originate from diverse
cellular types: pre-existent fibroblasts, adipocytes and preadipocytes, smooth muscle cells, endothelial cells, and bonemarrow-derived cells (Figure 1). From all these cellular types,
bone marrow-derived cells are a representative source of myofibroblasts within tumor stroma. Recent studies showed that 28
days after implantation of tumor pancreatic cells within a mousemodel, approximately 40% of total myofibroblasts from tumor
stroma derive from bone marrow cells (1).
Although during the normal fibrosis process, epithelial cells are
the ones transforming into activated fibroblasts, there are not sufficient data demonstrating whether normal or malignant epithelial
cells are transforming into myofibroblasts within tumor tissue.

Fig. 1. Origin of tumor-associated fibroblasts (TAF). Mesenchymal
stem cells (MSCs) transmigrate and can differentiate into TAFs at thelevel of tumor tissue. Other cells can also transform into TAFs: normal
fibroblasts (fibroblast-mesenchymal transition-FMT), tumor cells (epithelial-mesenchymal transition-EMT), smooth muscle cells, adipocytes
and endothelial cells (endothelial-mesenchymal transition-EndMT)

Tumor-associated fibroblasts role in tumorigenesis andmetastasis
Proliferation of tumor cells is an important step in invasion andmetastasis processes. In order to ensure an optimal environment
for proliferation and development, tumor cells are recruiting
stromal cells, which will support tumor development throughsecretion of growth factors and cytokines.
FSP-1 (fibroblast secreted protein-1) secreted by tumorassociated fibroblasts is an important factor for tumor cells
development. FSP-1 knockout mice presented a decreased
incidence in tumor formation and did not developed metastases when tumor cells were injected, while co-injection of FSP1 overexpressing fibroblasts induced tumor formation (2).
The results of this experiment showed that tumor-associatedfibroblasts have an important influence on tumor microenvironment, stimulating tumor development. SDF-1 (stromal
derived factor 1) is a cytokine secreted by tumor-associatedfibroblasts, with a role in recruiting endothelial cells precursors, which will further be involved in angiogenesis (3). SDF-1
is also known as CXCL12, ligand for CXCR4, overexpressedin tumor cells. CXCR4 activation can stimulate tumor cells
proliferation (4).
In some of the malignant tumors, such as breast cancer (5) andcolon cancer (6), an increased expression level for TGFβ was
reported, TGFβ being a growth factor which stimulates normal
fibroblasts differentiation into tumor-associated fibroblasts andmyofibroblasts.
Considering the important role played by tumor-associatedfibroblasts (TAFs) in tumor development and progression, we
tried to investigate in this study the influence of metformin on
TAFs’ phenotype. Changes induced in TAFs could switch their
behavior towards a less aggressive phenotype, which will notprovide support for tumor cells.

Received 10th of January 2014. Accepted 25th of February 2014. Address for correspondence: MedaDeica, MD, PhD student, Department of Functional Sciences,
Physiology, “Victor Babes” University of Medicine and Pharmacy Timisoara, EftimieMurgu Square No. 2A, RO-300041, Timisoara, Romania, Phone/fax: + 40
256490507; email: bravameda@yahoo.com

44

Fiziologia - Physiology  2014.24.1 (81)

MATERIALS AND METHODS
Isolation and culture of tumor-associated fibroblasts
Human tumor-associated fibroblasts were isolated using both
explant method and enzymatic digestion with Collagenase type
IV-S obtained from Clostridium histolyticum (Sigma-AldrichCompany, St. Louis, MO, USA). Surgical rezection pieces wereobtained from patients with diagnosis of infiltrative ductal mammary carcinoma. Isolated cells were washed with saline solution(PBS, Phosphate Buffer Saline, Sigma-Aldrich) and successively
filtered through 0.7 and 0.4 μm cell strainers. Cellular suspension
was centrifuged (1500 rpm, 10 min), and cells were resuspendedin culture medium - Dulbecco’s Modified Eagle Medium (DMEM,
Sigma), supplemented with 10% Fetal Calf Serum (FCS; PromoCell, Heidelberg, Germany) and 1% antibiotic solution containing
Penicillin/Streptomycin (Pen/Strep, 10,000 IU/mL, PromoCell).
TAFs were cultured in adherent culture flasks (T75 culture flasks),
at a cellular density of 10,000 cells/cm2, and expanded at 37°C,
in 5% CO2 atmosphere.
All tissue samples were obtained from human subjects after
signing the Informed Consent Form elaborated by Ethics Committee of “Victor Babes” University of Medicine and Pharmacy
Timisoara, according to Helsinki declaration of World healthOrganization.
Immunophenotypical analysis
TAFs were cultures in 6-well adherent culture plates. 24 hours
after plating, metformin was added in 2 concentrations – 5 mM
and 10 mM – in culture medium, and after 4 days of culture,
the cells were detached from the culture plates using 0.25%
Trypsin-EDTA (Sigma-Aldrich Company), centrifuged for 10
minutes at 1500 rpm, and washed twice with PBS. Cells were
further resuspended in 100 µl PBS for each tube of flowcytometric analysis, together with 4 µl of fluorochrome-conjugatedmouse anti-human antibodies. Cells are vortexed together withthe antibodies and left to incubate for 30 minutes, in the dark, atroom temperature. After incubation, cells are washed with 2 mlwash solution (Cell Wash Solution, BD Biosciences), centrifuged
10 minutes at 1500 rpm, the supernatant is discarded, and thepellet is resuspended in 500 µl wash solution and are ready for
flowcytometric acquisition and analysis. Flowcytometric acquisition was performed using FACSCalibur flowcytometer (BectonDickinson), endowed with 2 lasers and capable to differentiate
4 distinct colors. The fluorochrome-conjugated antibodies were:PE - TGFβ-RII, FITC - CD44.
Viability assay Annexin-V/PI
In order to determine metformin capacity to induce cellularapoptosis, we performed Annexin-V/PI flowcytometric assay.
Annexin-V binds phosphatidylserine on injured cell membrane.
For Annexin-V assay, the cells were washed with 1 ml AnnexinV Binding Buffer and centrifuged 10 minutes at 1500 rpm. The
supernatant is discarded and cells are resuspended in 100 µl deAnnexin-V Binding Buffer, and 10 µl of FITC-conjugated AnnexinV are added. After 15 minutes of incubation at room temperature,in the dark, cells are washed again, and resuspended in 500 µl of
2014.24.1 (81)  Fiziologia - Physiology

the same buffer, while PI solution (1 µg/ml) is added just before
the flowcytometric acquisition procedure.
Analysis of flowcytometric graphs was performed using FCS
Express 4 Flow Research Edition software (De Novo Software),while for statistic analysis we used GraphPad Prism 6 (GraphPad
Software).
RESULTS
Annexin V-FITC / PI assay
Flowcytometric analysis of Annexin-V/PI assay performed on
TAFs revealed a decrease of live cells, after addition in culture
medium of metformin in concentrations of 5 mM and 10 mM, for
72 hours. Compared to control cells (untreated TAFs), this assay
showed that a larger cell number are entering in early apoptotic
stage, when treated with metformin 5 mM; in case of using
higher metformin concentrations (10 mM), the cells found in late
apoptotic stages are in higher amount than for lower metformin
concentrations (5 mM) (Figures 2 and 3).

Fig. 2. Dot plot graphs showing distribution of metformin-treated
TAFs in different viability stages: live cells, early or late apoptotic
cells. Only few cells are dead after metformin treatment, for bothconcentrations used.

Fig. 3. Annexin-V/PI assay performed on tumor-associated fibroblasts showed a decrease of live cells when treated with metforminin different concentrations. The remaining cells are in early or ate
apoptotic stages, correlated with metformin concentration

Flowcytometric analysis of CD44 and TGFβ RII markers
Flowcytometric analysis of CD44 and TGFβ RII on TAFs sur45

face showed that the cells treated with metformin in cancentrationof 5 mM and 10 mM present an increased expression of CD44.
For TGFβ RII, the analysis revealed an increased expression
when 5 mM metformin was added and a significant decrease of
its expression when the cells were treated with 10 mM metformin(Figures 4, 5, 6, and 7).
CD44 is a transmembrane glycoprotein which functions as
ligand for hyaluronic acid, but also for other ligands, such as
matrix metalloproteinases, involved in tumor metastasis. CD44
is involved in cellular interactions, cellular adhesion and migration. Moreover, this protein participates in lymphocytes activaton,recirculation, homing, hematopoiesis, and tumor metastasis.
CD44 interact with osteopontin and leads to initiation of tumor
process (7). This glycoprotein interacts with laminin, collagen,and fibronectin, but the results of their physiological interactions
are not completely understood (8). CD44 expressed on surfaceof tumor cells is involved in systemic migration of tumor cells,
through interactions with selectins L and (9). This molecule isalso involved in signaling pathways of tumorigenesis initiation
through interaction with tyrosine kinase receptors (10). CD44 islargely used as surface marker for isolation of cancer stem cells
in breast cancer, prostate, ovary, and colon cancer (11). Usingother additional surface markers, we can differentiate different
tumor types (12). There are still contradictions related to correlation between CD44 and cancer prognostic. In many cancer
types, CD44 present a major role in initiation of tumorigenesis
and metastasis. Other studies performed on prostate tumor cells(13) and breast cancer cells (14), CD44 overexpression is notcorrelated with carcinogenesis.
TGFβ RII is part of TGFβ receptors family. Dependant on
structure and properties, there are three types of receptors. TypeI and type II receptors have similar affinity for ligands, higher incase TGF-β1, and decreased for TGF-β2. Type III receptors
have increased affinity, both for TGF-β1, as well as for TGF-β2.

Fig. 5. Contour plot of 5 mM metformin-treated TAFs

Fig.6. Contour plot of 10 mM metformin-treated TAFs

Fig. 4. Contour plot of TAFs control (untreated cells)
46

Fig. 7. Comparative graphs depicting expression of CD44 and TGFβ
RII in control TAFs (untreated cells) and metformin-treated TAFs

Fiziologia - Physiology  2014.24.1 (81)DISCUSSION AND CONCLUSION
The results of flowcytometric assay using Annexin V/PI employed to investigate metformin influence on cellular apoptosis
showed that TAFs present a decreased in live cell population,
while large number of cells are in different apoptotic stages,
thus indicating that metformin is capable of activating apoptotic
pathways in TAFs.
In order to better understand metformin effect on TAF, weused immunophenotypical assays. We investigated expressionof CD44 and TGFβ RII surface markers on TAFs. Interestingly,
we showed a significant decrease of TGFβ RII on TAF surface
when 10 mM metformin was added in the culture medium.
There are several studies sustaining that metformin is able toinduce apoptosis, but other studies are supporting the opposite
idea. It was shown that metformin has the capacity ot induce
apoptosis in lung cancer cells by activating MAPK pathway (15),in ovary tumor cells by activating Bcl-2 protein family (16), while
in mammary tumor cells (MCF-7) on the ERK signaling pathway(17). Contradicting these studies, Alimova et al. showed that
metformin is not inducing apoptosis in several cell lines, including
breast cancer cells MCF7, MCF7/713, BT474 and SK-BR3 (18).
In our experiments we support the hypothesis that metformin has
the capacity to induce apoptosis in tumor-associated fibroblasts(TAF).
Although some studies suggest that metformin reduces CD44+
CD24- cell population in tumor cells, in case of tumor-associatedfibroblasts we noticed an increased in CD44 expression andmaintenance of vimentin expression (data not shown). We may
conclude that TAFs maintained mesenchymal characteristics as aresult of metformin treatment. Interestingly, metformin induces adecrease in TGFβ-RII expression on TAF surfce. In other studies
performed on breast cancer cells, TGFβ transcription factor was
suppressed, which was correlated with epithelial-mesenchymaltransition. So far there are not enough data to associate TGFβ-RII
inhibition with a certain biological activity, but further studies will
include more thorough analysis of metformin effects on TGFβ
activity at the level of tumor-associated fibroblasts, performing
co-cultures of TAFs and breast cancer cells.
Acknowledgements
This work was supported by the Sectorial Operational Programme for Human Resources Development, financed from theEuropean Social Fund, FSE POSDRU 107/1.5/S/78702 and by
UEFISCDI, PNII-Idei, Project No. 318/2011.
REFERENCES

1. Ishii G, Sangai T, Oda T, Aoyagi Y, Hasebe T, et al. Bone-marrow-

2014.24.1 (81)  Fiziologia - Physiology

derived myofibroblasts contribute to the cancer-induced stromal reaction. Biochem. Biophys. Res. Commun., 2003; 309(1): 232-240.
2. Grum-Schwensen B, Klingelhofer J, Berg CH, El-Naaman C, Grigorian M, Lukanidin E, Ambartsumian N. Suppression of tumor development and metastasis formation in mice lacking the S100A4(mts1) gene.
Cancer Res., 2005; 65(9): 3772-80.
3. Goh PP, Sze DM, Roufogalis BD. Molecular and cellular regulators ofcancer angiogenesis. Curr Cancer Drug Targets, 2007; 7(8): 743-758.
4. Orimo A, Gupta PB, Sgroi DC, Arenzana-Seisdedos F, Delaunay T,
et al. Stromal fibroblasts present in invasive human breast carcinomas
promote tumor growth and angiogenesis through elevated SDF-1/
CXCL12 secretion. Cell, 2005; 121(3): 335-348.
5. SerraR, Crowley MR. TGF-beta in mammary gland development andbreast cancer. Breast Dis, 2003; 18: 61-73.
6. Tsushima H, Kawata S, Tamura S, Ito N, Shirai Y, et al. High levelsof transforming growth factor beta 1 in patients with colorectal cancer:
association with disease progression. Gastroenterology, 1996; 110(2): 375-382.
7. Rangaswami H, Bulbule A, Kundu GC. Osteopontin: role in cell signaling and cancer progression. Trends in Cell Biology, 2006; 16(2): 79-87.
8. Ponta H, Sherman L, Herrlich PA. CD44: From adhesion molecules
to signalling regulators. Nat Rev Mol Cell Biol, 2003; 4(1): 33-45.
9. Napier SL, Healy ZR, Schnaar RL, Konstantopoulos K. Selectin ligand
expression regulates the initial vascular interactions of colon carcinoma
cells. J. Biol. Chem., 2007; 282 (6): 3433-41.
10. Bourguignon LYW. CD44-mediated oncogenic signaling and cytoskeleton activation during mammary tumor progression. J Mammary
Gland Biol Neoplasia, 2001; 6(3): 287-297.
11. Bapat SA. Human ovarian cancer stem cells. Reproduction, 2010;
140(1): 33-41.
12. Strauss R, Li ZY, Liu Y, Beyer I, Persson J, et al. Analysis of epithelial and mesenchymal markers in ovarian cancer reveals phenotypic
heterogeneity and plasticity. PLoS ONE, 2011; 6(1): e16186.
13. Gao AC, Lou W, Dong JT, Isaacs JT. CD44 is a metastasis suppressor gene for prostatic cancer located on human chromosome 11p13.
Cancer Res, 1997; 57(5): 846-849.
14. Lopez JI, Camenisch TD, Stevens MV, Sands BJ, McDonald J,
Schroeder JA. CD44 attenuates metastatic invasion during breast
cancer progression. Cancer Res, 2005; 65(15): 6755-63.
15. Wu N, Gu C, Gu H, Hu H, Han Y, Li Q. Metformin induces apoptosisof lung cancer cells through activating JNK/p38 MAPK pathway and
GADD153. Neoplasma, 2011; 58(6): 482-490.
16. Yasmeen A, Beauchamp MC, Piura E, E. Segal, Pollak M, Gotlieb
WH. Induction of apoptosis by metformin in epithelial ovarian cancer:
involvement of the Bcl-2 family proteins. Gynecol. Oncol., 2011; 121(3):492-498.
17. Malki A, Youssef A. Antidiabetic drug metformin induces apoptosis
in human MCF breast cancer via targeting ERK signaling. Oncol. Res.,
2011; 19(6): 275-285.
18. Alimova IN, Liu B, Fan Z, Edgerton SM, Dillon T, Lind SE, Thor
AD. Metformin inhibits breast cancer cell growth, colony formationand induces cell cycle arrest in vitro. Cell Cycle, 2009; 8(6): 909-915.

47

MODIFICARI INDUSE DE ADAUGAREA METFORMINULUI IN MEDIUL DE
CULTURA AL FIBROBLASTELOR PERI-TUMORALE (TAF)
REZUMAT

Considerand rolul important pe care il au fibroblastele peri-tumorale (TAF) in dezvoltarea si progresia tumorala, am investigat inacest studiu influenta metforminului asupra fenotipului acestor celule. TAF au fost izolate din piese de rezectie chirurgicala de lapaciente diagnosticate cu carcinom mamar ductal infiltrativ. Celulele cultivate au fost tratate cu metformin in doua concentratii (5 mM
si 10 mM) timp de 72 de ore, celulele fiind ulterir analizate flowcitometric pentru expresia markerilor de suprafata CD44 si TGFβ-RII.
Proceduri flowcitometrice aditionale au inclus testul de viabilitate care utilizeaza Annexin-V/PI. Rezultatele au aratat ca fibroblastele
peri-tumorale tratate cu metformin au prezentat expresie crescuta a CD44 si o scadere a expresiei TGFβ-RII la nivelul suprafetei
celulare. Se poate astfel concluziona ca metfromin poate induce modificari fenotipice la nivelul TAF, care ar putea modifica comportamentul acestor celule spre un fenotip mai putin agresiv, care sa nu ofere suport pentru dezvoltarea tumorala.
Cuvinte cheie: fibroblaste peri-tumorale, metformin, markeri de suprafata, apoptoza

48

Fiziologia - Physiology  2014.24.1 (81)