Official Journal of the Romanian Society of Physiological Sciences

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CO-CHIEF EDITORSASSOCIATE EDITORSEXECUTIVE EDITORSFRANCISC SCHNEIDERIOANA SISKACARMEN TATUMIHAI NECHIFORSORIN RIGA
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Fiziologia- Physiology 2010.20.3(67)
Fiziologia- Physiology 2010supplement
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CONTENTS
1. REVIEW - Stem Cells for Heart Repair.................................................................................................................................................................................................................. 4
Carmen Tatu, Gabriela Tanasie, Florina Bojin, Rf Tatu, Daniela Puscasiu, Carmen Bunu
2. Apelin Involvement on Angiotensin II – Induced Venoconstriction in Obese Rats............................................................................................................................................... 8
Mihai Ana Camelia, Dinca Maria, Dumitriu Irina Luciana, Gurzu Mihaela Beatrice, Slătineanu Simona Mihaela, Foia Liliana Georgeta, Vata Luminita Gina, Cojocaru Elena, Petrescu
Gheorghe
3. Contribution of Intrapulmonary Renin Angiotensin System on Modulation of Rat Pulmonary Arteries Reactivity............................................................................................ 12Maria Dincă, Irina Luciana Dumitriu, Simona Mihaela Slătineanu, Luminiţa Gina Vâtă, Mihaela Beatrice Gurzu, Gheoghe Petrescu
4. Oxidative Stress in Chronic Venous Hypertension – Insights from an Animal Model.......................................................................................................................................... 17
Cristina Maria Vaida Voevod, Teodora Mocan, Remus Moldovan, Nicoleta Decea, Adriana Mureşan
5. Extended Spectrum Beta Lactamase Producing Klebsiella Pneumoniae Strains Isolated from Intensive Care Unit............................................................................................ 22
Delia Muntean, Monica Licker, Liliana Dragomirescu, F. Horhat, Luminiţa Bădiţoiu, Elena Hogea, Delia Berceanu Văduva, C. Piluţ, Dorina Dugăeşescu, Roxana Moldovan
6. Risk Assessment Study on Occupational Methyl-Ethyl Ketone Exposure ........................................................................................................................................................... 25
Delia Cheptanariu, Corneluta Fira-Mladinescu, Liliana Sarb, Stefania Rosca, Carmen Oros, Sorina Doroftei, Oana Suciu, Brigitha Vlaicu
7. Major Factors Influencing Life Quality – Rational Nutrition and Fitness ........................................................................................................................................................... 28
Cristian Negrea

CUPRINS
1. REVIEW - Celulele stem si regenerarea cardiaca.................................................................................................................................................................................................. 4
Carmen Tatu, Gabriela Tanasie, Florina Bojin, Rf Tatu, Daniela Puscasiu, Carmen Bunu
2. Implicarea apelinului în venoconstricţia indusă de angiotensină II la şobolanii obezi......................................................................................................................................... 8
Mihai Ana Camelia, Dinca Maria, Dumitriu Irina Luciana, Gurzu Mihaela Beatrice, Slătineanu Simona Mihaela, Foia Liliana Georgeta, Vata Luminita Gina, Cojocaru Elena, Petrescu
Gheorghe
3. Participarea sistemului renina angiotensina in modularea reactivitatii vaselor pulmonare............................................................................................................................... 12Maria Dincă, Irina Luciana Dumitriu, Simona Mihaela Slătineanu, Luminiţa Gina Vâtă, Mihaela Beatrice Gurzu, Gheoghe Petrescu
4. Stresul oxidativ in insuficienţa venoasă cronică – model experimental............................................................................................................................................................. 17
Cristina Maria Vaida Voevod, Teodora Mocan, Remus Moldovan, Nicoleta Decea, Adriana Mureşan
5. Tulpini de Klebsiella pneumoniae producătoare de beta lactamaze cu spectru extins izolate dintr-o secţie de terapie intensivă...................................................................... 22
Delia Muntean, Monica Licker, Liliana Dragomirescu, F. Horhat, Luminiţa Bădiţoiu, Elena Hogea, Delia Berceanu Văduva, C. Piluţ, Dorina Dugăeşescu, Roxana Moldovan
6. Studiu de evaluare a riscului determinat de expunerea la metil-etil-cetonă în mediul profesional................................................................................................................... 25
Delia Cheptanariu, Corneluta Fira-Mladinescu, Liliana Sarb, Stefania Rosca, Carmen Oros, Sorina Doroftei, Oana Suciu, Brigitha Vlaicu
7. Factori de influeţă majori ai calităţii vieţii – alimentaţia raţională şi fitnessul................................................................................................................................................... 28
Cristian Negrea
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REVIEWSTEM CELLS FOR HEART REPAIRCARMEN TATU, GABRIELA TANASIE, FLORINA BOJIN, RF TATU, DANIELA PUSCASIU,CARMEN BUNU“Victor Babes” University of Medicine and Pharmacy Timisoara, Romania

ABSTRACT
Cell therapy has emerged as an attractive therapeutic modality to repair damaged myocardium. A variety of stem and progenitor
cell populations could be used for cardiac repair. Each cell type has its own profile of advantages, limitations, and practicability
issues in specific clinical settings. Cells differ markedly in regard to their site of origin as well as to their anatomy and function, as
characterized by surface markers, transcription factors, and expressed proteins. They also differ in their ability to form one or moredifferentiated cell types.
Key words: mesenchymal stem cells, endothelial progenitor cells, hematopoietic stem cells, embryonic stem cells

INTRODUCTION
Cardiac repair can be considered as the outcome of threemajor processes: replacement (tissue transplant), rejuvenation
or restoration (activation of resident cardiac stem cells or otherstem cells via paracrine or autocrine mechanisms; modulationof apoptosis, inflammation, angiogenesis, or metabolism),
and regeneration (progenitor or stem cell engraftment forming
differentiated myocytes)(6,7). These different entities may beinterlinked in that modulation of myocardial injury, may also
benefit subsequent therapy directed at myocardial regeneration. In preclinical and clinical studies, a variety of cells havebeen considered as candidates for cell repair therapy. Cells
differ markedly in regard to their site of origin as well as to theiranatomy and function, as characterized by surface markers,transcription factors, and expressed proteins. They also differ
in their ability to form one or more differentiated cell types(7).
The bone marrow acts as a reservoir, housing diverse types ofprogenitor cells (e.g., endothelial progenitor cells, mesenchymal
stem cells, and hematopoietic stem cells), which are differentiallyregulated by growth factors and cytokines, affecting their retention, self-renewal, cell-cycle status, and mobilization (5). These
distinct subsets of progenitor cells are multipotent, although morecommitted, arising from a parent stem cell, and developmentally,
hold a hierarchy between stem cell and a fully differentiatedadult cell. On the contrary, stem cells define a class of multipotent or pluripotent cells fully capable of self-renewal and clonal
expansion. Although, literature is replete with instances of both
autologous stem and progenitor cell therapies for the treatmentof ischemic cardiomyopathies and their improved therapeutic
outcomes have been widely accepted, these trials have led
to a less precise use of terms to identify multipotent cells andtrue stem cells. Myocardial infarction engenders an irreversible
loss of cardiomyocytes. It has been reported that a typicalhuman infarct involves a loss of 1–1,5 billion cardiomyocyteswith a consequent loss in myocardial cell mass (approximately
one third of the total volume) (1,5). Therefore, progenitor celltherapies that can contribute to an increase in the number of

viable and functional cardiomyocytes population behold considerable clinical significance. As a consequence, many stemand progenitor cell types have been subjected to challenges
to demonstrate a cardiomyocytes-differentiation potential. Celltherapy has emerged as an attractive therapeutic modality torepair damaged myocardium. The recent identification of various
bone marrow-derived adult stem or progenitor cells capable ofcontributing to tissue regeneration has opened the possibility
that these cells can be used to repair damaged heart tissue (8).
Although bone marrow transplantation has long been used in
the treatment of hematologic diseases, bone marrow also hasstem or progenitor cells that can give rise to nonhematopoietic
cells, including endothelium, neural cells, skeletal myoblasts,
cardiomyocytes, and epithelium of lung, gut, and skin. Various
bone marrow cells have been examined for their ability to repair
myocardial damage, and initial animal and limited human studies
to date have shown therapeutic benefit. Controversy exists about
the fate of transplanted cells and the therapeutic mechanism ofbone marrow cell therapy in the heart.
A variety of stem and progenitor cell populations could be
used for cardiac repair. Each cell type has its own profile ofadvantages, limitations, and practicability issues in specificclinical settings (23).
Endothelial Progenitor Cells (EPCs)
EPCs have originally been defined by their cell surface
expression of the hematopoietic marker proteins CD133 andCD34 and the endothelial marker vascular endothelial growth
factor receptor-2, and their capacity to incorporate into sites ofneovascularization and to differentiate into endothelial cells insitu (23). Endothelial progenitor cells are a subpopulation within
the bone marrow that can promote vasculogenesis leading tovessel regrowth, thereby improving oxygenation to tissue after
damage. Preclinical trials indicate that endothelial progenitor cells
contribute to 1–25% of vessel formation after ischemic injury forseveral diseases. Additionally, endothelial progenitors may secrete
growth factors and paracrine signals that prevent cardiac myocyte

Received June 2010. Accepted July 2010. Address for correspondence: Carmen Tatu MD, PhD, Physiology Department, “Victor Babes” University of
Medicine and Pharmacy Timisoara, 2A Eftimie Murgu Square, Timisoara 300041, Romania, e-mail: carmen.tatu@umft.ro4
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Fiziologia - Physiology  2010.20.3 (67)4cell death. These cells are capable of vascular differentiation andsecretion of growth factors promoting angiogenesis and increasing
blood supply to the damaged heart (3). Improved neovascularization can play a crucial role in rescuing the failing ischemic
myocardium. Transdifferentiation of EPCs and whether this transdifferentiation plays a main role in the therapeutic benefit of EPCs
in recovering cardiovascular function is controversial. Endothelial
progenitor cells are potential candidates for cardiovascular repair,as they have been shown to mediate neovascularization at theinfarct border zone. Bone marrow–derived hemangioblasts andperipheral blood–derived monocytes can differentiate into EPCs,
which in turn can differentiate into functional endothelial cells(5). Soon after the discovery of EPCs, it was realized that tissue
ischemia and cytokines can mobilize EPCs, thereby contributing
to neovascularization of the ischemic tissue (21). Several recent
studies demonstrated transdifferentiation of human peripheral
blood CD34+ cells into endothelial lineage cells with incorporation
into blood vessels (8,18). However, other investigators claim thatbone marrow-derived cells do not transdifferentiate or incorporate
into vessel walls (8,26). These discrepancies may be due to thedifference in cell types, the use of different animal models used
to look for transdifferentiation, or the application of less or more
rigorous criteria to define transdifferentiation. Considering thesecontroversies, more-sophisticated methods with rigorous criteria,such as z-stacked confocal imaging and long-termin vivo tracking,are needed to elucidate the role of transdifferentiation of EPCs ormore fundamentally to address the nature and identity of EPCs.
Therapeutic mechanisms of EPCs in cell therapy in vivo also
remain to be better characterized.
Other studies have demonstrated that EPCs can mediatevasculogenesis under diverse pathophysiologic conditions likewound healing, limb ischemia, and after myocardial infarction,
under the influence of the released cytokines (5). These EPCs
are characterized by their ability to take up acetylated low-density
lipoprotein and are CD34+, CD133+, Flk-1+. Quite interestingly,
EPCs share many common characteristics and overlapping expression of surface markers with diverse cell types. For instance,
EPCs derived from the bone marrow as well as the circulating
EPCs demonstrate the expression of endothelial markers Flk-1,
Tie-2, VE-cadherin, CD34, CD146, and E-selectin, just as do themature circulating endothelial cells. Moreover, the uptake of acetylated LDL, a property associated with EPCs, can be mimickedby monocytes and certain hematopoietic cells. However, despite
these complexities related to the purification and characterizationof EPCs (22), EPCs are the most commonly studied candidates
in clinical trials for reendothelialization and neovascularizationof the myocardium. Differentiation of stem cells into functional
cardiac myocytes is a main goal of stem cell therapy.
Mesenchymal Stem Cells (MSCs)
MSCs represent a rare population of CD34- and CD133- cells
present in bone marrow stroma, 10-fold less abundant than hematopietic stem cells and other mesenchymal tissues. MSC arepresent in adult tissues including the bone marrow and adipose
tissues, MSCs are characterized by an absence of hematopietic
2010.20.3 (67)  Fiziologia - Physiology

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stem cells markers. They can be isolated and expanded easily
and, can transdifferentiate into functional cardiomyocytes anda variety of other cells, resulting in an improvement in left ventricular function and remodeling. MSCs can readily differentiate
into osteocytes, chondrocytes, and adipocytes. Differentiation of
MSCs to cardiomyocyte-like cells has been observed under specific culture conditions and after injection into healthy or infarcted
myocardium in animals. The magnitude of transdifferentiation iscurrently under investigation, but accumulating evidence supports the active engraftment and differentiation of transplanted
human MSCs within the healing myocardium in sheep (7). Thisfinding has been corroborated in vitro, with cardiogenic MSC
guidance demonstrating a capacity for sarcomerogenesis andelectromechanical coupling. Noninvasive multimodality imaging
indicates that therapy after myocardial infarction with allogeneic
MSCs promotes active cardiac regeneration in vivo. When injected into infarct tissue, MSCs may enhance regional wall motion
and prevent remodeling of the remote, noninfarcted myocardium. Little is known about the effects of MSCs on myocardial
perfusion (23). It is interesting to note however, that cultured
MSCs secrete angiogenic cytokines, which improve collateralblood flow recovery in a murine hind limb ischemia model. One
group of reasercher demonstrated that, in a model of murine
hindlimb ischemia, MSC transplantation enhanced tissue repair
via secretion of multiple cytokines, including vascular endothelial
growth factor (VEGF), fibroblast growth factor-2 (FGF-2), andplacental growth factor (PlGF), rather than incorporation of MSCs
into new or remodeling tissues (8,11,12). Others reported that
MSC viability can be enhanced with gene transfection (8,17) withgreater therapeutic effect on the injured myocardium. Recently, it
was reported that direct MSC injection by using catheter is safe,
reduces fibrosis, and augments myocardial regeneration (8). The
advantage of MSCs for potential clinical application lies in theease of acquisition and isolation, high expansion capability inculture, and low immunogenicity. The low immunogenicity canrender MSCs more-attractive candidate cells for treatment of
myocardial infarction because of their capability for modulating
inflammatory reactions in acute myocardial infarction and theutility of the allogeneic approach. This extensive capacity forclinical scale expansion in vitro has facilitated the developmentof preclinical models as well as clinical studies designed to assess the safety, feasibility, and efficacy of MSC transplantation
in a variety of diseases. However, the use of animal serum for
culture expansion restricts the growth of pilot clinical trials with
MSCs in humans (8). Because MSC clones can be expanded
in vitro, and reportedly have a low immunogenicity, they might
be used in an allogeneic setting in the future. They can also
modulate immune responses. It has been argued that these
multipotent MSCs can mediate an immunosuppressive effect.
Although MSCs do express MHC class I, they lack the B7 costimulatory molecule and escape recognition via both CD4+ T
helper (naive and memory) and CD8+ cytotoxic T cell subsets.
Moreover, MSCs can induce a bias toward T helper 2 rather
than T helper 1 (two stable differentiation states of T helper that
produce different lymphokines and different effector functions
5via divergent signaling mechanisms), owing to reduced IFN-g
secretion (4,5,13). Surprisingly, the expression of surface markers associated with lymphocyte activation, like CD25, CD38,
and CD69, have been shown to decrease in the presence of
MSCs (5,13). Therefore, they can inhibit lymphocyte proliferationby B-cell mitogens. Adipose tissue derived from the embryonicmesenchyme contains MSCs and endothelial progenitor cells aswell as adipose cells. Experimental data suggest that adipose
tissue – derived cells may transdifferentiate into cells with thecharacteristics of cardiomyocytes and perhaps blood vessels,
or at least neovascular tissue. Adipose cells are attractive because access to them is easy and they are not in short supply
in most societies. Nonetheless, additional characterization anddemonstration of efficacy in animal models are needed before
a clinical role for these cells can be established (7).
Hematopoietic Stem Cells (HSCs)
HSCs probably define a class of the oldest progenitor cells
that are still in use for cell-based therapeutics. Initial studies
using HSCs can be traced back to the 1970s. A hematopoietic
stem cell (HSC) is a cell that can self-renew and give rise to all
blood cells. The transplantation of a single HSC can constituteall hematopoietic cells in an organism, and a HSC fulfills therequirements for being a stem cell (i.e., selfrenewal capacityand potential to generate differentiated progeny)(8). Commonly
identified by the expression of CD34+ and CD133 cell surface
antigens, HSCs have been studied extensively and used clinically for bone marrow transplant for a variety of hematologic
disorders. Cells capable of assuming an endothelial phenotype
have been identified in the blood and bone marrow. Endothelial progenitor cells, a heterogenous population of cells that
also reside predominantly in the bone marrow, likely promote
neovascularization by secreting proangiogenic growth factors
and by stimulating re-endothelialization; both functions could
contribute to vascular homeostasis and perhaps myogenesis(7,19). The potential regenerative role of these cell types isbased on findings in experimental animal models, not on clinicalexperience. The use of HSCs to repair an infarcted myocardium
in a murine model has been investigated (8,16). Recent studies
have suggested the possibility that specific stem cell populations
derived from murine bone marow could regenerate most of the
key elements of myocardium and ameliorate ischemic cardiac
dysfunction. Studies involving an injection of Lin-/c-Kit+ HSCs
in the periinfarct area of female C57BL/6 mice showed de novo
cardiac regeneration (68% of the infarct volume) (16).
The more-versatile potential of bone marow-derived HSCs
has been documented with the use of side-population cells (8)and with lineage-negative, c-kit–positive cells (cells that express
a specific protein known as a tyrosine kinase, which adds phosphate groups to the amino acid tyrosine in other proteins); bothof which differentiated into endothelial cells, vascular smoothmuscle cells, and cardiomyocytes in a murine myocardial infarction model (15,16). In another attempt, intravenous injectionof GFP-tagged Lin-ScaI+ HSCs in murine model revealed intercellular connections of the administered cells with the resident
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cardiomyocytes. In the same study, Lin-CD34+CD38- human
HSCs demonstrated intercellular connections with the resident
cardiomyocytes in immunodeficient mice (NOD/SCID) (9,16).
However, subsequent studies failed to show any differentiationof the labeled HSCs to cardiomyocytes or endothelial cells in theinfarcted myocardium (16). It remains to be determined whetherbone marrow cells or cell lines from other sources will be favoredfor myocyte formation in the future. Unless the efficacy of existing bone marrow cell preparations is substantially enhanced,
attention will likely shift to other sources of cells. However, otherstudies suggested that HSCs do not readily acquire a cardiac
phenotype in the injured heart, despite the improvement inventricular function in the HSC-transplanted group (15). Thesestudies were all performed with murine HSCs, and currently
no studies have been performed with the human counterpartof these stem cells. In terms of clinical application, the lack ofthese HSCs in bone marrow and the unavailability of cultureexpansion techniques may limit the therapeutic application of
HSCs in ischemic heart diseases.
Embryonic Stem Cells (ES)The embryonic stem cell, derived from the inner mass ofthe developing embryo during the blastocyst stage, has thegreatest potential for organ regeneration and is the prototypicalstem cell (7,20). ESCs posses all the qualities of a prototypicalstem cell: clonal expansion, self-renewal and multipotentiality(5). Embryonic stem (ES) cells are totipotent stem cells. Under
specific culture conditions, ES cells differentiate into multicellular
embryoid bodies containing differentiated cells from all threegerm layers including cardiomyocytes; however, their innate
aptitude for pluripotent proliferation also presents an increased
risk of teratoma (7). Transcriptional and functional profiling of
human ES cell (hESC)-derived cardiomyocytes revealed asimilarity to those found in 20-week fetal heart cells, thereby
appearing as a strong candidate for the in vivo replacementof apoptotic cardiomyocytes in the failing myocardium (2,5).
Human ES cell–derived cardiomyocytes display structural andfunctional properties of early-stage cardiomyocytes that couple
electrically with host cardiomyocytes when transplanted into
normal myocardium (10,23). In electrophysiologic studies of
ESC-cardiomyocytes exhibit diverse electrophysiologic signatures that include cells with distinct nodal/pacemaker-, atrial, andventricular-like action potential properties (25). In theory, infinite
numbers of cardiomyocytes could be obtained from human
ES cell clones. In animal models of experimental myocardial
infarction and nonischemic cardiomyopathy, embryonic stem celltransplant has resulted in a remarkable improvement in cardiac
function and structure, and the cells appear to be electricallyintegrated (7,24). At least at early stages of in vitro maturation,
ESC-cardiomtocytes exhibit electrophysiologic characteristics
of primitive myocardium, including a comparatively low action
potential upstroke velocity and a depolarized maximum diastolicpotential, but these parameters appear to transition toward somewhat more mature values with increasing duration in culture(25). Voltage-clamp studies indicate that several cardiac-specificFiziologia - Physiology  2010.20.3 (67)6

currents are present in these cells, including fast sodium current,
L-type calcium current, pacemaker currents, as well as transient
outward and inward rectifier potassium currents (14,25).
Acknoledgements:
The work in this paper was supported by PNCDI-II –Parteneriate, project number 41-075/2007.
REFERENCES

1. Bonaros N, Rauf R, Schachner T, Laufer G, and Kocher A, Enhanced cell therapy for ischemic heart disease, Transplantation,
2008;86:1151–1160.
2. Cao F, Wagner RA, Wilson KD, Xie X, Fu JD, Drukker M, Lee A, Li
RA, Gambhir SS, Weissman IL, Robbins RC, Wu JC, Transcriptionaland functional profiling of human embryonic stem cell-derived cardiomyocytes, PLoS ONE, 2008;3:e3474.
3. Collins JM, Russell B, Stem Cell Therapy for Cardiac Repair, J
Cardiovasc Nurs. 2009 ; 24(2): 93–97.
4. Dazzi F and Marelli-Berg FM. Mesenchymal stem cells for graftversus-host disease: close encounters with T cells, Eur J Immunol,
2008;38:1479–1482.
5. Debayon P, Samson M, Nilanjana M, Mesenchymal Stem Cell:
Present Challenges and Prospective Cellular Cardiomyoplasty Approaches for Myocardial Regeneration, Antioxidants & Redox Signaling,2009;11(8):1841-1855.
6. Forrester JS, White AJ, Matsushita S, Chakravarty T, Makkar RR,
New paradigms of myocardial regeneration post-infarction: tissuepreservation, cell environment, and pluripotent cell sources. JACC
Cardiovasc Interv. 2009;2(1):1-8.
7. Gersh BJ, Simari RD, Behfar A, Terzic CM, MD, Terzic A,
Cardiac Cell Repair Therapy: A Clinical Perspective, Mayo Clin
Proc.2009;84(10):876-892.
8. Hyongbum K, Sung-Whan K, Douglas N, Sinae K, Young-sup Y,
Cell Therapy with Bone Marrow Cells for Myocardial Regeneration,
Antioxidants & Redox Signaling, 2009, 11;8;1897-1911.
9. Ishikawa F, Shimazu H, Shultz LD, Fukata M, Nakamura R, Lyons B,
Shimoda K, Shimoda S, Kanemaru T, Nakamura K, Ito H, Kaji Y, Perry
AC, Harada M, Purified human hematopoietic stem cells contributeto the generation of cardiomyocytes through cell fusion, FASEB J,
2006;20:950–952.
10. Kehat I, Khimovich L, Caspi O, Gepstein A, Shofti R, Arbel G, Huber
I, Satin J, Itskovitz-Eldor J, Gepstein L, Electromechanical integrationof cardiomyocytes derived from human embryonic stem cells, Nat
Biotechnol, 2004;22:1282–1289.
11. Kinnaird T, Stabile E, Burnett MS, Lee CW, Barr S, Fuchs S, andEpstein SE, Marrow-derived stromal cells express genes encoding
a broad spectrum of arteriogenic cytokines and promote in vitro andin vivo arteriogenesis through paracrine mechanisms, Circ Res,
2004;94:678–685.
12. Kinnaird T, Stabile E, Burnett MS, and Epstein SE, Bonemarrowderived cells for enhancing collateral development: mechanisms, animal
data, and initial clinical experiences, Circ Res, 2004;95: 354–363.
13. Le Blanc K and Ringden O, Immunomodulation by mesenchymal
stem cells and clinical experience, J Intern Med, 2007;262: 509–525.
14. Mummery C, Differentiation of human embryonic stem cells to
cardiomyocytes: role of coculture with visceral endoderm-like cells,
Circulation, 2003; 107(21):2733–40.
15. Murry CE, Soonpaa MH, Reinecke H, Nakajima H, Nakajima HO,
Rubart M, Pasumarthi KB, Virag JI, Bartelmez SH, Poppa V, Bradford
G, Dowell JD, Williams DA, Field LJ, Haematopoietic stem cells do nottransdifferentiate into cardiac myocytes in myocardial infarcts, Nature,
2004;428:664–668.
16. Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B,
Pickel J, McKay R, Nadal-Ginard B, Bodine DM, Leri A, Anversa
P, Bone marrow cells regenerate infarcted myocardium, Nature,2001;410:701–705.
17. Ott HC, Matthiesen TS, Goh SK, Black LD, Kren SM, Netoff TI, andTaylor DA, Perfusion-decellularized matrix: using nature’s platform toengineer a bioartificial heart, Nat Med, 2008;14: 213–221.
18. Peters BA, Diaz LA, Polyak K, Meszler L, Romans K, Guinan EC,
Antin JH, Myerson D, Hamilton SR, Vogelstein B, Kinzler KW, Lengauer
C, Contribution of bone marrow-derived endothelial cells to human tumor
vasculature, Nat Med, 2005;11: 261–262.
19. Rehman J, Li J, Orschell CM, March KL, Peripheral blood “endothelial progenitor cells” are derived from monocyte/macrophages and secreteangiogenic growth factors, Circulation, 2003;107(8):1164-1169.
20. Segers VF, Lee RT, Stem-cell therapy for cardiac disease, Nature,2008; 451(7181):937-942.
21. Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney
M, Magner M, Isner JM, Asahara T, Ischemia- and cytokine-induced
mobilization of bone marrow-derived endothelial progenitor cells for
neovascularization, Nat Med, 1999;5:434–438.
22. Timmermans F, Plum J, Yoder MC, Ingram DA, Vandekerckhove B,
Case J, Endothelial progenitor cells: identity defined? J Cell Mol Med,
2008;13: 87–102.
23. Wollert KC and Drexler H, Clinical Applications of Stem Cells for
the Heart, Circ. Res. 2005;96;151-163.
24. Yamada S, Nelson TJ, Crespo-Diaz RJ, et al, Embryonic stem
cell therapy of heart failure in genetic cardiomyopathy, Stem Cells,
2008;26(10):2644-2653.
25. Zhu WZ, Hauch K, Xu C, Laflamme MA, Human Embryonic StemCells and Cardiac Repair, Transplant Rev, 2009;23(1):53–68.
Ziegelhoeffer T, Fernandez B, Kostin S, Heil M, Voswinckel R, Helisch A, Schaper W, Bone marrow-derived cells do not incorporate into
the adult growing vasculature, Circ Res, 2004;94: 230–238.

CELULELE STEM SI REGENERAREA CARDIACAREZUMAT
Terapiile celulare au fost dezvoltate ca o modalitate atractiva de a repara miocardul afectat de diverse injurii. O mare varietate depopulatii de celule stem si celule progenitoare ar putea fi utilizate pentru repararea cardiaca. Fiecare dintre aceste tipuri celulareprezinta avantaje, limitari si aspecte legate de aplicarea practica in anumite circumstante clinice. Aceste celule prezinta diferentesemnificative in ceea ce priveste locul de origine, anatomia si functionalitatea, fiind caracterizate prin prezenta markerilor de suprafata,a factorilor de transcriptie si a expresiei proteice. Un alt aspect care le diferentiaza este capacitatea acestora de a forma unul sau
mai multe tipuri celulare diferentiate.
Cuvinte cheie: celule stem mezenchimale, celule progenitoare endoteliale, celule stem hematopoetice, celule stem embrionare
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APELIN INVOLVEMENT ON ANGIOTENSIN II – INDUCEDVENOCONSTRICTION IN OBESE RATSMIHAI ANA CAMELIA1, DINCA MARIA1, DUMITRIU IRINA LUCIANA1, GURZU MIHAELABEATRICE2, SLĂTINEANU SIMONA MIHAELA1, FOIA LILIANA GEORGETA2, VATA LUMINITA
GINA1, COJOCARU ELENA1, PETRESCU GHEORGHE1
1
Functional Sciences Department, Faculty of Medicine,2
Department of Semiology and Prevention, Faculty of Dental Medicine,“Gr. T. Popa” University of Medicine and Pharmacy, Iasi, Romania

ABSTRACT

Apelin (AP), the endogenous ligand of the recently de-orphanized APJ receptor is a relatively newly identified adipokine. Despite thishigh level of expression of both AP and APJ in the vascular wall, the vasomotor effects of AP are still under investigation. This study
was undertaken to evaluate comparatively the in vitro effects of apelin on angiotensin II (Ang II) – induced constriction of various
veins from normal vs. obese rats. Rings of the inferior vena cava (IVC), portal vein (PtV), femoral vein (FV), and pulmonary vein (PV)from male Sprague Dawley rats (control rats), obese prone (OP) rats and obese resistant (OR) rats were mounted between wires
and isometric contraction induced by Ang II (1µM) was measured. These results showed that reactivity for Ang II was increased in
IVC and FV in OR. The AP13 treatment decreased the Ang II- induced contractions by more than 30% on IVC and PtV from OR. On
OP, the F13A treatment increased the contraction induced by Ang II on all studied veins. These results suggested that AP could playprotective roles by antagonizing Ang II – induced venoconstriction.
Key words: apelin, angiotensin II, veins, obesity, rats

INTRODUCTION
The last decades has brought many data about white adipose
tissue capacity to secrete hormones, named adipokines, which
could be involved on the regulation of vascular reactivity and themodulation of local inflammatory responses (1). Taking into accountthe published studies about the obesity roles in the developmentof pulmonary diseases we studied the apelin implications on Ang II
contractile effects on pulmonary veins from male Sprague Dawley
rats (control rats), obese prone (OP) rats and obese resistant (OR)rats.
We have recently shown that apelin (AP), the endogenous ligandof the recently de-orphanized APJ receptor and a newly identified
adipokine, could have vasodilatatory and NO-dependent effects (2).
Even more, apelin modulated vasoconstrictor tone in rat pulmonary
vessels (3) and reduced the lipopolysaccharide-increased pulmonary
permeability in rats (4).
This study aims to investigate the role of apelin/APJ receptors
system in agonist-mediated pulmonary venoconstriction in normal
and obese rats, in order to accomplish a greater understandingof the involvement of adipokine in the regulation of pulmonary
vascular tone.
MATERIAL AND METHODSThe experiments were conducted in Sprague Dawley rats
(control rats), the OP-CD (Obese Prone) and the OR-CD (Obese
Resistant) that were obtained from Charles River Laboratories(Wilmington, MA, USA). The animals were kept under conventional
laboratory conditions of temperature, humidity and light, and allowed

free access to water and standard rat chow (for control rats) or ahigh fat diet (D12266B, 32.5% fat, Research Diets, NJ, USA). Rats
were given daily intraperitoneal injections of AP13 or F13A (100nmol/kg) for 2 weeks (5). After finishing the protocol, the inferior
vena cava (IVC), portal vein (PtV), femoral vein (FV), and pulmonaryvein (PV) veins were quickly removed, cleaned and cut into 1-2 mmwide rings as previous described (6,7). Individual rings were thenmounted in a MYO-01 MYOGRAPH SYSTEM (Experimetria LTD.,
Budapest, Hungary) and changes in vessel tension were recorded
and analyzed by ISOSYS data acquisition system (Experimetria
LTD., Budapest, Hungary). The tissue organ bath contained theKrebs–Henseleit solution containing (mM): NaCl 118, KCl 4.8,
CaCl2 2.5, MgSO4 1.6, KH2PO4 1.2, NaHCO3 25, glucose 5.5.
The Krebs-Henseleit buffer was maintained at 37°C, and bubbled
continuously with a mixture of 95% O2 and 5% CO2 (pH=7.2–7.4).
A resting tension of 0.5g for JV, FV and PtV, and 0.2g for PV wasapplied to each ring and then allowed to equilibrate for 45-60 minutes
before starting the experiment. The bathing medium was renewed
every 15 minutes. After the equilibration period, vessel rings were
initially stimulated twice with 40 mM KCl as a standard stimulus. The
functional integrity of the endothelium was assessed by testing thedegree of relaxation produced by adding 10 µM acetylcholine (ACh)to KCl pre-contracted rings. The rings that produced less than 70 %relaxation in response to ACh were discarded. After re-equilibration,
the rings were stimulated with the Ang II (1 µM) and the peak
contractile responses were measured. Results are expressed aspercentage of control contraction induced by 40 mM KCl (mean ±S.E.M, n=6). The statistical significance was tested using one-way

Received May 2010. Accepted June 2010. Address for correspondence: PETRESCU Gheorghe, MD, PhD, Functional Sciences Department, Faculty of Medicine,
Gr T Popa University of Medicine and Pharmacy, 700115, Iasi, Romania, e-mail: petrescugh@yahoo.com

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Fiziologia - Physiology  2010.20.3 (67)8analysis of variance (ANOVA), completed by the Bonferroni method(SigmaStat software, Jandel Corporation). p<0.05 was considered
statistically significant.
Ang II (rat), KCl, ACh, were all obtained from Sigma-Aldrich Inc.
(Germany). Apelin and the antagonist Apelin-13 (Ala13, F13A) werepurchased from Phoenix Europe GmbH (Germany). All the othercompounds used were of analytical grade.
RESULTSThe F13A treatment amplified the Ang II – induced contractions
on IVC from control rats (88.78±5.71 vs. 62.97±7.91) and OP rats
(96.18±7.16 vs. 68.72±6.02). On OR rats Ang II contractille effects
were higher (90.84±7.25 vs. 62.97±7.91) as compared with control
rats. IVC from AP13 treated OR rats had a lower contractile response
to Ang II as compared with untreated OR rats (48.51±10.34 vs.
90.84±7.25).

Fig. 1. Contractions induced by Ang II on IVC from control rats (SD), obese
resistant (OR) rats and obese prone (OP) rats untreated or treated with AP13
(SD+AP13, OR+AP13, OP+AP13) or F13A (SD+F13A, OR+F13A, OP+F13A). *:p<0.05 as compared with SD from the same protocol.

Fig. 2. Contractions induced by Ang II on PtV from control rats (SD), obese
resistant (OR) rats and obese prone (OP) rats untreated or treated with AP13
(SD+AP13, OR+AP13, OP+AP13) or F13A (SD+F13A, OR+F13A, OP+F13A). *:p<0.05 and **: p<0.01 as compared with SD from the same protocol.

Both the AP13 treatment and F13A treatment significantly
decreased (by an average of 30%) and increased (by an averageof 20%), respectively, the Ang II – induced contractions on portal
vein from SD, OR and OP rats.
On FV (Figure 3), there are significant differences between
Ang II – induced contractions on OR vs. SD rats (45.42±3.61 vs.
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21.11±2.18) and on OP vs. OR rats (20.28±3.14 vs. 45.42±3.61).
The F13A treatment increased the Ang II effects on OP rats
(41.21±5.11 vs. 20.28±3.14).

Fig. 3. Contractions induced by Ang II on FV from control rats (SD), obese
resistant (OR) rats and obese prone (OP) rats untreated or treated with AP13
(SD+AP13, OR+AP13, OP+AP13) or F13A (SD+F13A, OR+F13A, OP+F13A). *:p<0.05 as compared with SD from the same protocol.

Neither AP13 nor F13A modified the Ang II – induced contractions on PV (Figure 4) of SD rats. On the contrary, on OR rats theAP13 treatment and F13A treatment significantly decreased (bymore than 50%) and increased (by than 30%), respectively. On OP
rats only the F13A treatment significantly modified the Ang II – induced contractions (28.55±2.41 vs. 20.90±1.38). On the other hand,even if there was no difference between Ang II – induced contractions
on OP and OR rats, after the AP13 treatment, the responses of PV
to Ang II was significantly lower on OR rats as compared with OPrats (10.50±1.24 vs. 19.82±1.79).

Fig. 4. Contractions induced by Ang II on PV from control rats (SD), obese
resistant (OR) rats and obese prone (OP) rats untreated or treated with AP13
(SD+AP13, OR+AP13, OP+AP13) or F13A (SD+F13A, OR+F13A, OP+F13A). *:p<0.05 and **: p<0.01 as compared with SD from the same protocol.

DISCUSSIONS
Apelin is the endogenous ligand of recently de-orphanised APJ
receptors. In rat tissues, the APJ receptor and apelin mRNAs were
found in the lung, heart, skeletal muscle, kidney, brain and liver (8).
Even more, in situ hybridisation histochemistry studies revealed
intense APJ receptor gene expression in the parenchyma of the lung(9). In rats, the highest expression of APJ mRNA was detected in
the lung, suggesting that APJ and its ligand play an important role in
the pulmonary system (10). Until now there are no references about9

functional importance of AP – APJ pulmonary system. Considering
our data about NO-dependent inhibitory effects of AP13 on Ang
II-induced venoconstriction, we previous showed that AP13 could
stimulate NO synthesis in both airways and pulmonary vessels (11).
More recently, apelin has been described as an adipocyte-secreted
factor (adipokine), markedly up-regulated in obesity. By acting as
circulating hormone or paracrine factor, adipokines are involved inphysiological regulations (fat depot development, energy storage,
metabolism or eating behavior) or in the promotion of obesityassociated disorders as vascular dysfunctions (12).
Taking into account (i) the increased of AP level in obesity (12)(ii) the functional importance of tissue synthesized AP (3) and (iii)
the impact of veins dysfunction on vascular beds circulation (13,14)
we comparatively assessed the isolated veins reactivity to Ang II on
control rats, OR rats and OP rats untreated or treated with either
AP13 or F13A (the blocker of APJ receptors).
The Ang II – induced contractions were significantly increased on
OP and OR rats, as compared with SD rats, only for FV (Figure 3).
Either APJ receptors stimulating or APJ receptors blocking prevent
differences between OP/OR rats and SD rats, emphasizing importance of AP/APJ receptors system in modulation of obesity – induced
modulation of FV reactivity. Administration of AP13 decreased Ang
II induced contraction of FV and PV (on OR rats) and PtV (on all
experimental models). Blocking of APJ receptors by ip administrationof F13A increased the venous reactivity for all studied veins but only
on OP rats. On control (SD) rats the F13A effects were significantly
only for IVC (Figure 1) and PtV (Figure 2). On OR rats the F13A
effects were significantly only for PtV (Figure 2) and PV (Figure4). These data suggested that on OP rats were the AP levels are
increased (12); the AP/APJ receptors system could have a vasorelaxant role. For PtV, the AP13 or F13A effects are significantly on all
studied experimental models sustaining the importance of the AP/
APJ receptors system in regulation of portal circulation.
Taking into account the well known contribution of obesity in the
progression of various venous diseases (15, 16) our data could suggest the involvement of apelin/APJ receptor system in dysregulationof venous tone and reactivity as a new component of obesity related
induced vascular diseases.
CONCLUSION
These results suggested that AP/APJ system could play protective roles by antagonizing Ang II – induced venoconstriction mainly onobese rats. The functional importance of AP/APJ system on venous
tone regulation is determined by vascular beds studied.
Acknowledgements
These studies were partially supported by funds from the
Romanian National Ministry of Education and Research (Grant
CNCSIS PN-II-ID-PCE-2007-1 No. 1273/2007 and PN–II–ID–
PCE–2008–2 No. 2670/2008). We gratefully acknowledge thesupport of Dr Cristin Coman, Senior Scientist, Head of Experimental Unit Baneasa, INCDMI Cantacuzino, Bucharest, for help
with rats’ acquisition.

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REFERENCES
1. Gurzu B, Zugun FE, Costuleanu M, Mihaescu T, Carasevici E, Petrescu G. Adipokines involvement in lung function. Rev Med Chir Soc
Med Nat Iasi. 2008;112(3):719-725.
2. Gurzu B, Petrescu BC, Costuleanu M, Petrescu G. Interactions between apelin and angiotensin II on rat portal vein. J Renin Angiotensin
Aldosterone Syst. 2006;7(4):212-216.
3. Andersen CU, Markvardsen LH, Hilberg O, Simonsen U. Pulmonary
apelin levels and effects in rats with hypoxic pulmonary hypertension.
Respir Med. 2009;103(11):1663-1671.
4. Petrescu BC, Gurzu B, Iancu RI, Indrei A, Dumitriu I, Chelaru L,Slătineanu SM, Petrescu G, Costuleanu M. Apelin effects on lipopolysaccharide-increased pulmonary permeability in rats. Rev Med Chir
Soc Med Nat Iasi. 2010;114(1):163-169.
5. Frier BC, Williams DB, Wright DC. The effects of apelin treatment
on skeletal muscle mitochondrial content. Am J Physiol Regul Integr
Comp Physiol. 2009 Dec;297(6):R1761-1768.
6. Gurzu B, Costuleanu M, Slatineanu SM, Ciobanu A, Petrescu G.
Are multiple angiotensin receptor types involved in angiotensin (1-7)
actions on isolated rat portal vein. J Renin Angiotensin Aldosterone
Syst. 2005 Sep;6(2):90-95.
7. Raffetto JD, Qiao X, Beauregard KG, Khalil RA. Estrogen receptormediated enhancement of venous relaxation in female rat: implications in sex-related differences in varicose veins. J Vasc Surg. 2010
Apr;51(4):972-981.
8. Katugampola SD, Maguire JJ, Matthewson SR, Davenport AP. [(125)
I]-(Pyr(1))Apelin-13 is a novel radioligand for localizing the APJ orphan
receptor in human and rat tissues with evidence for a vasoconstrictorrole in man. Br J Pharmacol. 2001;132(6):1255-1260.
9. O’Carroll AM, Selby TL, Palkovits M, Lolait SJ. Distribution of mRNA
encoding B78/apj, the rat homologue of the human APJ receptor, andits endogenous ligand apelin in brain and peripheral tissues. Biochim
Biophys Acta. 2000;1492(1):72-80.
10. Hosoya M, Kawamata Y, Fukusumi S, Fujii R, Habata Y, Hinuma
S, Kitada C, Honda S, Kurokawa T, Onda H, Nishimura O, Fujino M.
Molecular and functional characteristics of APJ. Tissue distribution ofmRNA and interaction with the endogenous ligand apelin. J Biol Chem.
2000;275(28):21061-21067.
11. Dumitriu IL, Petrescu BC, Gurzu MB, Gurzu B, Slatineanu SM. New
insight into nitric oxide involvement in regulation of airways smoothmuscle tone. Rev Med Chir Soc Med Nat Iasi. 2007; 111(2): 454-459
12. Carpéné C, Dray C, Attané C, Valet P, Portillo MP, Churruca I,
Milagro FI, Castan-Laurell I. Expanding role for the apelin/APJ system
in physiopathology. J Physiol Biochem. 2007;63(4):359-373.
13. Rocha SF, Adams RH. Molecular differentiation and specializationof vascular beds. Angiogenesis. 2009;12(2):139-147.
14. Gurzu B., Petrescu B.C., Dumitriu Irina Luciana, Mihai A.C., Slatineanu S.M. Local generation of angiotensin in isolated rat pulmonary
vessels. In: Strbak V (Ed). Selected Papers from the Joint Meeting of
the Slovak Physiological Society, the Physiological Society and theFederation of European Physiological Societies, Bratislava, Slovak
Republic, September 11- 14, 2007. Medimond International Proceedings 2007; 67-71.
15. Björntorp P. Obesity and adipose tissue distribution as risk factors for the development of disease. A review. Infusionstherapie.
1990;17(1):24-27.
16. Dela Cruz CS, Matthay RA. Role of obesity in cardiomyopathy andpulmonary hypertension. Clin Chest Med. 2009;30(3):509-523, ix.

Fiziologia - Physiology  2010.20.3 (67)10

IMPLICAREA APELINULUI ÎN VENOCONSTRICŢIA INDUSĂ DE
ANGIOTENSINĂ II LA ŞOBOLANII OBEZIREZUMAT

Apelinul (AP), ligand endogen al receptorilor APJ recent de-orfanizaţi este o adipokină nou identificată. Cu toate că s-a evidenţiat
la nivelul peretelui vascular o expresie crescută a apelinului şi a receptorului său, APJ, efectele vasomotorii ale apelinului nu s-au
descifrat în totaltate. Acest studiu a fost efectuat pentru a evalua comparative efectele in vitro ale AP asupra veoconstricţiei indusă
de angiotensina (Ang II) îndiferite teritorii venoase de la şobolanii normali vs. obezi. Inele de venă cavă inferioară (VCI), venă portă
(VPt), venă femurală (VF) şi venă pulmonară (VP) de la şobolani Sprague Dawley (lot de control), şobolani predispuşi la obezitate(OP), şobolani rezistenţi la obezitate (OR) au fost montate în miograf şi a fost măsurată contracţia izometrică indusă de Ang II. Rezultate obţinute au arătat că efectele venomotorii ale Ang II au fost mai crescute la nivelul VCI şi VF la OR. Tratamentul cu AP13 ascăzut contracţia indusă de Ang II cu mai mult de 30% la nivelul VCI şi PtV. La OP, tratamentul cu F13A a crescut contracţia indusă
de Ang II în toate inelele venoase studiate. Aceste rezultate sugerează că AP poate avea un roluri protective prin antagonizarea
venoconstricţiei indusă de Ang II.
Cuvinte cheie: apelin, angiotensina II, vene, obezitate, şobolani

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CONTRIBUTION OF INTRAPULMONARY RENIN
ANGIOTENSIN SYSTEM ON MODULATION OF RATPULMONARY ARTERIES REACTIVITYMARIA DINCĂ1, IRINA LUCIANA DUMITRIU1, SIMONA MIHAELA SLĂTINEANU1, LUMINIŢA
GINA VÂTĂ1, MIHAELA BEATRICE GURZU2, GHEOGHE PETRESCU1
1
Discipline of Physiology –Functional Exploration, Department of Functional Sciences, Faculty of Medicine
2
Discipline of Immunology and General Pathology, Faculty of Dental Medicine
University of Medicine and Pharmacy “Gr T Popa” Iasi, Romania

ABSTRACT
Published data sustain the involvement of urotensin II (U_II) in the pathogenesis of pulmonary vascular diseases. Taking into consideration the importance of renin angiotensin system (RAS) into regulation of pulmonary vessels reactivity we investigated if themodulation of intrapulmonary RAS could modify the vasomotor effects U_II on rat pulmonary artery (RPA) rings from ovalbumin
sensitized (OSR) or pulmonary hypertensive (PHR) rats. U_II induced contractions were recorded and data obtained from OSR andPHR were compared with those from normal rats (NR). In order to modulate intrapulmonary RAS, the losartan, chymostatin andamastatin were administered by tracheal instillation. The U_II – induced contractions are higher on RPA from OSR (with at least a
fifth) and PHR (with more than 30%) as compared with NR. The blocking of intrapulmonary RAS by tracheal administration of losartan
decreased the differences between OSR or PHR and NR. Reducing of angiotensin degradation significantly amplified U_II contractile
effects on both experimental models. These data sustain the implication of intrapulmonary RAS on increasing reactivity of pulmonary
arteries during some pathological situations and suggest possible positive effects of intrapulmonary administrated angiotensin II type
1 receptor antagonists on pulmonary vessels diseases.
Keywords: urotensin II, angiotensin, pulmonary artery, asthma, pulmonary hypertension, rats

INTRODUCTION
Urotensin-II (U_II), a peptide isolated from fish urophysis,
more than four decades ago, is the endogenous ligand of the
mammalian recently deorphanized receptor GPR14. The initialstudies proclaimed the U_II as the “new endothelin” (1).
U_II has potent vasodilator and vasoconstrictor actions and issynthesized in the pulmonary endothelium (2). Plasma and lungtissue homogenate levels of U_II (1) and expression of U_II bypulmonary arterial endothelial and smooth muscle cells (3) areelevated in different models of pulmonary hypertension. U_II canstimulate proliferation of vascular smooth muscle cells and vascular remodeling processes that, in conjunction with enhanced
vasoconstriction, are involved in the pathogenesis of pulmonary
vascular diseases (4).
There has been accumulating evidence which suggests theinteraction among the renin angiotensin system (RAS) and U_II
could have important functional implications (5, 6). On isolated rat
aortic rings with intact adventitia and endothelium the vasoconstrictor effects of U_II and angiotensin (Ang) II have synergistic
vasoconstrictor effects (5, 6). On the oher hand, Ang II mediates
U_II increased cell proliferation and intracellular reactive oxygen
species levels in rat cardiac fibroblasts (8).
Based on the well known roles of the RAS on diseased pulmonary vessels (9, 10) and the already discussed collaboration
between U_II and RAS we investigated if and to what degree theblockade of RAS could modulate the vasomotor effects U_II on
pulmonary artery rings from OVA sensitized rats (OSR) and pulmonary hypertensive rats (PHR). To modulate the intrapulmonary
RAS losartan (LOS, an AT1 receptor blocker), chymostatin (CST,a chimase inhibitor) and amastatin (AMA, an aminopeptidase
inhibitor) were given by tracheal instillation.
MATERIALS AND METHODSThe experiments were conducted in age-matched male
Wistar rats with body weights of 200-250 g housed under standard laboratory conditions, with free access to standard rodent
chow and tap water. Three groups of animals were used: (1)untreated control rats (NR, n=18), (2) OVA sensitized rats (OSR,n=18), (3) rats with monocrotaline – induced pulmonary hypertension (PHR, n=18). This study was approved by the Ethics
Committee of the University of Medicine and Pharmacy “Grigore
T. Popa” Iasi and it was performed according to the Helsinki
convention for the use and care of animals, and to the European
Communities Council Directive 609 of 24 November 1986.
Sensitization: The rats were sensitised against OVA by s.c.
and i.p. injection of 0.2 mL physiological saline, containing 100
mg OVA and 8 mg aluminium hydroxide. The protocol was repeated 2 weeks later (8). In vitro challenge: After 7 days, before
starting administering of studied substances, vascular rings werepre-treated with OVA (100 mg/mL) (11).

Received May 2010. Accepted June 2010. Address for correspondence: Irina Luciana Dumitriu, Discipline of Physiology –Functional Exploration, Department of
Functional Sciences, Faculty of Medicine, University of Medicine and Pharmacy “Gr T Popa” Iasi, Romania, e-mail: dumitriu_irinaluciana@yahoo.com

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Fiziologia - Physiology  2010.20.3 (67)12Monocrotaline(MCT) – induced pulmonary hypertension.
To induce pulmonary hypertension, the rats from PHR group
received a subcutaneous injection of MCT (60 mg/kg body wt)
as previous described (12).
Modulation of intrapulmonary RAS: In the last week of experimental protocol (third week for OSR or the forth week for PHR or
in the week before euthanasia for NR) the LOS (0.1mM, a blocker
of angiotensin II type 1 (AT1) receptors), CST (0.1mM) or AMA(0.1mM) were administered by tracheal instillation (50microL/day, once every two days), as previous described (13).
Wire myography. Rats were decapitated and exsanguinated.
The first branches of rat pulmonary arteries (RPA) were rapidly removed, cleaned and cut into rings, 1-2 mm wide. The endothelium
was removed mechanically by gently rubbing the luminal surface
with a tungsten wire. Individual rings were then mounted between
tungsten wires in a MYO-01 MYOGRAPH SYSTEM (Experimetria
LTD., Budapest, Hungary) and changes in vessel tension wererecorded and analyzed by ISOSYS data acquisition system(Experimetria LTD., Budapest, Hungary). The tissue organ bath
contained the Krebs–Henseleit solution containing (mM): NaCl
118, KCl 4.8, CaCl2 2.5, MgSO4 1.6, KH2PO4 1.2, NaHCO3 25,
glucose 5.5. The Krebs-Henseleit buffer was maintained at 37°C,
and bubbled continuously with a mixture of 95% O2 and 5% CO2(pH=7.2–7.4). A resting tension of 0.7 g for RPA was applied toeach ring and then allowed to equilibrate for 90 minutes before
the start of the experiment. The bathing medium was renewedfrom 15 to 15 minutes. After the equilibration period, vessel rings
were stimulated twice with 40 mM KCl as a standard stimulus.
Endothelium absence was certified by absence of 1microM
acetylcholine (ACh) – induced relaxation on KCL precontracted
rings. After re-equilibration (60 min.) the dose response curvesof U_II – induced contractions were constructed.
Expression of results and statistical analysis: The results are
expressed as the percentage of the control contraction (KCl, 40
mM; mean ± S.E.M.). Statistical significance was tested using
one-way analysis of variance (ANOVA), completed with Bonferroni method (SigmaStat software, Jandel Corporation). p<0.05
was considered statistically significant.
KCl, U_II, LOS, CST, AMA, OVA, aluminium hydroxide and
MCT were all obtained from Sigma (Sigma-Aldrich Inc., St. Louis
MO). All the other compounds used were of analytical grade.
RESULTS
U_II cumulative doses (0.1 nM – 1 µM) induced dose – dependent contractile effects on RPA rings (Emax = 83.86±4.70%;-logEC50 = 8.52±0.03) from NR (Figure 1). The U_II – induced
contractions were significantly higher on RPA rings from both
OSR (Emax = 103.59±1.26%; -logEC50 = 8.51±0.06) and PHR(Emax = 110.00±4.86%; -logEC50 = 8.86±0.06). Even more,
the contractile responses of RPA from PHR were significantly
higher than from OSR for U_II doses between 5nM and 50nM:
15.58±0.77 vs. 9.82±0.94 (p<0.001), 33.44± 1.41 vs. 26.13±2.21(p<0.05), 67.17±4.60 vs. 52.21±3.32 (p<0.01), 95.26±4.09 vs.
78.27±3.52 (p<0.01), 106.67±3.80 vs. 91.41±3.13 (p<0.01).

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Fig. 1. Contractile effects of urotensin II (U_II, cumulative doses 0.1nM - 1μM) on rat pulmonary arteries rings from normal rats (NR), ovalbumin
sensitized rats (OSR) and rats with pulmonary hypertension (PHR). Data arepresented as percentage from control contraction with 40 mM KCl. *: p<0.05,^: p<0.01, #: p<0.005 as compared with NR.

The losartan treatment did not modify the U_II - induced
dose – dependent contractions (Emax = 81.64±3.69 %; -logEC50:
8.48±0.01) on NR (figure 2). LOS treatment reduced the contractile
effects on OSR (Emax = 96.68±2.68%; -logEC50:8.34±0.01) andPHR (Emax = 101.16±1.21%; -logEC50:8.30±0.24). Even if on
untreated rats the differences between OSR and PHR versus
NR were significantly for almost all doses, after LOS treatment
only for 50nM, 0.1microM, 0.5microM, 1 microM the contractilleresponses are higher on OSR (82.37±2.67, p<0.01; 89,39±3,20,
p<0.05; 96,68±2,68, p<0.01; 94,96±0,85, p<0.001) and PHR(84.80±5.98, p<0.05; 94.94±4.51, p<0.005; 101.16±1.21,
p<0.005; 99.61±1.67, p<0.001) as compared with NR (70.11±3.12;
71.89±4.97; 81.64±3.69; 81.40±1.89). Another interesting findingis the decreasing of U_II efficacy on LOS – treated OSR (4.59nM
vs. 3.09nM) and LOS – treated PHR (4.96nM vs. 1.38nM) as
compared with untreated OSR and PHR.

Fig. 2. Losartan (LOS) treatment effects on contractions induced by urotensin
II (U_II, cumulative doses 0.1nM - 1 μM) on rat pulmonary arteries rings fromnormal rats (NR), ovalbumin sensitized rats (OSR) and rats with pulmonary
hypertension (PHR). Data are presented as percentage from control contraction with 40 mM KCl. *: p<0.05, ^: p<0.01, as compared with NR.

13

The inhibition of chymostatin – sensitive Ang II formation did
not significantly modify the U_II – induced contractions on RPA
from NR (Emax: 83.81±3.13; -logEC50: 8.50±0.09), OSR (Emax:
98.03±2.85; -logEC50: 8.37±0.07) and PHR (Emax: 102.69±3.97;-logEC50: 8.64±0.11) as compared with untreated rats. On theother hand, the treatment with CST lowered the differences between contractile effects of U_II on OSR versus NR.

Fig. 3. Chymostatin (CST) treatment effects on contractions induced byurotensin II (U_II, cumulative doses 0.1nM - 1 μM) on rat pulmonary arteriesrings from normal rats (NR), ovalbumin sensitized rats (OSR) and rats withpulmonary hypertension (PHR). Data are presented as percentage from control contraction with 40 mM KCl. *: p<0.05, ^: p<0.01, as compared with NR.

Decreasing of pulmonary Ang II degradation by amastatintracheal instillation increased the U_II contractile effects in allrings. Even on NR after the AMA treatment, the U_II – induced
contractions (Emax: 98.46±3.72%; -logEC50: 8.52±0.06)
are significantly elevated for doses higher than 10nM. There
are no more significant differences between OSR (Emax:
121.94±2.03%; -logEC50: 8.81±0.18) and PHR (Emax:
124.35±4.48%; -logEC50: 8.96±0.17) after AMA treatment. As
expected, the EC50 for U_II is reduced too, 1.52nM on OSR
and 1.09nM on PHR.

Fig. 4. Amastatin (AMA) treatment effects on contractions induced byurotensin II (U_II, cumulative doses 0.1nM - 1 μM) on rat pulmonary arteriesrings from normal rats (NR), ovalbumin sensitized rats (OSR) and rats withpulmonary hypertension (PHR). Data are presented as percentage from control contraction with 40 mM KCl. *: p<0.05, ^: p<0.01, as compared with NR.

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DISCUSSIONSUrotensin-II (U_II) has been considered as the most potent
mammalian vasoconstrictor identified so far (3, 4). But, the vasoconstrictor activity of U_II was dependent upon the anatomical
origin of the vessel studied and the species from which it was
isolated (14). U_II was a potent vasoconstrictor of rat main
pulmonary arteries; the U_II contractile effects were increasedin the absence of the endothelium or in presence of NG-nitroL-arginine methyl ester (L-NAME) (15, 16). The U_II – induced
contractions were powerful on rat main and small pulmonary
arteries from chronic hypoxic rats as compared with normal rats
(16). On the other hand, published results about the U_II effects
on human pulmonary artery are unclear. U_II does not induce
vasoconstriction of isolated human pulmonary arterial or in lungpreparations (17), but in human small pulmonary arteries U_II
was a potent vasodilator (18).
Taking into account the endothelium dependence of U_II
effects and different degree of endothelial disease on OSR
versus PHR we used on this paper only data from RPA without
endothelium. Our results showed that in absence of endothelium, U_II could induce powerful contractions on OSR and PHT
than in NR (fig. 1). As compared with published data (15, 16),
our results clearly present the dose dependent vasoconstrictory
activity of U_II.
Both experimental models used by us, the OVA sensitizationand MCT – induced PHT, are producing approximating the same
effects (but different degree) on pulmonary arteries: increase
reactivity to vasoconstrictor agonists and initiate the vascularinflammation (followed by endothelial disease and vascularremodeling) (7,19,20).
As we supposed based on published data, the U_II contractile effects on RPA were modified by the modulation of
intrapulmonary RAS. The LOS, CST and AMA were deliveredby tracheal instillation in order to be active mainly at pulmonary
level (21).
Blocking AT1 receptors decreased the U_II contractile effects
on OSR and PHR. In experimental models used in this study,
OSR and PHR, both the efficacy and potency of U_II are lower(figure 1). These results confirm the hypothesis that, duringsome pathological circumstances, the intrapulmonary RAS could
sustain the U_II reactivity of pulmonary arteries. These results
are agreed with published data about the benefic effects of AT1
blockade on pulmonary arterial disease (22, 23).
Taking into account our previous results about chimase–dependent Ang II formation on vascular walls of pulmonary
arteries from OSR we delivered intratracheally the CST. The only
modification that we observed on CST – treated rats as comparewith untreated rats was the increase of EC50 on OSR. So, theexistence of multiple ways to synthesize Ang II (24) prevents the
CST treatment effects on the U_II contractile effects.
Even if we can not significantly modify the U_II – induced
contractions by decreasing Ang II formation, the inbition of aminopeptidases and subsequently decrease of Ang II degradation had
significantly amplificatory effects on U_II – induced contractions.
On this case, we obtain an increase of Emax to U_II even on
Fiziologia - Physiology  2010.20.3 (67)14

RPA from NR (the EC50 did not modify). On RPA from OSR and
PHR, after CST treatment, both the Emax and EC50 (with 50%
for OSR) were increased.
CONCLUSION
The isolated pulmonary arteries from PHR and OSR arereacting more powerfully to U_II as compared with vessels obtained from NR. Blocking of intrapulmonary RAS by intratracheal
administration of LOS decreased U_II-induced contractions on
OSR and PHT and the reducing of Ang II degradation had opposite effects. So, our data sustain the implication of intrapulmonary
RAS on increasing reactivity of pulmonary arteries during somepathological situations and suggest possible positive effects of local blocade of AT1 receptors on pulmonary vessels diseases.
Acknowledgements
These studies were partially supported by funds from the
Romanian National Ministry of Education and Research (Grant
CNCSIS PN-II-ID-PCE-2007-1 No. 1273/2007).
REFERENCES
1. Maguire JJ, Davenport AP. Is urotensin-II the new endothelin? Br J
Pharmacol. 2002;137(5):579-588.
2. Ames RS, Sarau HM, Chambers JK, Willette RN, Aiyar NV, Romanic
AM, Louden CS, Foley JJ, Sauermelch CF, Coatney RW, Ao Z, Disa
J, Holmes SD, Stadel JM, Martin JD, Liu WS, Glover GI, Wilson S,
McNulty DE, Ellis CE, Elshourbagy NA, Shabon U, Trill JJ, Hay DW,
Ohlstein EH, Bergsma DJ, Douglas SA. Human urotensin-II is a potent
vasoconstrictor and agonist for the orphan receptor GPR14. Nature.
1999;401(6750):282-286.
3. Qi J, Du J, Tang X, Li J, Wei B, Tang C. The upregulation of endothelial nitric oxide synthase and urotensin-II is associated with pulmonary
hypertension and vascular diseases in rats produced by aortocaval
shunting. Heart Vessels. 2004;19(2):81-88.
4. Djordjevic T, Görlach A Urotensin-II in the lung: a matter for vascular remodelling and pulmonary hypertension? Thromb Haemost.
2007;98(5):952-962.
5. Lamarre NS, Tallarida RJ. A quantitative study to assess synergistic
interactions between urotensin II and angiotensin II. Eur J Pharmacol.
2008; 586(1-3):350-351.
6. Takahashi K, Hirose T, Mori N, Morimoto R, Kohzuki M, Imai Y, Totsune
K. The renin-angiotensin system, adrenomedullins and urotensin II in
the kidney: possible renoprotection via the kidney peptide systems.
Peptides 2009;30(8):1575-1585.
7. Wang YX, Ding YJ, Zhu YZ, Shi Y, Yao T, Zhu YC. Role of PKC in
the novel synergistic action of urotensin II and angiotensin II and inurotensin II-induced vasoconstriction. Am J Physiol Heart Circ Physiol.
2007;292(1):H348-359.
8. Chen YL, Liu JC, Loh SH, Chen CH, Hong CY, Chen JJ, Cheng TH.
Involvement of reactive oxygen species in urotensin II-induced proliferation of cardiac fibroblasts. Eur J Pharmacol. 2008;593(1-3):24-29.
9. Gurzu B, Petrescu BC, Dumitriu IL, Mihai AC, Slătineanu SM. Local
generation of angiotensin in isolated rat pulmonary vessels. Proceedings of the Joint Meeting of the Slovak Physiological Society, thePhysiological Society and the Federation of European Physiological
Sciences 2007. Monduzzi Editore, Bologna, 67-71.
10. Han SX, He GM, Wang T, Chen L, Ning YY, Luo F, An J, Yang T,

2010.20.3 (67)  Fiziologia - Physiology

cmyb

15

Dong JJ, Liao ZL, Xu D, Wen FQ. Losartan attenuates chronic cigarettesmoke exposure-induced pulmonary arterial hypertension in rats: possible involvement of angiotensin-converting enzyme-2. Toxicol Appl
Pharmacol. 2010;245(1):100-107.
11. Cavalher-Machado SC., Tavares de Lima W., Damazo A., Frias
Carvalho V., Martins MA, Silva PMR, Sannomiya P. Down-regulation ofmast cell activation and airway reactivity in diabetic rats: role of insulin.
Eur Respir J 24: 552–558; 2004.
12. Lourenço AP, Roncon-Albuquerque R Jr, Brás-Silva C, Faria B,
Wieland J, Henriques-Coelho T, Correia-Pinto J, Leite-Moreira AF.
Myocardial dysfunction and neurohumoral activation without remodelingin left ventricle of monocrotaline-induced pulmonary hypertensive rats.
Am J Physiol Heart Circ Physiol. 2006;291(4):H1587-1594.
13. Semmler-Behnke M, Takenaka S, Fertsch S, Wenk A, Seitz J,
Mayer P, Oberdörster G, Kreyling WG. Efficient elimination of inhaled
nanoparticles from the alveolar region: evidence for interstitial uptake
and subsequent reentrainment onto airways epithelium. Environ Health
Perspect. 2007;115(5):728-733.
14. Douglas SA, Sulpizio AC, Piercy V, Sarau HM, Ames RS, Aiyar
NV, Ohlstein EH, Willette RN. Differential vasoconstrictor activity ofhuman urotensin-II in vascular tissue isolated from the rat, mouse,dog, pig, marmoset and cynomolgus monkey. Br J Pharmacol.
2000;131(7):1262-74.
15. McLean MR, Alexander D, Stirrat A, Gallagher M, Douglas SA,
Ohlstein EH, Morecroft I, Polland K. Contractile responses to humanurotensin-II in rat and human pulmonary arteries: effect of endothelial
factors and chronic hypoxia in the rat. Br J Pharmacol. 2000;130(2):2014.
16. Deuchar GA, Morecroft I, Dempsie Y, Herold N, Nilsen M, Hicks MN,
McLean MR. The in vivo effects of human urotensin II in the rabbit and
rat pulmonary circulation: effects of experimental pulmonary hypertension. Eur J Pharmacol. 2006;537(1-3):135-142.
17. Bennett RT, Jones RD, Morice AH, Smith CF, Cowen ME. Vasoconstrictive effects of endothelin-1, endothelin-3, and urotensin II in isolated
perfused human lungs and isolated human pulmonary arteries. Thorax.
2004;59(5):401-407.
18. Stirrat A, Gallagher M, Douglas SA, Ohlstein EH, Berry C, Kirk A,
Richardson M, MacLean MR. Potent vasodilator responses to humanurotensin-II in human pulmonary and abdominal resistance arteries. Am
J Physiol Heart Circ Physiol. 2001;280(2):H925-928.
19. Schmiedl A, Tschernig T, Luhrmann A, Pabst R. Leukocyte infiltrationof the periarterial space of the lung after allergen provocation in a rat
asthma model. Pathobiology. 2005;72(6):308-315.
20. Huang J, Kaminski PM, Edwards JG, Yeh A, Wolin MS, Frishman
WH, Gewitz MH, Mathew R. Pyrrolidine dithiocarbamate restores endothelial cell membrane integrity and attenuates monocrotaline-induced
pulmonary artery hypertension. Am J Physiol Lung Cell Mol Physiol.
2008;294(6):L1250-1259.
21. Dumitriu IL, Gurzu B, Ciureanu I, Dinca M, Plamadeala P, Costuleanu
M, Slatineanu SM, Petrescu Gh. Involvement of the intrapulmonary rennin angiotensin system in the development of allergic lung disease. The
Journal of Physiological Sciences. 2009; 59(Suppl. 1): 269-269.
22. Cernucan DL, Gurzu B, Slătineanu S, Dumitriu IL, Slătineanu SM,
Costuleanu M, Petrescu G. Contractile effects of angiotensinogen andits multiple metabolization pathways on vessel walls. Rev Med Chir Soc
Med Nat 2007;111(3):717-721.
23. Kato T, Nasu T, Sonoda H, Ito KM, Ikeda M, Ito K. Evaluation ofolmesartan medoxomil in the rat monocrotaline model of pulmonary
hypertension. J Cardiovasc Pharmacol. 2008;51(1):18-23.

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PARTICIPAREA SISTEMULUI RENINA ANGIOTENSINA IN MODULAREA
REACTIVITATII VASELOR PULMONAREREZUMAT
Datele publicate susţin participarea urotensin II (U_II) in patologia vaselor pulmonare. Luând în considerare importanţa sistemului renină
angiotensină (SRA) în reglarea reactivitaţii vaselor pulmonare am investigat posibila influenţă a modularii activitaţii SRA intrapulmonar
asupra efectelor contractile ale U_II la nivelul inelelor de arteră pulmonară (IAP) de la şobolani sensibilizaţi la ovalbumină (SSO) şişobolani cu hipertensiune pulmonară (SHP). Contracţiile induse de U_II au fost înregistrate şi datele obţinute de la SSO şi SHP au
fost comparate cu cele de la şobolani normali (SN). Losartan, chimostatin si amastatin au fost administrate prin intubaţie traheală
pentru a modula SRA intrapulmonar. Contracţiile induse de U_II sunt mai mari la nivelul IAP de la SSO şi SHP comparativ cu SN.
Blocarea SRA intrapulmonar prin administrarea intratraheală de losartan reduce diferenţele dintre SSO sau SHP şi SN. Scăderea
degradării angiotensinelor amplifică semnificativ efectele semnificative ale U_II în ambele modele experimentale. Aceste date susţin
implicarea SRA intrapulmonar în creşterea reactivităţii arterelor pulmonare în unele situaţii patologice şi susţin efecte benefice aleadministării pulmonare a antagonistilor receptorilor angiotensinei II de tip 1 (AT1) în afecţiuni ale arterelor pulmonare.
Cuvinte cheie: urotensina II, angiotensina, artera pulmonara, astm, hipertensiune pulmonara, sobolani

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Fiziologia - Physiology  2010.20.3 (67)16

OXIDATIVE STRESS IN CHRONIC VENOUS HYPERTENSION– INSIGHTS FROM AN ANIMAL MODEL
CRISTINA MARIA VAIDA VOEVOD, TEODORA MOCAN, REMUS MOLDOVAN, NICOLETADECEA, ADRIANA MUREŞAN
Department of Physiology, University of Medicine and Pharmacy ”Iuliu Haţieganu” Cluj-Napoca

ABSTRACT
Objective: Endothelial disorders of the veins are considered to play a role in the pathogenesis of primary varicose veins and oxidative
stress is implicated in these disorders. With this aim, we investigated the levels of metabolic markers of oxidative stress after creating
partial venous obstruction on an experimental animal model.
Methods: Chronic venous hypertension was produced by partial ligation of the superficial and common femoral vein in Wistar rats
( 2 groups: group 1- ligation of the superficial femoral vein, n=30 animals,10 animals/lot; group 2 – ligation of the common femoralvein, n=30 animals,10 animals/lot). At specified intervals blood was collected from the retroorbital sinus of the animals. We investigated the levels of malondialdehyde, carbonilated proteins, glutathion, hydrogen donors and thiol radicals in the study groups andcompared the results with a control group.
Results: There was statistically significant difference between the study groups and the control group concerning the levels of oxidative
stress markers. Plasma levels of malondialdehyde and carbonilated proteins were found higher than in control group at each time
point for both study groups.Total antioxidant capacity of the blood was found at decreasing levels compared with controls.
Conclusions: The current model of induced venous hypertension demonstrates early changes in the oxidative status of study animals
not only in the local area but also in the sistemic circulation.
Key words: oxidative stress, free oxygen radicals, antioxidants, inflammation, chronic venous insufficiency.

INTRODUCTION
Chronic venous disease (CVD) is common. Its manifestations
include varicose veins, skin changes such as dermatitis, hyperpigmentation and lipodermatosclerosis and chronic leg ulcers.
The pathogenesis of chronic venous disease is still unclear.
The mechanisms regulating varicose vein development andthe subsequent skin sequelae seen in chronic venous disease(CVD) have been investigated recently. Despite the diversityof signs and symptoms associated with the disease, it seems
likely that they are related to venous hypertension (1). Valvularincompetence is the most important cause of venous hypertension. It seems that initial valvular lesion is followed by reflux inthe superficial venous system and dilation of superficial veins (2).
Other authors suggest that valvular damage occurs secondary
to the initial venous wall lesion generating venous hypertension(3). Recent findings suggest that inflammatory processes are
involved in the structural remodeling in venous valves and in
the vein wall, leading to valvular incompetence and the development of varicose veins. Vein wall remodeling occurs during
the inflammatory reaction produced by hypertension. Venous
hypertension alters the laminar shear stress and stimulates theinteraction between leucocytes and endothelium which initiates
migration, adhesion and activation of white blood cells. There
has been shown infiltration of valve leaflets and the venous wallby leukocytes (monocytes and tissue macrophages) in all valve

specimens from patients with CVD and in none from controls(4). The leukocyte activation is accompanied by the expressionof integrins and by synthesis and release of many inflammatory
molecules, including proteolytic enzymes, leukotrienes, prostaglandin, bradykinin, free oxygen radicals, cytokines, and possibly
other classes of inflammatory mediators (5). Neutrophils, mast
cells, tissue macrophages, endothelial cells and fibroblasts are
believed to play an important role in the initiation of the inflammatory response in CVD, being responsible for the productionof free oxygen radicals (6,7) Vein wall remodeling is likely to
involve the complex interplay of a range of factors, including
an altered ratio between metalloproteinases (MMPs) and theirtissue inhibitors (TIMPs), and elevated levels of cytokines andgrowth factors favor an alteration of the extracellular matrix(8). Among the mediators of vessel wall damage free oxygen
radicals play an important role, acting through two mechanisms.
The first is the oxidation of cell membrane components, followed
by endhotelium damage, which eventually leads to increased
vascular permeability. The second mechanism mechanism isthe chemotaxis and activation of leucocytes and platelets (9).
As these mechanisms work together, the inflammatory reaction
perpetuates itself, cell damage is amplified and venous stasis
is increased (10).
Studies in humans can generally give information only about

Received May 2010. Accepted August 2010. Address for correspondence: Cristina Maria Vaida Voevod, Physiology Department, “Iuliu Hatieganu” University of Medicine
and Pharmacy Cluj-Napoca, Romania

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processes operating in established disease, whereas the very
early stages may be helpful in understanding pathogenesis andtrigger mechanisms. Animal models have a role to play in thisregard (11). The aim of this study is to investigate the levelsof metabolic markers of oxidative stress after creating partial
venous obstruction on an experimental animal model.

trolled flow hood, and maintained with standard pellet
diet and water ad libitum.
All the studied rats were in the custody of the Physiology
Department Animal House and the experiments were performed
respecting international rules concerning experiments on
animals.

MATERIAL AND METHODS
We produced venous hypertension by partial ligation of thecommon or superficial femoral vein under general anesthesia.
The experiments were performed on 2 groups (30 animals/group)of white Wistar rats, males, with a mean weight of 200 grams,
from the University of Medicine Animal House. A third groupof 10 animals was used as control. Each group was divided inthree lots, 10 animals/lot, based on three different time points
for collecting blood samples.
Group I – ligation of the superficial femoral vein.
Lot 1 – 4 weeks
Lot 2 – 8 weeks
Lot 3 – 10 weeks
Group II – ligation of the common femoral vein.
Lot 4 – 1 weekLot 5 – 2 weeks
Lot 6 – 3 weeks
Blood samples were collected from the retroorbital sinusof the rats at different time points as described earlier andconserved by freezing at -80 degrees until examination. Laboratory tests were performed at the Physiology Department in
the Laboratory for Oxidative Stress Study. We investigated thelevels of malondialdehyde (MDA)(12), carbonilated proteins (PC)(13), glutathion (GSH)(14), hydrogen donors (DH)( 15) and thiol
radicals (SH)(14) in the study groups and compared the resultswith a control group.
Operating protocol included the following stages:
the day of the surgery animals were prevented from food and watereach animal was given general anesthesia(Ketamine 80mg/kg and Xylasine 8mg/kg Ketamine/
Xylazine = 2/1, intramuscular) with supplemental doses
when needed
an incision was performed on the medialsurface of the groin, from the inguinal ligament to the knee
and the femoral vessels were exposed. We dissected thesuperficial, common femoral vein. We placed a sutureon the common femoral vein just above the confluenceof the superficial and profound femoral veins, we also
ligated the superficial epigastric vein in order to prevent
back flow; we ligated the superficial femoral vein justbelow the confluence using monofilament sutures (8-0).
Monofilament sutures (4-0) were used as a guide in orderto prevent the total ligation of the veins and to ensure arestant blood flow
the skin incision was closed, and the rats
were given intensive postsurgical care
the rats were housed in a light-cycle con-Statistical methods:- Values were expressed as percentage calculated fromcontrol values- Mean ± standard deviation as centrality and dispersion
indexes- Non-parametric tests: Mann-Whitney U Test, Kruskall-Wallis
Test, Spearman r calculus.

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RESULTS
1. Analysis of the oxidative stress markers in the firstgroup, with ligation of the superficial femoral vein (Table I)
MDA expressed higher levels when compared with the control lot (Figure 1) and the values were found significantly higher
in the second and third lot (p=0.000, p=0.000)(Table II).

Fig. 1. Protein carbonyl values in groups included in our experiment

Regarding the protein carbonyl we found increased values
compared to those from controls and the peak was found in thefirst lot (Figure 1). All detected values were significantly higher
than those found in controls (p=0.000, p=0.000, p=0.000)(TableII).
The dynamical assessment of the values indicated a decrease of PC values, but they still remain significantly higher thancontrols (p=0.87, p=0.000, p=0.001). MDA values were foundrising in dynamics (p=0.001, p=0.000, p=0.01) (Table V).
The antioxidant capacity of the blood presented lower
levels then those found in control groups (Figures 2 and 3). All
SH detected values were found significantly lower than control
values (p=0.004, p=0.003, p=0.002)(Table II). Total antioxidant
capacity of the blood, expressed by DH values was significantly
lower in all studied lots when compared to controls (p=0.001,
p=0.049, p=0.001) (Table II). GSH detected levels were higher
than in control group. When we performed the analysis between
studied lots we remarked a significant decrease in the antioxidantFiziologia - Physiology  2010.20.3 (67)18

capacity in dynamics (Table V)(p=0.22), and the second and third lots had significantly lower
values than the first one (p=0.005, p=0.001) but their values werestill higher than those found in controls (Table VI).
Regarding the antioxidant capacity, SH levels were foundlover in all study groups compared to controls (Figure 5), withstatistical significant values in the first two weeks after ligation(p=0.002, p=0.002)(Table IV) but there was no significant difference between study lots in dynamics (Table VI).

Fig. 2. Antioxidant capacity of the blood – SH and HD

Fig.5. Antioxidant capacity – SH and HD levels

Fig. 3. Gluthatione levels in all groups considered for our experiment

2. Analysis of the oxidative stress markers in the second
group, with ligation of the common femoral vein (Table III)We found significant higher levels of MDA only in the third
week after vein ligation (p=0.000) (Table IV), for the first two
weeks the values were lower than those from controls (Figure4). Concerning PC, all values were found higher then controls
with a peak in the second week after surgery (p=0.000) (Table
IV) (Figure 4).

The total antioxidant capacity of the blood, reflected in DH
values showed a significant decrease in the first two weeks(p=0.002, p=0.000) and a slightly increase in the third week, but
without statistical significance when compared in dynamics withthe previous lot (p=0.25) (Table VI).
When compared between lots, SH values expressed a significant lower value in dynamics (p=0.004, p=0.001)(Table VI).
3. Analysis of the oxidative stress markers between
the two study groups: ligation of the superficial femoral
vein versus ligation of the common femoral vein (Table VII,
Table VIII)
We found significantly increased levels of MDA in the second group (p=0.000). PC values were lower in the group withligation of the common femoral vein compared to the first group(p=0.001).
Antioxidative defense was found significantly lower in thesecond group, regarding all three studied markers: SH (p=0.01),
DH (p=0.016), GSH (p=0.001). )(Table VII, Table VIII).

Fig.4. Oxidative stress markers in all groups considered for our experiment –MDA and PC

The analysis between lots confirmed that in dynamics after
the initial lower values of MDA, without statistical significance(p=0.7), the ulterior values were significantly rising (p=0.000,
p=0.002), with linear increase. Regarding PC levels the second
lot expressed a nonsignificant rise compared to the first one
2010.20.3 (67)  Fiziologia - Physiology

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19

Fig.6. Gluthatione levels in all groups considered in our experiment

19

Table I. Comparative analysis between lots with ligation of superficial
femoralvein
Table I. Comparative analysis
between lots
withandligationcontrolsof superficial femoral vein and controls
Lot

MDA
Mean/SD
1.25 ± 0.3
1.44 ± 0.22
2.04 ± 0.23
2.78 ± 0.55

Controls
Lot 1
Lot 2
Lot 3

PC
Mean/SD
0.74 ± 0.06
2.18 ± 0.41
2.11 ± 0.43
1.30 ± 0.11

SH
Mean/SD
0.19 ± 0.05
0.14 ± 0.01
0.12 ± 0.08
0.11 ± 0.01

DH
Mean/SD
37.17 ± 5.6
27.26 ± 3.73
32.15 ± 3.85
25.55 ± 5.49

GSH
Mean/SD
2.88 ± 1.12
14.33 ± 0.41
10.33 ± 1.05
12.02 ± 1.8

Table II. Statistical significanceLot
Controls
Lot 1
Lot 2
Lot 3

MDA
p

PC
p

0.15
0.000
0.000

MDA
Mean/SD
1.25 ± 0.3
1.14 ± 0.1
1.23 ± 0.85
2.52 ± 0.42

Table II. Statistical significance
SH
p

DH
p

GSH
p

0.000
0.000
0.000

0.004
0.003
0.002

0.001
0.049
0.001

0.000
0.000
0.000

PC
Mean/SD
0.74 ± 0.06
1.96 ± 0.44
2.25 ± 0.46
1.38 ± 0.18

SH
Mean/SD
0.19 ± 0.05
0.13 ± 0.01
0.13 ± 0.02
0.12 ± 0.02

DH
Mean/SD
37.17 ± 5.6
28.44 ± 2.55
20.46 ± 6.41
23.48 ± 3.89

GSH
Mean/SD
2.88 ± 1.12
20.23 ± 8.19
15.14 ± 1.27
12.24 ± 1.71

Table III. Comparative analysis between lots with ligation of common
femoralcontrols
Table III. Comparative analysis
betweenveinlots withandligationof common femoral vein and controls

Lot
Controls
Lot 4
Lot 5
Lot 6

Table IV. Statistical significanceLot
Controls
Lot 4
Lot 5
Lot 6
Lot

PC
p

SH
p

DH
p

GSH
p

0.88
0.4
0.000

0.000
0.000
0.000

0.002
0.002
0.01

0.002
0.000
0.001

0.000
0.000
0.000

MDA
p

PC
p

SH
p

DH
p

GSH
p

0.001
0.000
0.01

0.87
0.000
0.001

0.023
0.01
0.49

0.013
0.40
0.007

0.000
0.005
0.023

MDA
p

PC
p

SH
p

DH
p

GSH
p

0.7
0.000
0.002

0.22
0.005
0.001

0.65
0.13
0.36

0.004
0.001
0.25

0.007
0.000
0.001

Table V. Dynamical assessment between lots with ligation of superficial
Table V. Dynamical assessment femoral
between lots veinwith ligation of superficial femoral vein

Controls
Lot 1 vs lot 2
Lot 1 vs lot 3
Lot 2 vs lot 3
Lot

Table IV. Statistical significance

MDA
pTable VI. Dynamical assessment between lots with ligation of common
Table VI. Dynamical assessmentfemoral
between lotsveinwith ligation of common femoral vein

Controls
Lot 4 vs lot 5
Lot 4 vs lot 6
Lot 5 vs lot 6

Table VII. Comparative analysis between lots with ligation of superficial

Table VII. Comparative
analysis between
with ligationfemoralof superficialvein
femoralandvein, common
femoral vein and controls
femoralvein,lots
commoncontrols

Lot

MDA
Mean/SD
1.25 ± 0.3
1.44 ± 0.22
2.52 ± 0.42

Controls
Lot 1
Lot 6

PC
Mean/SD
0.74 ± 0.06
2.18 ± 0.41
1.38 ± 0.18

SH
Mean/SD
0.19 ± 0.05
0.14 ± 0.01
0.12 ± 0.02

DH
Mean/SD
37.17 ± 5.6
27.26 ± 3.73
23.48 ± 3.89

GSH
Mean/SD
2.88 ± 1.12
14.33 ± 0.41
12.24 ± 1.71

Table
VIII. Statistical significance
Table VIII. Statistical significance
Lot

MDA
p

PC
p

SH
p

DH
p

GSH
p

Lot 1 vs lot 6

0.000

0.001

0.01

0.016

0.001

DISCUSSIONS
Numerous experimental studies using animals were
performed, most of them indicating the levels and activity ofleucocytes, MMPs, cytokines and adhesion molecules. Only
few studies were concerned about the role of free radicals inthe development of local venous damage. All reviewed data
revealed the role of inflammatory response in chronic venous
disease. Many questions need to be answered, such as: Are thefree oxygen radicals responsible for activation the inflammatorycascade, or they are only a consequence of the activation of whiteblood cells and their interaction with the endothelium? Increased
production of free oxygen radicals after venous hypertension andstasis are the result of hypoxia or of inflammation? What is the
DISCUSSIONS

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role of free radical scavengers in the treatment of CVD?
CONCLUSIONS
1. The current model of induced venous hypertension demonstrates early changes in the oxidative status of study animals
not only in the local area but also in the sistemic circulation.
2. The increase of the venous pressure that we inducedby ligating the superficial femoral vein is followed by increased
levels of oxidative stres markers and decreased values of antioxidant markers.
3. The higher increase of the venous pressure in the lover
limb that we induced by ligating the common femoral vein
determined a more agressive oxidative stress, that appeard inearly stages after the surgery, as well as an important decreaseof the antioxidative defence capacity of the blood.
4. Comparing the two procedures we performed we can conclud that the more important the obstructed blood vessel is for thephysiology of the lower limb circulation, the more important andagressive is the damage of the oxidative/antioxidative balance.
REFERENCES

1. Nicolaides AN. Chronic venous disease and the leukocyte-endotheliuminteraction: from symptoms to ulceration. Angiology, 2005; 56(1): S11-9.
2.Goldman MP, Fronek A. Anatomy and pathophysiology of varicose
veins. Dermatol Surg 1989;15:138-45.
3.Raffetto, J D, Khalil, R A. Mechanisms of varicose vein formation: valve
dysfunction and wall dilation. Phlebology 2008;23(2): 85-98
4. Ono T, Bergan JJ, Schmid-Schönbein GW, Takase S. Monocyte
infiltration into venous valves. J Vasc Surg 1998;27:158-166.
5. Cheng JJ, Wung BS, Chao YJ, Hsieh HJ, Wang DL. Cyclic strain inducesredox changes in endothelial cells. Chin J Physiol 1999;42:103-11.
6. Wlaschek M, Scharffetter-Kochanek K. Oxidative stress in chronic
venous leg ulcers. Wound Rep Reg 2005; 13: 452-461
7.Coleridge Smith PD. The microcirculation in venous hypertension.
Vasc Med 1997;2:203-213
8.Pascarella L, Schmid-Schönbein GW, Bergan J. An animal model ofvenous hypertension: the role of inflammation in venous valve failure.
J Vasc Surg 2005;41:303-311.
9. Cordts PR, Kaminski MV, Raju S, et al. Could gut-liver function
derangements cause chronic venous insufficiency? Vasc Surg
2001;35:107-14
10. Takase S, Pascarella L, Lerond L, Bergan JJ, Schmid-Schönbein
GW. Venous hypertension, inflammation and valve remodeling. Eur J
Vasc Endovasc Surg 2004;28:484-493.
11. Bergan JJ, Pascarella L, Schmid-Schönbein GW. Pathogenesis ofprimary chronic venous disease:Insights from animal models of venous
hypertension. J Vasc Surg 2008;47:183-92
12. Conti M, Moran PC, Levillain P, et al. Improved fluorimetric determination of malondialdehyde. Clin.Chem. 1991, 37, 1273 - 1275.
13. Reznick AZ, Packer L. Oxidative damage to proteins: spectrophotometric method for carbonyl assay. In: Methods in enzymology 1994,
233: 357 - 363.
14. Hu ML. Measurement of protein thiol groups and glutathione inplasma. In :Methods in Enzymology vol.233, Academic Press, Inc.
1994, pp.380-384.
15. Janazsewska A, Bartosz G. Assay of antioxidant capacity: comparison of four methods as applied to human blood plasma. Scand.
J. Lab. Invest. 2002, 62: 231-236.

Fiziologia - Physiology  2010.20.3 (67)20

STRESUL OXIDATIV IN INSUFICIENŢA VENOASĂ CRONICĂ – MODEL
EXPERIMENTAL
REZUMAT
Obiectiv: Alterarea endoteliului venos se consideră a juca un rol important în patogeneza bolii varicoase. Stresul oxidativ pare a fi
implicat în producerea tulburărilor endoteliale. Pornind de la această premisă am investigat nivelurile markerilor stresului oxidativ
după crearea experimentală a unei obstrucţii venoase parţiale pe un model animal.
Metodă: Hipertensiunea venoasă cronică a fost obţinută prin crearea unor ligaturi parţiale pe vena femurala superficială şi comună laşobolani rasa Wistar. Experimentele s-au efectuat pe două grupuri de animale: grupul I - ligatură de venă femurală superficială, n=30
animale, 10 animale/lot; grupul 2 – ligatură de venă femurală comună, n=30 animale, 10 animale/lot. La intervale de timp specificate
s-a recoltat sânge din sinusul retroorbital şi s-au determinat nivelurile serice de malondialdehidă, proteine carbonilate, glutation,
donori de hidrogen şi grupări tiol. Rezultatele au fost analizate între loturi precum şi faţă de un lot martor.
Rezultate: S-au obţinut diferenţe semnificative statistic între loturile studiate şi lotul martor în ceea ce priveşte nivelurile markerilor
de stres oxidativ, cu creşteri semnificative ale malondialdehidei şi proteinelor carbonilate, insoţite de scăderea capacităţii antioxidante.
Concluzii: Hiperpresiunea venoasă creată prin ligaturi parţiale ale venelor mari ale membrelor inferioare de şobolan determina
amplificarea stresului oxidativ nu doar la nivel local ci şi în circulaţia generală.
Cuvinte cheie: Stres oxidativ, radicali liberi, insuficienţă venoasă cronică, hiperpresiune venoasă, antioxidanţi

2010.20.3 (67)  Fiziologia - Physiology

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21

EXTENDED SPECTRUM BETA LACTAMASE PRODUCINGKLEBSIELLA PNEUMONIAE STRAINS ISOLATED FROMINTENSIVE CARE UNIT
DELIA MUNTEAN1, MONICA LICKER1, LILIANA DRAGOMIRESCU1, F. HORHAT1, LUMINIŢA
BĂDIŢOIU2, ELENA HOGEA1, DELIA BERCEANU VĂDUVA1, C. PILUŢ1, DORINA DUGĂEŞESCU1,ROXANA MOLDOVAN111
Microbiology Department, University of Medicine and Pharmacy “Victor Babes” Timisoara
2
Epidemiology Department, University of Medicine and Pharmacy “Victor Babes” Timisoara

ABSTRACT
Aims: The aim of our study was to determine the prevalence of ESBL producing germs, especially of Klebsiella strains, isolated frompatients hospitalised in Intensive Care Unit (ICU), and their associated resistance patterns.
Methods: Identification of germs and extensive antimicrobial tests (by dilution antimicrobial susceptibility tests) were performed by
the Vitek2 system (bioMerieux France). For ESBL detection we performed the Vitek ESBL test (AST-GN27 cards), which includes
cefotaxime and ceftazidime, alone and in combination with clavulanic acid. Results: From 835 pathological samples we isolated 548
microbial strains with nosocomial potential. 285 from these strains were represented by Enterobacteriaceae, from which, 48,07%(137 strains) were extended spectrum beta-lactamase (ESBL) producers. The highest percentage was noticed in the case of ESBL
producing Klebsiella pneumoniae- 78 strains (56,93%).
Conclusions: The routine detection of the beta-lactam resistant phenotypes Klebsiella pneumoniae clinical isolates is particularly
important because of therapeutical problems related to acquired resistance in this species.
Keywords: Klebsiella pneumoniae, infection, resistance phenotypes

INTRODUCTION
The introduction of the third-generation cephalosporins into
clinical practice in the early 1980s was heralded as a major
breakthrough in the fight against beta-lactamase-mediatedbacterial resistance to antibiotics. These cephalosporins had
been developed in response to the increased prevalence ofbeta-lactamases in certain organisms, for example, ampicillinhydrolyzing TEM-1 and SHV-1 beta-lactamases in Escherichia
coli and Klebsiella pneumoniae (1).
Beta-lactamases are most commonly classified according
to two general schemes: the Ambler molecular classification
scheme and the Bush-Jacoby-Medieros functional classification
system (2, 3, 4). The Ambler scheme divides beta-lactamases
into four major classes (A to D). The basis of this classification
scheme rests upon protein homology (amino-acid similarity),
and not phenotypic characteristics. In the Ambler classification
scheme, beta-lactamases of classes A, C and D are serine betalactamases. In contrast, the class B enzymes are metallo-betalactamases. The Bush-Jacoby-Medeiros classification schemegroups beta-lactamases according to functional similarities(substrate and inhibitor profile). There are four main groups
and multiple subgroups in this system. This classification
scheme is of much more immediate relevance to the physician
or microbiologist in a diagnostic laboratory because it considers
beta-lactamase inhibitors and beta-lactam substrates that areclinically relevant.

A commonly used working definition is that the ESBLs arebeta-lactamases capable of conferring bacterial resistance to the
penicillins, first-, second-, and third-generation cephalosporins,
and aztreonam (but not the cephamycins or carbapenems) byhydrolysis of these antibiotics, and which are inhibited by betalactamase inhibitors such as clavulanic acid (3).
OBJECTIVES
The aim of our study was to determine the prevalence of
ESBL producing germs, especially of Klebsiella strains, isolated
from patients hospitalised in Intensive Care Unit (ICU), and theirassociated resistance patterns.
METHODS
We collected from ICU (County Hospital, Timisoara, Romania), between 01.03.2009-04.12.2009 a total number of
835 samples (538 bronchoalveolar aspirates, 101 urines, 3
peritoneal fluid, 4 cerebrospinal fluid, 16 blood, 173 woundsecretions), from 207 patients with various diagnosis, the mostprevalent ones being polytrauma, cerebral stroke, neoplasia,
acute abdomen, burns.
Isolation on conventional culture media and identificationof germs were performed at the hospital laboratory. Confirmation of identification tests (Vitek2 system-bioMerieux France),
as well as extensive antimicrobial tests (by dilution antimicrobial susceptibility tests) were performed at the University

Received July 2010. Accepted September 2010. Address for correspondence: Delia Muntean, Microbiology Department, “Victor Babes” University of Medicine andPharmacy Timisoara, Eftimie Murgu Square No. 2A, 300041, Timisoara, Romania

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Fiziologia - Physiology  2010.20.3 (67)22Microbiology Department’s laboratory. For ESBL detection
we performed the Vitek ESBL test (AST-GN27 cards), which
includes cefotaxime and ceftazidime, alone and in combination
with clavulanic acid. A predetermined reduction in the growthof the cefotaxime or ceftazidime wells containing clavulanic
acid, compared with the level of growth in the well with the
cephalosporin alone, indicates a positive result. Sensitivity
and specificity of the method exceed 90% (5).

285 from these strains were represented by Enterobacteriaceae, from which, 48, 07% (137 strains) were extended spectrum beta-lactamase (ESBL) producers. The highest percentage
was noticed in the case of ESBL producing Klebsiella pneumoniae- 78 strains (56,93%). The majority of these strains wereinvolved in respiratory and urinary tract nosocomial infections
and all of them have associated other resistance phenotypes
as well (table 3).

RESULTS
From 835 samples (bronchoalveolar fluids, wound secretions, urines, etc.) we isolated 548 microbial strains with nosocomial potential (Figure1, Tables I and II).

Table III. Resistance phenotypes in Klebsiella pneumoniae isolates

76.53%
positivesam ples100%

Klebsiellapneumoniae

23.47% sterile
sam ples50%
0%Fig. 1. Positive pathological samples
Table I. Klebsiella pneumoniae in positive samples

Pathologicalsamplesbronchoalveolar
aspirates
urineswound secretions
peritoneal fluid
cerebrospinal fluid
blood

Positive samples
514

95.53%

Klebsiellapneumoniae
117
22.76%

32
84
3
1
5

31.68%
48.55%
100%25%
31.25%

146-

43.75%
7.14%-

Table II. Isolated germs from ICU department

isolated germs

Strains
No.
%
139

25.36

E.coli66

12.04

Klebsiellapneumoniae
Klebsiella oxytoca

137

25.00

7

1.27

Pseudomonas
aeruginosa

51

9.30

Acinetobacter baumannii

75

13.68

Other
Enterobacteriaceae

75

13.68

S. aureus

MDR strains
No.
%

MRSA
61
ESBL
21
ESBL
78
ESBL
3
Carbapenems
resistant
22
Carbapenems
resistant
44
ESBL
35

2010.20.3 (67)  Fiziologia - Physiology

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23

43.88

31.81
56.93
42.85
43.13

58.66

47.94

137

ESBL+ KTGANt+cross resistance Fq + TER+SXTR
ESBL+ impermeability
cephamycins + KTG+ partiallyresistant Fq + TER
78
ESBL+ KTGANt+cross
ESBL
resistance Fq + TER
56.93%
ESBL + KTANt + crossresistance Fq
ESBL (CTX-M) + KTGNt +partially resistant Fq +SXTR
ESBL+ KTGANt+crossresistance Fq
ESBL+ impermeability cephamycins+ partially resistantFq + TER+SXTR
ESBL+TER+SXTR
ESBL+SXTR

19
24.35%
4
5.12%
11
14.11%
22
28.21%
33.84%
14
17.94%
3
3.84%
1
1.28%
1
1.28%

Legend: ESBL-extended spectrum beta-lactamase, K-resistance to kanamycine,
G-resistance to gentamycine, T- resistance to tobramycine, A-resistance to amikacin,
Nt-resistance to netilmicin, Fq-fluoroquinolone, SXT-trimethoprim-sulfamethoxazole,TEtetracycline

DISCUSSIONSIn our study 78 strains (56,93%), from 137 Klebsiellapneumoniae strains, were extended spectrum beta-lactamase
producing.
There is a considerable geographical difference in the occurrence of ESBLs in the European countries. Within countries,
hospital-to-hospital variability in occurrence may also be marked(6). In a SENTRY worldwide surveillance program report, ESBL
phenotypes were detected in 45% of K. pneumoniae strains from
Latin America, 23% from the western Pacific, 23% from Europe,
8% from the United States, and 5% from Canada (7).
The first ESBL was identified in Germany in 1983; sincethen, over 200 variants of the clavulanic acid-inhibited form of
the enzyme have been described worldwide. The most common
extended-spectrum phenotypes arise from point mutations in the
blaTEM, blaSHV, or blaCTX genes resulting in alterations of the primaryamino acid sequence of the enzyme (8). Since these genes are
generally found on plasmids, many of the organisms that harbor
ESBLs also are resistant to other classes of antibiotics, such asaminoglycosides, fluoroquinolones, tetracyclines, chloramphenicol, and sulfonamides (9). Many strains were multiresistant, being
sensitive only to imipenem (7).
In this study, all ESBLs have been associated with other23

resistance patterns like: resistance to quinolones, trimethoprimsulphamethoxazole, aminoglycosides or tetracycline. Only 7
strains (8,97%) were cefoxitin resistance and we did not isolate
any strain with imipenem resistance.
CONCLUSIONS
1. The routine detection of the beta-lactam resistant phenotypes Klebsiella pneumoniae clinical isolates is particularly
important because of therapeutical problems related to acquired
resistance in this species. It can help clinicians prescribe empiric
antibiotic therapy and can help microbiologists monitor trends in
antimicrobial resistance, such as the dissemination of ESBLs.
2. Carbapenems remain active against all our ESBL producing Klebsiella pneumoniae strains. This information should beconsidered when prophylactic therapy is recommended in thisdepartment.
3. Alteration of antibiotic susceptibility breakpoints may
become necessary but need
to be carefully considered in combination with pharmacokinetic, pharmacodynamic and clinical data.
REFERENCES

1. Paterson DL, BonomonRA. Extended-Spectrum β-Lactamases:
a Clinical Update. Clinical Microbiology Reviews., 2005; 18(4):657-686.
2. Ambler RP, Coulson AF, Frere JM, Ghuysen JM, Joris B,
Forsman M, Levesque RC, Tiraby G, Waley SG. A standard
numbering scheme for the class A beta-lactamases. Biochem. J.
1991; 276:269-270.

3. Bush KG, Jacoby A, Medeiros AA. A functional classification
scheme for beta-lactamases and its correlation with molecular
structure. Antimicrob. Agents Chemother., 1995; 39:1211-33.
4. Rasmussen BA, Bush K. Carbapenem-hydrolyzing betalactamases. Antimicrob. Agents Chemother. 1997; 41:223-232.
5. Livermore DM, Struelens M, Amorim J, Baquero F, Bille J,
Canton R, Henning S, Gatermann S, Marchese A, Mittermayer
H, Nonhoff C, Oakton KJ, Praplan F, Ramos H, Schito GC,
Van Eldere J, Verhaegen J, Verhoef J, Visser MR. Multicentre
evaluation of the VITEK 2 Advanced Expert System for interpretive reading of antimicrobial resistance tests. J. Antimicrob.
Chemother., 2002; 49:289-300.
6. Babini G S, Livermore D M. Antimicrobial resistance amongst
Klebsiella spp. collected from intensive care units in Southernand Western Europe in 1997-1998. J. Antimicrob. Chemother.,
2000; 45:183-189.
7. Winokur PL, Canton R, Casellas JM, et al. Variations in theprevalence of strains expressing an extended-spectrum-betalactamase phenotype and characterization of isolates from Europe, the Americas, and the Western Pacific region. Clin InfectDis., 2001; 32(2): S94-S103.
8. Bush K. New ß-lactamases in Gram-negative bacteria: diversity and impact on the selection of antimicrobial therapy. Clin.
Infect. Dis., 2001; 32:1085-89.
9. Nathisuwan S, Burgess D S, Lewis J S. Extended-spectrum
beta-lactamases: epidemiology, detection, and treatment. Pharmacotherapy., 2001; 21:920-928.

Acknowledgments
These data are part of the PNII 42121 project: “Molecular characterization of multidrug resistant strains, hospital or community
acquired, collected from south-west Romania”.

TULPINI DE KLEBSIELLA PNEUMONIAE PRODUCĂTOARE DE BETA
LACTAMAZE CU SPECTRU EXTINS IZOLATE DINTR-O SECŢIE DE TERAPIEINTENSIVĂREZUMAT
Scop: În studiul nostru am urmărit determinarea prevalenţei tulpinilor producătoare de BLSE, în special a celor din genul Klebsiella,izolate de la pacienţi internaţi într-o secţie de Terapie Intensivă.
Metode: Pentru identificarea germenilor şi testarea sensibilităţii la antibiotice am utilizat sistemul automat Vitek2 (bioMerieux France).
Detecţia tulpinilor producătoare de BLSE s-a realizat cu ajutorul cardurilor AST GN27 pentru sistemul Vitek 2, care includ testarea
simultană la cefotaxim şi ceftazidim asociate sau nu cu acid clavulanic.
Rezultate: Din 835 produse patologice am izolat 548 tulpini microbiene cu potenţial nosocomial. 285 dintre aceste tulpini au fost
reprezentate de Enterobacteriaceae, din care, 48,07% (137 tulpini) au fost producătoare de beta-lactamaze cu spectru extins (BLSE).
Cel mai mare procent de tulpini BLSE a fost înregistrat pentru Klebsiella pneumoniae- 78 tulpini (56,93%).
Concluzii: Detectarea de rutină a fenotipurilor de rezistenţă la beta-lactamine pentru tulpinile de Klebsiella pneumoniae izolate este
importantă datorită problemelor terapeutice create de rezistenţa dobândită pentru această specie.

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Fiziologia - Physiology  2010.20.3 (67)24

RISK ASSESSMENT STUDY ON OCCUPATIONAL
METHYL-ETHYL KETONE EXPOSURE
DELIA CHEPTANARIU1,2, CORNELUTA FIRA-MLADINESCU2, LILIANA SARB3, STEFANIAROSCA3, CARMEN OROS3, SORINA DOROFTEI2, OANA SUCIU2, BRIGITHA VLAICU21OMNISAN Polyclinic Timisoara
2
Research Centre in Preventive Medicine, „Victor Babes” UMF Timisoara
3
Institute of Public Health “Prof. Dr. Leonida Georgescu” Timisoara

ABSTRACTThe study assesses the presence of methyl-ethyl-ketone in a factory producing polyurethanic foam and estimates the potential healthrisk associated to its inhalation. Values obtained at the workplace were compared to legal occupational exposure limits (OELs) and tothe maximal exposure values not involving health risks (HBEL - health-based exposure limits). The hazard index was calculated (HI).
When reported to OELs, a maximal HI of 1.2 is observed in case of 15 minutes exposure and of 1.48 in case of 8 hours exposure.
When reported to HBEL, the hazard index is much higher than the cut-off of 1, even when the lowest recorded value is taken into
account. Thus, HI values are in the range of 4.47 and 21.35 for records on 15 minutes, whereas for 8 hours exposure HI is between
5 and 17.41. Even though there is no risk for acute effects, the possibility of some chronic effects imperatively requires the identification of factors which favour concentration increase in the occupational environment.
Key words: occupational exposure limits, health-based exposure limits, hazard index

INTRODUCTION
Methyl-ethyl-ketone (MEK) is used as a solvent in the application of protective coatings (varnishes) and adhesives (glues
and cements), in magnetic tape production, in smokeless powder manufacture, in the dewaxing of lubricating oil, in vinyl film
manufacture, and in food processing. Its use as a component inadhesives used to join PVC pipes is a potential route for entry of
the chemical into potable water (1). It is also commonly used inpaint removers, cleaning fluids, acrylic coatings, pharmaceuticalproduction, and colorless synthetic resins, and as a printing catalyst
and carrier (2). MEK has been detected as a natural componentof numerous foods, including milk, nuts, cheese, bread and nectarines (1,9,21). MEK is also found in tobacco smoke and volatile
releases from building materials and consumer products (1).
Short-term inhalation exposure (4 hours) to MEK underexperimental conditions at or near 200 ppm (590 mg/m3) does
not appear to pose an increased risk of neurologic or irritation
symptoms (4,5,6,7).
Several occupational studies examined the effects of chronic
exposure to MEK. Freddi et al. reported that MEK exposure was associated with slightly, but not statistically significant, reduced nerve
conduction velocities (distal axonopathy) and other symptoms suchas: headache, loss of appetite and weight, gastrointestinal upset,dizziness, dermatitis, and muscular hypotrophy, but no clinicallyrecognizable neuropathy (8). The human case reports and somestudies provide limited and equivocal evidence that repeatedexposure to MEK in the workplace increases the hazard for persistent neurological impairment (13,14). The available occupational
studies are limited by inadequate characterization of exposure,
multiple solvent exposure, and study design problems.

Although there is some suggestion of increased risk forsome cancers (including bone and prostate) and multiple solvent
exposures that includes MEK, there is no clear evidence for a
relationship between these cancers and MEK exposure alone.
According to EPA’s (United States Environmental Protection
Agency) draft revised cancer guidelines, the hazard descriptor“data are inadequate for an assessment of human carcinogenic
potential” is appropriate for MEK (17,18).
The present study assesses the presence of MEK in occupational environments and estimates the potential health risk associated to its inhalation. For this, the targeted objectives were:- determining MEK concentration at the workplace;- comparing obtained levels to limits stated by various organisms;- evaluation of chronic carcinogenetic and non-carcinogenetic
risks determined by occupational MEK exposure.
MATERIAL AND METHODS
The study was performed in a polyurethanic foam producing
factory, during the period between 2001 and 2005, as a part of an
occupational harmful agents exposure assessment programme.
The investigated departments were as follows: foaming, foamprocessing, injection equipments, rigid, casting, confections,valuing, mechanical-energetically workshop, technical quality
control department, research department.
Substance detection procedures were performed according
to methodological specifications edited by the Ministry of Health(10,11,12,22). MEK was determined by spectrophotometry, using
an UV absorption spectrophotometer.
MEK concentrations are presented as point concentrations(C15 min, resulting from collections made within 15 minutes) andweighted mean concentrations (C 8 hours, representing mean

Received August 2009. Accepted October 2009. Address for correspondence: Delia Cheptanariu, MD, Research Centre in Preventive Medicine, V. Babes no 16,Timişoara 300226, phone/fax. 0256-490412, e-mail: dorodel@yahoo.com

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exposure during an 8 hours working period). Result analysis
was made by:
1. Comparing obtained concentrations with
- OELs (loccupational exposure limits) in Romania, according
to present legislation (23);- RfC (inhalation reference concentration). This is defined as
the concentration to which human population may be exposed byinhalation, during a lifetime, without a considerable risk regarding
non-carcinogenetic effects (19,20) and it is included in IRIS-EPA
monographs (Integrated Risk Information System - United States
Environmental Protection Agency)- HBEL; health-based exposure limits (limits of exposures
without health risk). HBEL was calculated by the Santa Clara
Centre for Occupational Safety and Health (SCCOSH) using themethodology for environmental risk assessment, developed by theUnited States Environmental Protection Agency (EPA) (16).
2. Calculation of the hazard index (HI), for the assessment ofchronic non-carcinogenetic effects, by reporting the concentrations of substances detected at the workplace to limits imposedby the Organisms involved in environmental and workplace
protection. Hazard index higher than 1, represents a high healthrisk for chronic effects.
RESULTS
Chemical laboratory tests performed on samples collectedin the factory producing polyurethanic foam revealed the presence of MEK in one of the 10 investigated departments, namely
the department of casting. The limits of detected values arepresented in Table I.
Table I. MEK concentrations detected in the factory producing
polyurethanic foam (mg/m3)

Substance No. positive samples
MEK

36

C8hours

C15min
Min.
value
76

Max.
value
363

Min.
value
85.3

Max.
value
296

For occupational risk assessment determined by the presence of MEK, values were reported to concentrations which
certain authorities or legal provisions regard as without risk or
posing a minimal risk (Table II).
Table II. Exposure limits stated by various Public Regulatory
Organisms in the field (mg/m3)

Substance

OELs
15min

OELs
8hoursRfC

HBEL
noncancer

HBEL
cancer

MEK

300

200

5

17

-

Note: OELs – occupational exposure limits (Romania); RfC (inhalation reference
concentration) – concentration without chronic non-carcinogenetic effects in the general
population; HBEL (health-based exposure limits) – exposure limits without occupational
health risk.

Table III presents the hazard index (HI), calculated withreference to various norms, established by different Organisms.
When reported to occupational exposure limits OELs, a hazard
index slightly over 1 is observed. On the contrary, in case ofreporting to HBEL, the hazard index is well over the cut-off ofone, even when the lowest recorded concentration value istaken into account. Thus, HI values for records on 15 minutes
are between 4.47 and 21.35 and for the 8 hours exposure, it isbetween 5 and 17.41. Reporting to RfC shows high values overthe unitary limit for the hazard index.
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Table III. The hazard index (HI) calculated according to exposure limits
stated by various Public Regulatory Organisms in the field
Substance

MEK

HIOELs15min0.251.21

8hours
0.421.48

HIRfC
15
min
15.272.6

HIHBEL

8 hours 15 min
17.0659.2

4.4721.35

8
hours
517.41

Note: OELs – occupational exposure limits (Romania); RfC (inhalation reference
concentration) – concentration without chronic non-carcinogenetic effects in the general
population; HBEL (health-based exposure limits) – exposure limits without occupational
health risk.

DISCUSSION
Even if present scientific evidence do not support with certainty the harmful effects of MEK, the precaution principle andrisk assessment are imperative. This position is justified by thepercent of samples in our study with concentration values over
the occupational exposure limits (OELs) stipulated by Romanianlegislation. These are limits of time weighted mean, for a specified
period (8 hours or on short term – maximum 15 minutes) of the
air concentration of the chemical agent, in the breathing areaof the worker (23). Analysing the results obtained by our study,
from the perspective of these norms, we found that, even though
OELs15min was over the highest limit in 8.33% of the samples,
OELs8hours presented values over the limit in 33% of cases, thus
revealing the risk of chronic exposure.
Limits of occupational exposure are the highest legally acceptable values for human exposure to a toxic substance. Very
many of these values are very close to the maximal acceptablevalues, with a small safety margin for the risk of long term effects(3). General population exposure to the same air pollutant ismuch more strictly regulated. The proof is the RfC value which
is 40 times lower than OELs8 hours for MEK.
Santa Clara Centre for Occupational Safety and Health (SCCOSH) considers that exposure of workers to toxic substances
must not be higher at the workplace than in any other ordinary
place where exposure is possible. For this reason, values corresponding to a very low disease risk in an occupational environment
were calculated for a series of substances starting from the IRIS,HEAST and California EPA databases and adjusting RfC (inhalation reference concentration) by changing the exposure time inoccupational exposure (8 hours/day, 240 days/year, for a 40 years
period) (16). Analysing these data, the high difference between
legal provisions and health protection levels may be observed.
Risk assessment is the scientific, systematic characterization of potential adverse effects resulting from human exposures
to risk agents or situations. Risk assessment is the basis forregulating quality criteria, for elaborating standards and decision
making according to local realities (15).
For characterization of the risk of acute effects the point(momentary) exposure is used, whereas for chronic noncarcinogenetic effects is advisable to take into account the mean
exposure during a period of time. Our results show that thehazard index calculated by reporting to HBEL recorded values
much higher than the limit of 1, even in cases of the lowest concentrations detected. This aspect suggests a very high risk forchronic non-carcinogenetic diseases. In the case of calculation
by reporting to OELs15min, a maximal IH value of 1.2 is recorded,with no acute pathology problems. It may be observed that valFiziologia - Physiology  2010.20.3 (67)26ues for momentary exposure do not differ very much from thosedetermined by medium exposure, fact which may be consideredan alarm signal for the exposed staff members.
As for carcinogenetic effects, there is no assessment
methodology in the case of MEK, as there are not enough data
supporting the carcinogenicity of this substance and thus, URL(Unit Risk – the additional number of cancer cases determinedby inhalator exposure to a concentration unit during a lifetime).
CONCLUSIONSThe study identifies a critical point in the technologicalprocess regarding MEK exposure. Even though pollution withthis substance is limited, identification of factors favouring theincrease of its occupational concentration is mandatory.
REFERENCES

1. Agency for Toxic Substances and Disease Registry. Toxicological profile for
2-butanone. Atlanta, GA: U.S. Department of Health and Human Services,
Public Health Service, 1992.
2. Budavari S. The Merck Index: an encyclopaedia of chemicals, drugs,
and biologicals. 13th edition. Merck & Co., Inc., Whitehouse Station, New
Jersey, 2001.
3. California Environmental Protection Agency, Reproductive and Cancer
Hazard Assessment, Branch Office of Environmental Health Hazard
Assessment. Occupational Health Hazard Risk Assessment Project for
California: Identification of Chemicals of Concern, Possible Risk Assessment Methods, and Examples of Health Protective Occupational Air
Concentrations, 2007. Online at: http:// www.cdph.ca.gov/ programs/ hesis/
documents/riskreport.pdf
4. Dick R, Setzer J, Wait R, et al. Effects of acute exposure of toluene andmethyl ethyl ketone on psychomotor performance. Int Arch Occup Environ
Health 1984; 54: 91-109.
5. Dick RB, Brown WD, Setzer JV, et al. Effects of short duration exposuresto acetone and methyl ethyl ketone. Toxicol Lett 1988; 43: 31-49.
6. Dick R, Setzer J, Taylor B, et al. Neurobehavioral effects of short duration
exposures to acetone and methyl ethyl ketone. Br J Ind Med 1989; 46:
111-121.
7. Dick RB, Krieg Jr EF, Setzer J, et al. Neurobehavioral effects from acuteexposures to methyl isobutyl ketone and methyl ethyl ketone. Fundam Appl
Toxicol 1992; 19: 453-473.
8. Freddi A, Paci A, Vittori O, et al. Clinical and electromyographic study ofworkers exposed to methyl ethyl ketone vapour. Ann Med Perugia 1982;
73: 111-136.
9. Hazardous Substance Data Bank. National Library of Medicine,
Bethesda, MD, 1999. Online at: http://toxnet.nlm.nih.gov/cgi-bin/sis/

htmlgen?HSDB
10. Ministerul Sănătăţii Publice. Caiet metodologic – Metode de investigareîn domeniul Medicinii Muncii. Bucureşti, 1991.
11. Ministerul Sănătăţii Publice. Atmosfera locului de muncă - cerinţe
generale pentru performanţa procedurilor de măsurare a agenţilor chimici
SR EN 482. Bucureşti, 2002.
12. Ministerul Sănătăţii Publice. Metode de referinţă pentru măsurarea
concentraţiei agenţilor chimici şi fizico-chimici în mediul profesional.
Bucureşti, 2005.
13. Mitran E, Callender T, Orha, B, et al. Neurotoxicity associated with occupational exposure to acetone, methyl ethyl ketone, and cyclohexanone.
Environ Res 1997; 73: 181-188.
14. Oleru UG, Onyekwere C. Exposures to polyvinyl chloride, methyl
ketone and other chemicals; The pulmonary and non-pulmonary effect. Int
Arch Occ Environ Health 1992; 63: 503-507.
15. Omenn GS, Faustman EM. Risk assessment, risk communication andrisk management, in Detels R (ed): Oxford Textbook of Publics Health Third
Edition, Oxford University Press, 1997; 971-988.
16. Santa Clara Centre for Occupational Safety and Health, Health Based
Exposure Limits Committee. Chemical Exposure Guidelines, version 9,
Internet Edition 1995.
17. United States Environmental Protection Agency. Guidelines for carcinogen risk assessment [review draft]. Risk Assessment Forum, Washington,
DC; NCEA-F0644, 1999. Online at: http://www.epa.gov/ncea/raf/cancer.
htm
18. United States Environmental Protection Agency. Toxicological review ofMethyl ethyl ketone: In Support of Summary Information on the Integrated
Risk Information System (IRIS), September 2003.
19. United States Environmental Protection Agency. Integrated Risk
Information System (IRIS); Compare IRIS Values. Online at: http://cfpub.
epa.gov/ncea/iris/compare.cfm
20. United States Environmental Protection Agency, Office of Researchand Development, Office of Health and Environmental Assessment. Methods for derivation of inhalation reference concentrations and application of
inhalation dosimetry. Washington DC: EPA/600/8-90/066F, 1994.
21. World Health Organization. Methyl ethyl ketone. Environmental HealthCriteria, volume 143, 1992.
22. ***. H.G. 1218/2006 privind stabilirea cerinţelor minime de securitateşi sănătate în muncă pentru protecţia lucrătorilor împotriva riscurilor legate
de prezenţa agenţilor chimici. Monitorul Oficial al României nr. 845 din 13
octombrie 2006.
23. ***. Ordinul Ministerului Muncii şi Protecţiei Sociale 508/2002 şi Ordinul
Ministerului Sănătăţii Publice 933/2002 privind aprobarea Normelor
generale de protecţie a muncii. Monitorul Oficial al României nr. 880 din 6
decembrie 2002.

STUDIU DE EVALUARE A RISCULUI DETERMINAT DE EXPUNEREA LA
METIL-ETIL-CETONĂ ÎN MEDIUL PROFESIONALREZUMAT

Studiul evaluează prezenţa metil-etil-cetonei într-o întreprindere de producere a spumelor poliuretanice şi estimează riscul potenţial
pentru sănătate asociat cu inhalarea acesteia. S-au comparat valorile obţinute la locul de muncă cu valorile limită de expunere
profesională (VLE) prevăzute de legislaţie şi cu valorile limită de expunere fără risc pentru sănătate (HBEL - health-based exposure
limits). S-a calculat indicele de hazard (IH). Când raportarea s-a făcut la valorile limită de expunere profesională (VLE), se observă
un indice de hazard de maximum 1,2 în cazul expunerii pe 15 min şi 1,48 în cazul expunerii pe 8 ore. În cazul raportării la HBEL,
indicele de hazard depăşeşte cu mult pragul de 1, chiar dacă se ia în calcul valoarea minimă a concentraţiei înregistrate. Astfel,
valorile IH se situează la înregistrările pe 15 minute între 4,47 şi 21,35 iar pentru expunerea timp de 8 ore, acesta este cuprins între
5 şi 17,41. Deşi nu există riscul unor efecte acute, posibilitatea efectelor cronice impune identificarea factorilor care favorizează
creşterea concentraţiei în mediul ocupaţional.
Cuvinte cheie: valori limită de expunere profesională, valorile limită de expunere fără risc pentru sănătate, indice de hazard
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MAJOR FACTORS INFLUENCING LIFE QUALITY –RATIONAL NUTRITION AND FITNESSCRISTIAN NEGREA„Victor Babeş” University of Medicine and Pharmacy, Timişoara, Romania

ABSTRACTIn the present paper we revealed the synergic link between rational nutrition and fitness, for the prevention, improvement and treatment
against obesity. Foods represent one of the most important environmental factors influencing human health. They have the peculiarity
of continuous and slow action upon the human body, influencing physical, psychological development and work capacity. Together
with rational nutrition, physical activity is a major factor participating in the accomplishment of a balanced lifestyle management,
preventing some risk behaviors. Medical statistics show that the number of obese individuals is constantly increasing. Overweight
trigger the increased risk for many diseases such as diabetes mellitus, high blood pressure, atherosclerosis and especially ischemic
cardiopathy, gallbladder diseases and even some forms of cancer. Fitness is presented as a health related concept connected to
keeping and promoting health, with reference to the beneficial effects upon the organism.
Key words: rational nutrition, obesity, fitness

INTRODUCTION
The human body develops and maintains its complex structure by a permanent substance exchange with the environment.
From the environment, man receives the oxygen required forburning processes occurring in living cells, as well as nutritional
factors or trophins from which the energy needed for maintaining
body functions and activities is obtained, and also reparatory andreconstruction materials used for repairing used tissues or for
growth during childhood. Every second, within the human body,
millions of cells die and are born.
Aliments represent one of the most important factors of theenvironment influencing human health. They characteristicallyact continuously and slowly upon human organisms, influencing
physical, psychological development and work capacity. Fromfoods entering the composition of a daily portion, the body only
keeps and uses a certain percent, the rest being eliminated.
This percent of foods needed for the organism to maintain life
and work capacity is designated as nutritive factors or trophins.
The richer in nutritive factors the more useful and precious an
aliment is for man.
All nutritional factors needed by the organism are now known,
as well as the foods containing them and the respective proportions, their chemical composition, and the necessary quantity
during various physiological states or environmental conditions.
Because no food contains all nutritional factors needed by theorganism, the necessity for a complete and varied nutrition, also
known as rational nutrition, becomes imperative (1).
For maintaining the energetic balance, physical activity is
indispensable, being the most effective way to consume ingested
calories. Studies show that physical exercises are especially
useful for maintaining a normal body weight, together with anadequate diet.
A beneficial physical activity program may be achieved byintensive walking sessions of 30-60 minutes a day. The movement may also be performed in more rounds of 15-30 minutes.
Other forms of aerobic physical exercise may be used, such as
gardening, running, bicycle riding, swimming, and fitness.
Sport is recommendable, especially for young people andchildren predisposed to obesity, it being extremely useful in preventing obesity. The exclusive preoccupation of obese people forfood may be replaced with something else, such as sports. These
individuals must not be isolated from their peers because thecompany of other young people creates relations and preoccupations susceptible of distracting the attention from the obsessive
need for sweets and candies. When regularly practiced, sport
may have effects against the accumulation of fat tissue and it may
also create a new preoccupation and a different rhythm of living
which may replace sedentary life and the overrating of pleasuresprovided by meals. The possibilities of practicing sports must be
used starting during childhood, especially in the case of childrenof obese families. Different preoccupations, such as artistic ones,
may also distract the attention from the perpetual obsession of
feeding. They do not pose the disadvantage of sports, namely
the danger of stimulating the already increased appetite of theobese or obesity predisposed individual, due to physical efforts
which increase the calories and food needs, implicitly (2).
OBESITY. OBESOGENIC RISK FACTORS. OBESITY
RISKS
Obesity is the excessive increase of the fat tissue mass,
manifested by the body weight increase with more than 20% of

Received August 2009. Accepted January 2010. Address for correspondence: Dr. Cristian Negrea, „Victor Babeş” University of Medicine and Pharmacy, Timişoara, Eftimie
Murgu Square No. 2A, 300041, Romania

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Fiziologia - Physiology  2010.20.3 (67)28the normal weight. Body weight values between the upper limitof the ideal weight and the inferior limit of obesity are considered as overweight or excess weight. Statistics show that the
number of obese persons is continuously increasing, especiallyin developed societies.
Genetical factors may play a major role in the occurenceof obesity or in the increased susceptibility to its occurence. In
many severe obesity forms, starting during childhood, the total
number of adipocytes is increased.
Among changeable factors, the first and most obvious isoverfeeding, i.e. the use of an excessive quantity of food. It has
been demonstrated that an increase of the calories intake leads
to the increase of the fat deposits. A second important factor isthe lack of physical activity, a sedentary, inactive lifestyle. The
low energy consumption at work and during free time highly
contributes to the occurrence or increase of the genetic misbalance. In the occurrence of obesity, an important role is played bypsychological, family and socio-professional factors. For somepeople, food ingestion represents a psychological support, away of coping with the problems they are confronted with or withvarious negative emotions. On the other hand, the family environment may constitute the transmission route for some wrong
habits and attitudes towards foods. Sedentary professions, theincreased use of cars, the excessive TV watching indirectly exerts
a significant influence on the alarming spread of obesity. One of
the rather frequently incriminated factors is alcohol consumption.
This is due to the excess of calories that it determines, and whichusually is added to a food excess.
Extra kilos cause an increased risk for many diseases,
such as diabetes mellitus, arterial hypertension, aterosclerosis
in general and ischemic heart disease in particular, gallbladderdiseases and even some forms of cancer. In fact, not only the
degree of ponderal excess, but also the regional distributionof the fatty tissue influences to an important extent the risk of
disease. Obesity is frequently accompanied by the premature
wear of joints and by varicose veins disease of the lower limbs.
Unfortunately, the estetic inconvenience cannot be excluded
either. Very often, obese people suffer emotionally due to theirstigmatization by society. Last but not least, the physical andsocial handicap plays a role. Many obese people reach the point
when they cannot help themselves, they move with great difficultyor are even in the impossibility to move and suffer intensely due
to their isolation from the rest of the community (3).
FITNESS AND ITS EFFECTS
Fitness is a health related concept which refers to its maintaining and promoting. Being healthy does not only mean not
being ill. Many people are content if nothing hurts, but healthdoes not only refer to the physical, but also to the psychical,
mental and social status. Moreover, if the aging process cannot
be stopped, it can certainly be slowed down.
The favorable effects of sports on the health of the individualand community and on human activity in general, are known andscientifically proven. Many studies describe the mechanisms by
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which physical exercise influences the functions and systemsof the human body. “The European Chart of Sport”, in article 2,
defines sport as being “the total of physical activities aiming at:
the expression or improvement of physical condition (fitness) andof mental wellbeing; construction of social relationships; obtaining
sport results in competitions at various levels” (4).
Regularly practiced physical activity determines maintaining or improving of the structure of various tissues and organs(muscles, tendons, heart), improves functions and counterweights deteriorations occurring due to inactivity, sedentary life
and aging.
The term fitness defines physical condition, and it is quasisimilar with health. When speaking of health producing effects offitness, reference is made to positive psychological changes aswell as to obvious and beneficial influences on some diseases,
best known being the diseases of vertebral disks, endocrineand especially cardiovascular diseases. In practice, because it
best responds to the effect of physical effort, the ischemic heartdisease must constitute the main objective of any motion based
programme, regardless of age, gender and of the way physicalactivity is being practiced on an individual basis or under specialized supervision. The beneficial effects of fitness are describedon various apparatuses and systems:- The cardiovascular apparatus: increases the quantity ofblood which the heart can pump; increases the quantity of bloodin the blood vessels; blood becomes more fluid and circulateseasier through arteries and veins (beneficial in arteriosclerosis,
ischemic heart disease, arterial hypertension).
- The respiratory apparatus: the lung becomes able toventilate a larger quantity of air per minute (helps in chroniclung diseases).
- The skeletal muscles: increases the force, resistance andpower; muscles”dissolve” more slowly with aging (helps in lumbar
diseases, fractures produced by falling, in elderly people).
- The adipose tissue: decreases the total fat mass aroundviscera.
- The glucidic metabolism: increases the capacity of themuscle to take glucose from the blood (helps in diabetes).
- The lipidic metabolism: increases the capacity of the muscle
to take fats from the blood and to use them for procuring energy(helps in arteriosclerosis)- The defense function of the organism: the capacity of theimmune system to respond to biotic and abiotic aggressions
improves.
- Digestive processes: the intestinal transit improves, eliminating constipation.
- The nervous system: the reaction speed and the promptitude of responses to various stimuli are improved (helps in
fractures by falling, in elderly persons).
- The cognitive functions: the reaction speed and promptitude
of responses to various stimuli improve.
- The psycho-social behavior: the self image, professional
efficacy, family behavior improves and the state of wellbeing andjoy of living occur (helps in depression and anxiety).
Fitness is not synonym with body building. It shares some29exercises with body building but the work intensity and volume
are different. Fitness means a good physical condition and it
includes: walking, running, cycling, stretching, exercises on various devices, relaxation and stretching methods, all performed
under the guidance of a sports teacher (5,6).

REFERENCES

1. Chis O. Healthy nutrition guide, 2004, Ed. AnandaKali, Sibiu
2. Barzu M. Higene, 2002 Ed. Mirton, Timisoara
3. Dragan I. Obesity, 2005, Ed. Bogdana, Bucuresti
4. http://www.gov-sport.ro
5. Barzu M. Exercise Physiology, 2002, Ed. Mirton, Timisoara
6. Barzu M. Medical control and self-control in Physical Educationand Sports, 2002, Ed. Mirton, Timisoara

FACTORI DE INFLUEŢĂ MAJORI AI CALITĂŢII VIEŢII – ALIMENTAŢIA
RAŢIONALĂ ŞI FITNESSULREZUMATÎn cadrul acestei lucrări am evidenţiat legătura sinergică dintre alimentaţia raţională şi fitness, în vederea prevenirii, ameliorării şi
combaterii obezităţii. Alimentele reprezintă unul din cei mai importanţi factori ai mediului extern care influentează sănătatea omului.
Ele au particularitatea de a acţiona continuu şi lent asupra omului, influenţându-i dezvoltarea fizică, psihică şi capacitatea de muncă.
Alături de alimentaţia raţională, activitatea fizică este un factor care participă la realizarea unui management echilibrat al modului deviaţă, prevenind unele comportamente cu risc. Statisticile medicale arată că numărul de obezi este în continuă creştere. Kilogrameleîn plus antrenează cu sine creşterea riscului faţă de numeroase boli cum ar fi diabetul zaharat, hipertensiunea arterială, aterosclerozaîn general şi cardiopatia ischemică în particualar, bolile vezicii biliare şi chiar anumite forme de cancer. Fitnessul este prezentat ca
fiind un concept legat de sănătate, care se referă la menţinerea şi promovarea acesteia, menţionându-se efectele benefice asupraorganismului.
Cuvinte cheie: alimentaţie raţională, obezitate, fitness

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PREVALENCE OF EXTENDED SPECTRUM BETA LACTAMASEPRODUCING KLEBSIELLA PNEUMONIAE STRAINS ISOLATEDFROM THE HOSPITAL AND COMMUNITY
C. PILUŢ1, MONICA LICKER1, ELENA HOGEA1, DORINA DUGĂEŞESCU1, DELIA
BERCEANU-VĂDUVA 1, MIHAELA CRĂCIUNESCU 1, LILIANA DRAGOMIRESCU 1, DELIA
MUNTEAN1, MIHAELA POPA1, ROXANA ZUGRAVU1, F. HORHAT1, MATILDA RĂDULESCU1,
LUMINIŢA BĂDIŢOIU2, CARMEN TATU3, ROXANA MOLDOVAN1
1
Microbiology Department, University of Medicine and Pharmacy “Victor Babes” Timisoara
2
Epidemiology Department, University of Medicine and Pharmacy “Victor Babes” Timisoara
3
Physiology Department, University of Medicine and Pharmacy “Victor Babes” Timisoara

ABSTRACT
Bacterial multidrug resistant (MDR) it is more and more frequently described not only in hospitals but in community as well, representing an alarming phenomenon for the modern medicine through the drastically restriction of therapeutic options. We isolated from ICU(Timisoara Emergency Clinical County Hospital), between March 2009 – December 2009, 137 Klebsiella pneumoniae strains, froma total number of 548 microbial strains, and between January 2009- November 2009 we isolated from community (processed at thelevel of Timisoara Bioclinica Laboratory) 303 Klebsiella pneumoniae strains, from the total number of 3002 microbial strains.
All the Klebsiella pneumoniae strains were phenotyped, with the help of automatic VITEK 2 compact system, using VITEK 2 GP/
GN identification cards and AST cards for antimicrobial sensitivity tests. For ESBL detection we performed the Vitek ESBL test
(AST-GN27 cards).
In both, hospital and community, Klebsiella pneumoniae showed the highest percentage of ESBL production, followed by otherenterobacteria.
Keywords: ESBL, Klebsiella pneumoniae, phenotype

INTRODUCTION
Since first reported in Europe in the early 1980s, extendedspectrum beta-lactamases (ESBLs) have spread worldwide.
When producing these broad-spectrum plasmid-encoded enzymes, organisms become highly effective at inactivating penicillins, most cephalosporins, and aztreonam. Mainly produced by
Klebsiella spp, ESBLs have been isolated worldwide in different
species, most of them belonging to the Enterobacteriaceae.
ESBL-producing bacteria can appear as in vitro susceptible tobeta-lactams by conventional laboratory methods, making thelaboratory diagnosis problematic. [1]In a SENTRY worldwide surveillance program report, ESBL
phenotypes were detected in 45% of K. pneumoniae strains from
Latin America, 23% from the western Pacific, 23% from Europe,
8% from the United States, and 5% from Canada [2].
MATERIAL AND METHOD
We isolated from ICU (Timisoara Emergency Clinical County
Hospital), between March 2009 – December 2009, 137 Klebsiella pneumoniae strains, from a total number of 548 microbial
strains, and between January 2009- November 2009 we isolated
from community (processed at the level of Timisoara Bioclinica
Laboratory) 303 Klebsiella pneumoniae strains, from the total
number of 3002 microbial strains.
Samples were sent to the Microbiology Department Labora-

tory’s of the University of Medicine and Pharmacy “Victor Babes”
Timisoara, where have been processed. All the samples were
cultured on Columbia 5% sheep blood agar (bioMerieux) andselective media Mac Conkey (BioRad). Identification of Klebsiellapneumoniae strains was generally based on morpho-tinctorial
character (gram negative bacilli), cultural and biochemical tests(API 20E galleries) and phenotyped, with the help of automatic
VITEK 2 compact system, using VITEK 2 GP/GN identification
cards and AST cards for antimicrobial sensitivity tests.
For ESBL detection we performed the Vitek ESBL test
(AST-GN27 cards), which includes cefotaxime and ceftazidime,alone and in combination with clavulanic acid. A predetermined
reduction in the growth of the cefotaxime or ceftazidime wells
containing clavulanic acid, compared with the level of growth in
the well with the cephalosporin alone, indicates a positive result.
Sensitivity and specificity of the method exceed 90% [3].
RESULTS AND DISCUSSION
From the total of 548 bacterial strains with nosocomial potential isolated from ICU, 264 were multidrug resistant (MDR)
strains. Distribution of bacterial strains was: 139 S.aureus, 137
Klebsiella pneumoniae, 75 Acinetobacter baumannii, 66 E.coli,
51 Pseudomonas aeruginosa, 7 Klebsiella oxytoca and 73 otherenterobacteria (Figure 1).

Received September 2010. Accepted October 2010. Address for correspondence: C. Pilut, Microbiology Department, “Victor Babes” University of Medicine and Pharmacy
Timisoara, Eftimie Murgu Square No. 2A, 300041, Timisoara, Romania

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1%

13%

S.aureus

26%

9%

Klebsiella pneumoniae
Acinetobacter baumannii
E.coli>

12%

Pseudomonas aeruginosa
Klebsiella oxytoca

25%

14%

Other enterobacteria

Fig. 1. Microbial strains isolated from ICU

283 from these strains were represented by Enterobacteriaceae, from which, 48.40% (137 strains) were ESBL producers.
The highest percentage was noticed in the case of Klebsiellapneumonia - 78 strains (56.93%).
Table I. Resistance phenotypes in Klebsiella pneumoniae isolated from ICU
ESBL+ KTGANt+cross resistance Fq 19+ TER+SXTR
24.35%
ESBL+ impermeability cephamycins + 4
KTG+ partially resistant Fq + TER
5.12%
ESBL+ KTGANt+cross resistance Fq 11+ TER
14.11%
78
K l e b s i e l l a 137 ESBL ESBL + KTANt + cross resistance Fq 22
28.21%
pneumoniae
56.93%
ESBL (CTX-M) + KTGNt + partially 3
resistant Fq +SXTR
3.84%
ESBL+ KTGANt+cross resistance Fq 14
17.94%
ESBL+ impermeability cephamycins+ 3
partially resistant Fq + TER+SXTR
3.84%
ESBL+TER+SXTR
1
1.28%
ESBL+SXTR
1
1.28%

Legend: ESBL-extended spectrum beta-lactamase, K-resistance to kanamycine,
G-resistance to gentamycine, T- resistance to tobramycine, A-resistance to amikacin,
Nt-resistance to netilmicin, Fq-fluoroquinolone, SXT-trimethoprim-sulfamethoxazole,TEtetracycline

In the community, from the total of 3002 bacterial strains
isolated, 435 were MDR strains and the distributions were:
1778 E.coli, 809 S.aureus, 303 Klebsiella pneumoniae, 103
Pseudomonas aeruginosa, 9 Acinetobacter baumannii (figure
2).
3%

S.aureus

27%

Klebsiella pneumoniae
Acinetobacter baumannii
E.coli
Pseudomonas aeruginosa

>

10%
60%

0%

Fig. 2. Microbial strains isolated from community

In the community 2081 from isolated strains were represented by Enterobacteriaceae, from which, 10.90% (227 strains)
were ESBL producers. The highest percentage was also noticed
in the case of Klebsiella pneumonia - 107 strains (35.31%).

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Table II. Resistance phenotypes in Klebsiella pneumoniae isolated from
community
ESBL+ impermeability cephamycins + 10
9.34%
KTGNt + TER+SXTR
ESBL+ impermeability cephamycins + 11
KTGANt10.28%
ESBL+ impermeability cephamycins + 6
KTGANt + TER+SXTR
5.60%
ESBL+ impermeability cephamycins + 6
RR
G + TE
5.60%
ESBL+ impermeability cephamycins + 1
TER +SXTR
0.94%
ESBL+ impermeability cephamycins + GR 1
+ TER + SXTR
0.94%
ESBL+ impermeability cephamycins 1
+ GR
0.94%
ESBL+ impermeability cephamycins 2+ SXTR
1.87%
ESBL+ impermeability cephamycins + 1
GR + SXTR
0.94%
ESBL + GR + TER + SXTR + cross resis- 18
tance Fq
16.81%
ESBL+ GR + TER + SXTR
7
6.54%
ESBL+ KTGNt + TER + SXTR
7
6.54%
ESBL+ KTGANt + TER+ SXTR
6
5.60%
R
R
3
K l e b s i e l l a 303 107 ESBL+ G + TE
2.80%
pneumoniae
ESBL
R
R
4
35.31% ESBL+ G + SXT
3.73%
ESBL+ TER + cross resistance Fq
54.67%
ESBL+ TER
2
1.87%
ESBL+ GR + partially resistant Fq
3
2.80%
ESBL+ GR2
1.87%
ESBL+ TER+ SXTR
2
1.87%
ESBL - wild type2
1.87%
ESBL+ SXTR
1
0.94%
ESBL+ TAR
1
0.94%
ESBL+ KTGANt + SXTR
1
0.94%
ESBL+ KTGANt
1
0.94%
R
ESBL+ KTGNt + TE
1
0.94%
ESBL+ KTGNt + SXTR
1
0.94%
ESBL+ KTGNt
1
0.94%

Legend: ESBL-extended spectrum beta-lactamase, K-resistance to kanamycine,
G-resistance to gentamycine, T- resistance to tobramycine, A-resistance to amikacin,
Nt-resistance to netilmicin, Fq-fluoroquinolone, SXT-trimethoprim-sulfamethoxazole,TEtetracycline

ESBLs are a rapidly evolving group of beta-lactamases whichshare the ability to hydrolyze third-generation cephalosporins andaztreonam yet are inhibited by clavulanic acid. Typically, they
derive from genes for TEM-1, TEM-2, or SHV-1 by mutations
that alter the amino acid configuration around the active siteof these beta-lactamases. This extends the spectrum of betalactam antibiotics susceptible to hydrolysis by these enzymes.
Fiziologia - Physiology  2010.20.3 (67)32

An increasing number of ESBLs nor TEM or SHV lineage,
have recently been described. The presence of ESBLs carries
tremendous clinical significance. The ESBLs are frequently
plasmid encoded. Plasmids responsible for ESBL production
frequently carry genes encoding resistance to other drug classes(for example, aminoglycosides).[4]In this study, all ESBLs have been associated with other
resistance patterns like: resistance to quinolones, trimethoprimsulphamethoxazole, aminoglycosides or tetracycline.
CONCLUSIONS
1. In both, hospital and community, Klebsiella pneumoniae showed the highest percentage of ESBL production,
followed by other enterobacteria.
2. These Klebsiella pneumoniae strains have been
isolated from bronchoalveolar fluids, wound secretions, urines.
3. Bacterial MDR it is more and more frequently described
not only in hospitals but in community as well, representing an
alarming phenomenon for the modern medicine through thedrastically restriction of therapeutic options.
4. Because of the very limited remaining alternatives fortreatment and ESBLs significant prevalence worldwide, infection
control remains the best way to deal with this bacterial resistancemechanism.
5. The increasing frequency of ESBL-producing enter-obacteria among hospitalized patients is an important problem
for both microbiologists and clinicians, because of the difficultyin the correct detecting, reporting, and treating of the infections
caused by these organisms.
Acknowledgments
These data are part of the PNII 42121 project: “Molecularcharacterization of multidrug resistant strains, hospital or community acquired, collected from south-west Romania”.
REFERENCES

1. Colodner R. Extended-spectrum beta-lactamases: a challenge forclinical microbiologists and infection control specialists. Am J InfectControl., 2005; 33(2):104-7.
2. Winokur PL, Canton R, Casellas JM, et al. Variations in theprevalence of strains expressing an extended-spectrum-betalactamase phenotype and characterization of isolates from Europe,
the Americas, and the Western Pacific region. Clin Infect Dis., 2001;
32(2): S94-S103.
3. Livermore DM, Struelens M, Amorim J, Baquero F, Bille J, Canton
R, Henning S, Gatermann S, Marchese A, Mittermayer H, Nonhoff C,
Oakton KJ, Praplan F, Ramos H, Schito GC, Van Eldere J, Verhaegen J,
Verhoef J, Visser MR. Multicentre evaluation of the VITEK 2 Advanced
Expert System for interpretive reading of antimicrobial resistance tests.
J. Antimicrob. Chemother., 2002; 49:289-300.
4. Paterson DL, Bonomo RA. Extended-spectrum beta-lactamases: aclinical update. Clin Microbiol Rev., 2005;18(4):657-86.

PREVALENTA TULPINILOR DE KLEBSIELLA PNEUMONIAE PRODUCĂTOAREDE BETA LACTAMAZE CU SPECTRU EXTINS IZOLATE DIN SPITAL SI
COMUNITATEREZUMAT
Bacteriile multirezistente sunt tot mai frecvent descrise nu doar în spital ci şi în comunitate reprezentând pentru medicina modernă
un fenomen alarmant ce reduce drastic posibilităţile de tratament. Am izolat în perioada martie – decembrie 2009 de pe secţia deTerapie Intensivă a Spitalului Clinic Judeţean un număr de 137 tulpini de Klebsiella pneumoniae din totalul de 548 tulpini microbiene,
iar în perioada ianuarie – noiembrie 2009 am izolat din comunitate (Laboratorul Bioclinica) un număr de 303 Klebsiella pneumoniae
din totalul de 3002 tulpini.
Toate tulpinile de Klebsiella pneumoniae au fost identificate şi fenotipate cu ajutorul aparatului VITEK 2.
Atât în spital cât şi în comunitate, tulpinile de Klebsiella pneumoniae au reprezentat procentul cel mai mare de tulpini
betalactamazoproducătoare cu spectru extins(ESBL), fiind urmată de alte enterobacterii.
Cuvinte cheie: ESBL, Klebsiella pneumoniae, fenotip de rezistenţă

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NORMAL, EARLY AND DELAYED PUBERTYBAGIU RADU
„Victor Babeş” University of Medicine and Pharmacy, Timişoara, Romania

ABSTRACT
Puberty is defined as the period during which reproductive capability is acquired. Normal puberty starts around 10 years, girls having the tend to start puberty earlier than boys. Puberty lasts 3-4 years, with the transition from one stage to another, almost every
year. Chronological pubertar evaluation is made with reference to Tanner stages, and biological evaluation, depending on bone age.
Precocious puberty is defined as puberty before the normal age for the population, under 8 years old for girls and under 9 years oldfor boys. It affects about 4-5% of girls and is about five times more commonly seen in girls than boys. Delayed puberty is defined
as no pubertal changes in a girl aged 13 years old or boys aged 14 years old, or failure of progression of puberty over 2 years. It
affects about 2% of the population.
Keywords: puberty, chronological and biological evaluation

Normal puberty
Puberty is defined as the period during which reproductive
capability is acquired. Frequently, the terms puberty and adolescence are used interchangeably. Puberty is different fromadolescence which is the whole period of growth from childhood to adult and is expanding more than the period of puberty,
between 10-19 years old.
There is a wide range of normality in puberty. Normal puberty
starts around 10 years old, whereas girls tend to start puberty
earlier than boys. Physical manifestations of puberty, in particular
the emergence of secondary sexual characters, begin to occur at
11 years old to 50% of girls and 11 ½ years old to 50% of boys.
Regular manifestation in female puberty, breast growth, it is more
visible than the first signs of male puberty, increased testicles.
Increase in height during puberty in girls occurs earlier, while inboys is installed later, with a difference of two years between thetwo sexes in the sequence of events in time (6, 11).
Puberty lasts 3-4 years, with the transition from one stage
to another, almost every year. Puberty vary greatly not only as
the match in time, but even in length. For example, 50% of girls
need 4 years to go through all stages of puberty, some need
only 18 months, while others need more than five years. Installation of the beginning of puberty can’t predict the duration ofpuberty (8). Pubertar evaluation is usualy made with reference
to Tanner stages (9, 10).
Assessment of the biological age of the child, as opposed tochronological age, is performed using the bone age. This uses
assessment of epiphyses on an X-ray of the left hand and allows assessment of remaining growth and correlates better withpubertary stages than chronological age. Puberty is manifested
in both sexes by the development of pubic and axillary hair;in girls, between the ages of 8 and 14 years old, by growth of
the breasts and menstruation; in boys, between the ages of 9
and 15 years old, by growth of the penis, testes and scrotum,
deepening of the voice, initiation of spermatogenesis, and by
growth of facial hair.
Increase of height and other body dimensions, begin at boys
one year after testicular growth, and reach maximum about ayear after that, on average at the age of 14 years old. In girls,
height increase starts once with breasts development, sometimes
even before, and only when it passes the tip of height growth,
menarche occurs (5).
The body changes that occur with puberty are due to theaction of the sex hormones. These sex hormones include
testosterone and other androgens for both sexes, together withoestrogen and progesterone in girls. The secretion of these
hormones is from the testes in boys and ovaries in girls and toa lesser extent from the adrenal glands in both sexes. Someoestrogen is also produced in both sexes from peripheral conversion of sex hormones. Secretion of sex hormones is under thecontrol of gonadotrophins, released from the anterior pituitary
gland at the base of the brain. There are two gonadotrophin
hormones, follicle stimulating hormone (FSH) and luteinizing
hormone (LH).
The release of gonadotrophin hormones from the anterior
pituitary gland is in turn controlled by gonadotrophin-releasing
hormone(GnRH) from the hypothalamus. A negative feedback
loop prevents excess hormone production. If there is sufficientsex hormone circulating, then this feeds back to both the pituitary
gland and hypothalamus to reduce the amount of FSH and LH.
At birth, the circulating levels of FSH and LH are similar to thoseof an adult. Soon after birth, however, both GnRH secretion andReceived April 2010. Accepted May 2010. Address for correspondence: Dr. Radu Bagiu, “Victor Babes” University of Medicine and Pharmacy Timisoara, No. 2A Eftimie
Murgu Square, Timisoara 300041, Romania

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levels of circulating FSH and LH reduce to very low levels. Asa child progresses towards puberty, the hypothalamus starts tosecrete pulses of GnRH. The frequency and size of these pulses
increase with time until there is constant secretion of GnRH, asin adulthood (3).
Puberty is a time of great physical and emotional change
and can be a difficult time in any family. It can cause a great dealof anxiety both to the child and the parents if puberty does notprogress as expected and is either too advanced or delayed forthe age of the child. This makes assessment and managementof disorders of puberty particularly challenging.
Precocious puberty
Precocious puberty results mostly from the precocious
activation of the gonadotropic axis. Most physicians consider
that onset of pubertal development before the age of 8 years
old in girls and 9 years old in boys as precocious puberty andimpose at least a clinical and bone age evaluation by a paediatricendocrinologist. The major concern in precocious puberty is theunderlying condition, and central nervous system or gonadal
neoplasm have to be formally excluded as a first step in the
diagnosis. A secondary concern is height, since precocious
puberty leads to accelerated growth, accelerated bone maturation and ultimately reduced stature. Precocious puberty affects
about 4-5% of girls and is about five times more commonly seenin girls than boys (7). Precocious puberty is heterogeneous andstrict criteria should be used to define it, both in terms of age
and in terms of potential for progression (8).
Central or true precocious pubertyPremature activation of the hypothalamic – pituitary – gonadal axis results in a gonadotrophin-driven early puberty. In allcases, refer for further investigation to exclude hypothalamic
tumours and other central nervous system pathology. No pathological cause for this pattern of precocious puberty is absent in
50 – 60% of males and 90% of females (1). In these individuals,
there may be a family history of precocious puberty. Obesity
may also be involved in development of precocious puberty ingirls (2).
Pseudoprecocious puberty
Pseudoprecocious puberty, or gonadotrophin-independent
precocious puberty, accounts for 20% of all cases of precocious
puberty. There is an increased level of sex hormones in theabsence of excess FSH or LH.
Causes of pseudoprecocious puberty:- sex hormones secreting tumors- hepatoma, hepatoblastoma- congenital disorders- familial male precocious puberty- McCune-Albright syndrome- hypothyroidism.
Social effects of precocious puberty
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Children who are taller than other children of the same age
will often be treated as older than they actually are. This can resultin children being punished or teased for behaviour appropriate
to their age or considered stupid or behind when in fact they are
attaining expected developmental milestones (5). Children withprecocious puberty may show more awareness of sexual partsand masturbate which can create embarrassment, especiallyin adults, and confusion in the child. Underarm deodorants andmore frequent baths and hair washing, may be necessary as
sweating and body odour can be a problem. All children want tolook and act like friends of their own age. Although early puberty
can give a child distinct physical advantages when playing manysports, being different from other children of the same age canlead to embarrassment for the child, teasing or bullying, andsocial exclusion that can lead to poor self-esteem. Parents haveto face their own reactions to the changes in their child which canbe alarm, distress, distaste, guilt and confusion (4).
Delayed puberty
Delayed puberty is defined as no pubertal changes in agirl aged 13 years old or boy aged 14 years old, or failure of
progression of puberty over 2 years. It affects about 2% of the
population.
The most common cause of delayed puberty in boys isconstitutional delay, more than 50%. 80% of girls have a pathological cause for delayed puberty. Pathological causes can bedivided into those caused by failure of the ovaries or testes
also termed primary or hypergonadotrophic hypogonadism
and those caused by failure of stimulation of normal gonads to
produce sex hormones termed secondary or hypogonadotrophic
hypogonadism (6).
Hypergonadotrophic delayed puberty
There are many possible causes of testicular or ovarian
failure. Lack of sex hormone production results in low levels of
circulating sex hormones, reduced negative feedback on the
hypothalamus and pituitary, and high levels of circulating FSH
and LH.
Causes of testicular and ovarian failure are:- Klinefelter syndrome- testicular dysgenesis syndrome- absent or undescended testes- testicular cancer, leukaemia- epidemic parotidite- testicular injury- chemotherapy, glucocorticoids therapy- Turner syndrome- ovarian tumours- polycystic ovarian syndrome.
Hypogonadotrophic delayed puberty
If the hypothalamus does not secrete enough GnRH or
the pituitary gland does not secrete enough LH or FSH, then
the normal gonads will not be stimulated to secrete sufficientsex hormones for pubertal changes to occur. Biochemically,35

circulating levels of sex hormones, FSH and LH are all reduced.
Depending on whether the underlying cause of the problem isin the hypothalamus or pituitary, GnRH levels may be reduced
or increased.
Causes of hypogonadotrophic delayed puberty are:- sporadic or familial constitutional delay- destructive tumours or lesions of the hypothalamus or
pituitary- hypothalamic syndromes such as Prader – Willi syndromeor Laurence – Moon – Biedl syndrome- thyroid disease, hyperthyroidism, rarely hypothyroidism- suppression of the hypothalamic – pituitary – gonadal axis
as a result of excess secretion of hormones negatively feedingback onto that axis — hyperprolactinaemia, Cushing’s syndrome,
hyperthyroidism or congenital adrenal hyperplasia- serious systemic illnesses , including chronic renal failure,
blood disorders such as thalassaemia and sickle cell disease,
Crohn’s disease- malnutrition due to food shortage, anorexia nervosa or
malabsorption, for example as a result of cystic fibrosis orcoeliac disease- excessive exercise particularly at gymnasts (12).
Social effects of delayed puberty
Many children, particularly boys, have great difficultyin coping with delayed puberty. They may be considerably
shorter, and appear much younger, than their friends. Lateron their lack of sexual development may cause them embarrassment and make them feel left out. In some, this may
result in immature behavior, while in others, it may manifest as
aggressive and/or antisocial behavior. The reactions of others
to the child may also be affected by delayed puberty. Children
who are small are often treated according to their size and nottheir age. Parents may become hyperprotective. Immatureaspect and incapability to join in with group activities may thenlead to further exclusion by the child’s peer group. The small
teenagers can be a natural target for bullying and sometimes,
inappropriate skills are developed to compensate in order tosurvive with decline of self-esteem (5).
REFERENCES

1. Adan L, Chemaitilly W, Trivin C et al, Factors predicting adult heightin girls with idiopathic central precocious puberty: implications for treatment, Clin Endocrinol (Oxf), 2002, 56, 297-302
2. Antoniazzi F, Cisternino M, Nizzoli G, et al., Final height in girls withcentral precocious puberty: comparison of two different luteinizing
hormone-releasing hormone agonist treatments, Acta Paediatr, 1994,
83, 1052-1056
3. Bougneres PF, Lack of effect of GnRH agonists on height in girls withadvanced puberty: a randomized long-term pilot study, J Clin Endocrinol
Metab, 1999, 84, 3575-3578
4. Carel JC, Léger J, Clinical practice: Precocious puberty, New England
Journal of Medicine, 2008, 358 ( 22 ), 2366 – 77
5. Coste J, Ecosse E, Lesage C, et al., Evaluation of adolescent statural
growth in health and disease: reliability of assessment from heightmeasurement series and development of an automated algorithm,
Horm Res , 2002, 58, 105-114
6. Draper R, Puberty- normal and abnormal, PatientPlus, 2007, www.
patient.co.uk/ showdoc/40024607
7. Draper R, Precocious puberty, PatientPlus, 2007, www.patient.
co.uk/showdoc/40000365
8. Klein KO, Precocious puberty: who has it? Who should be treated?
J Clin Endocrinol Metab, 1999, 84,411-414
9. Marshall WA and Tanner JM, Variations in the pattern of pubertal
changes in girls. Arch Dis Child, 1969, 44, 291-303
10. Marshall WA and Tanner JM, Variations in the pattern of pubertal
changes in boys. Arch Dis Child, 1970, 45, 13-24
11. Sadler C, White J, Everitt H, et al., Women’s health, Oxford University Press, 2007
12. Van Dorp F, Simon C, Child health, Oxford : Oxford University
Press, 2007

PUBERTATEA NORMALĂ, PRECOCE ŞI ÎNTÂRZIATĂREZUMAT
Pubertatea este definită ca perioada în care este dobândită capacitatea reproductivă. Pubertatea normală începe în jur de 10 ani,
fetele având tendinţa de a începe pubertate ceva mai devreme decât baieţii. Pubertatea durează 3-4 ani, cu trecerea de la o etapă laalta, aproximativ în fiecare an. Evaluarea pubertară cronologică este efectuată cu referire la etapele Tanner, iar evaluarea biologică,în funcţie de vârsta osoasă. Pubertatea precoce este definită ca pubertatea înainte de vârsta puberală normală pentru populaţie,sub 8 ani pentru fete şi sub 9 ani pentru băieţi. Ea afectează aproximativ 4-5% dintre fete şi este de cinci ori mai frecventă la fete
decât la băieţi. Pubertatea întârziată este definită ca lipsa modificărilor pubertare la fetele de 13 ani sau băieţii de 14 ani, sau eşecul
de progresie al pubertăţii de peste 2 ani. Afectează aproximativ 2% din populaţie.
Cuvinte cheie: pubertate, evaluare cronologică şi biologică

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