FIZIOLOGIA
physiology
FOUNDING EDITORCHIEF EDITOR
CO-CHIEF EDITORSASSOCIATE EDITORSEXECUTIVE EDITORSFRANCISC SCHNEIDERCARMEN PANAITESCUCARMEN TATUFLORINA BOJINMIHAI NECHIFORSORIN RIGAGABRIELA TANASIEDACIANA NISTORCALIN MUNTEANEDITORIAL BOARD
ARDELEAN AURELBĂDĂRĂU ANCA
BENEDEK GYORGYBENGA GHEORGHECOJOCARU MANOLEGĂLUȘCAN ATENAIANCAU MARIAMIHALAŞ GEORGETA
MUNTEAN DANINAMUREŞAN ADRIANANESTIANU VALERIUOPREA TUDORPANAITESCU CARMEN(Arad)(Bucureşti)(Szeged)(Cluj)(Bucureşti)(Timișoara)(Craiova)(Timişoara)(Timişoara)(Cluj)(Craiova)(New Mexico)(Timişoara)PĂUNESCU VIRGILPETROIU ANAPODARIU ANGELA CODRUTA
RÂCZ OLIVERRIGA DANSABĂU MARIUS
SAULEA I. AURELSIMIONESCU MAIASWYNGHEDAUW BERNARDTANGUAY M. ROBERTTATU ROMULUS FABIANVLAD AURELIANVOICU VICTORZĂGREAN LEON

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Fiziologia - Physiology • 2018.28.2(96)CONTENTS
Real-Time Imagining of NK-92 Cells Interaction with Tumoral Cell Lines ................................................... 4
Zogoreanu R, Gavriliuc O, Bojin F, Paunescu V

Mechanisms of Bone Loss in Glucocorticoid Induced Osteoporosis ......................................................... 9
Barbulescu AL, Sandu RE, Vreju FA, Firulescu AC, Criveanu C, Chisalau BA, Parvanescu CD,
Dinescu SC, Ciobanu DA, Taisescu O, Tartea EA, Albu CV

Simultaneous Cancers of the Head and Neck – Case Presentation .......................................................... 14
Mot IC, Poenaru M, Balica NC, Horhat DI

Alergic Rhinnitis – Methods of Prevention ................................................................................................... 17Goanță CM, Tușaliu M, Stanciulescu A

Investigation of Neutral Polysaccharides from Fomes Fomentarius: A Preliminary Study ..................... 20
Bojin L, Cojocariu C, Georgescu M, Puiu M, Pascariu MC, Serb A, Puscasiu D, Tatu C, Sisu E

Management of Epistaxis in Nasal Tumors and Heart Diseases at the University Hospitalof Timisoara ..................................................................................................................................................... 28
Marin AM, Marin A, Poenaru M

CUPRINS
Imagistică temporizată pentru evidențierea interacțiunilor dintre celulele NK-92
și linii celulare tumorale ................................................................................................................................... 4
Zogoreanu R, Gavriliuc O, Bojin F, Paunescu V

Mecanismele pierderii osoase în osteoporoza glucocorticoid indusă ........................................................ 9
Barbulescu AL, Sandu RE, Vreju FA, Firulescu AC, Criveanu C, Chisalau BA, Parvanescu CD,
Dinescu SC, Ciobanu DA, Taisescu O, Tartea EA, Albu CV

Cancere simulatane de cap și gât – Prezentare de caz ............................................................................... 14
Mot IC, Poenaru M, Balica NC, Horhat DI

Rinita alergică – Metode de prevenție ........................................................................................................... 17Goanță CM, Tușaliu M, Stanciulescu A

Investigarea polizaharidelor neutre provenite din Fomes Fomentarius: un studiu preliminar ............... 20
Bojin L, Cojocariu C, Georgescu M, Puiu M, Pascariu MC, Serb A, Puscasiu D, Tatu C, Sisu E

Managementul epistaxisului în tumori nazale și afecțiuni cardiace la Spitalul Universitar
din Timișoara ................................................................................................................................................... 28
Marin AM, Marin A, Poenaru M

Fiziologia - Physiology • 2018.28.2(96)3REAL-TIME IMAGINING OF NK-92 CELLSINTERACTION WITH TUMORAL CELL LINESZOGOREAN ROXANA1, GAVRILIUC OANA1,2, BOJIN FLORINA1,2,PĂUNESCU VIRGIL1,2
1Clinical Emergency County Hospital “Pius Brînzeu” Timișoara, Centre for Cellular and Gene Therapies in the Treatment of Cancer –OncoGen2”Victor Babes” University of Medicine and Pharmacy Timisoara, Department of Functional Sciences, Immunology Discipline

ABSTRACTOne priority in developping better immunotherapies in cancer is the use of natural killer cells to treat different types ofcancer. The citotoxicity of natural killer cells to tumoral cells depends on the type of immunological synapse formed. By
establishing a litic immunological synapse, the effector cells is activated and release enzymes to destroy the target cells.
The termination of the interaction between two cells is as important as the engagement in immunological synapse, because
the detachment of the target cell can influence the serial killing of natural killer cells. Here, we followed the behavior of NK92 cells in interaction with 3 different types of tumoral cell lines. The diferences observed in the interaction of natural killer
cells with tumoral cells gives insight into the factors invovled in citotoxicity.
Keywords: immunological synapse, cytotoxicity, natural killer cells, tumor cells, time-lapse

INTRODUCTION
Natural killer cells are the first line of defense of theimmune system against viral infected cells and tumoral cells[1-3]. The cytotoxic activity of natural killer cells depends onthe close contact with the target cell. Detection of target cell
is followed by adhesion to the cell to form a stronger contact.
Signals inside the cell determine rearrangements of actin
filaments causing changes in the morphology of the cell [4].
Depending on the signals received from the activating andinhibitory receptors the NK cells form an inhibitory or a lytic
immunological synapse with the target cell [1]. The activatedNK cells induce apoptosis of target cells through release oflytic enzymes in the immunological synapse interface [5].
The initiation of immunological synapse starts with thebinding of integrin and selectin receptors to their ligands on
the surface of target cell. Adhesion molecules like LFA-1(CD11a/CD18) and MAC1 (CD11b/CD18) provide signals
for further recruitment of receptors to form a stronger cellularcontact and determines the clustering of activating andinhibitory receptors [6] Activating receptors NKG2D, Nkp30,
Nkp44, NKp46, DNAM-1, 2B4 and NTB-A activate the NK
cells through phosphorylation of the tyrosine kinase family
Src and Syc, phospholipase C and PI3K. The activation

signal induces the actin reorganization which allows the lytic
molecules to be transported at the immunological synapse
cleft. Degranulation of lytic vesicles with peforin andgranyzime results in death of connected target cell [7].
For the lytic immunological synapse to be established isessential for the NK cell to identify the target cell and toreceive activating signals. The cytolytic activity of NK cells
was highly studied in order to understand the process which
determine the activation stage and the evasion of tumor
cells [8]. The death of target cell determine the detachmentof an activated NK cell which can establish faster a new
immunological synapse [9]. The serial killing of NK cells
depends on the activation status and the type of target cells[10]. The former interaction with the target cell dictates theongoing activated signal and so the efficiency ofconsequently killing. Studies have shown the formation of a
new immunological synapse only after the detachment fromthe old target cells [11].
Challenges in obtaining sufficient NK cells from thedonors and to genetically manipulate NK cells lead to theuse of NK-92 cell line for immunotherapy. The NK-92 cellline is interleukin-2 dependent and tend to form clumps inculture [12]. The cell line has a higher degree of cytotoxicity

Received July 16th 2018. Accepted September 20th 2018. Address for correspondence: Gavriliuc Oana, PhD, ”Victor Babes” University of
Medicine and Pharmacy Timisoara, Department of Functional Sciences, Immunology Discipline, Clinical Emergency County Hospital “PiusBrînzeu” Timișoara, Centre for Cellular and Gene Therapies in the Treatment of Cancer – OncoGen; Eftimie Murgu Square No. 2A, RO300041, Timisoara, Romania; phone: +40728301083; e-mail: gavriliuc.oana@umft.ro4

Fiziologia - Physiology • 2018.28.2(96)than NK primary cells due to higher content of peforin andgranyzime B and the lack of almost all the KIR receptors
expressed on normal NK cells [13]. Also, NK-92 cells lackthe CD16 receptor involved in ADCC [12].
In this study we investigated the behavior of NK-92 andestablishment of immunological synapse with three different
types of target cells. Most cytotoxic assay analyzes theindividual contact between the cells and forget that the cellto-cell communication is relevant in activation andproliferation response. The tendency of clumping for NK-92
indicates the health and activation status of the cells [12].
We decided to follow the morphology and contact dynamics
of NK-92 cells in contact with target cells with focus on theclump formation and kinetics.

RESULTS
NK-92 cells have activated morphology on contact
with target cells (Fig.1).

MATERIAL AND METHODS
Cell lines
NK-92 cell line was maintained in X-vivo 10 [Lonza]
supplemented with 5% Human Plasma, 500 U IL-2
[Peptrotech]. The target cells NALM-6 and MCF-7 were
cultivated in RPMI 1640 [Lonza] with 10% FBS [Gibco] and
1% Penicillin/Streptomycin [Sigma], SKBR-3 cell line wascultivated in DMEM [Sigma] + 10% FBS [Gibco] and 1%
Penicillin/Streptomycin [Sigma].
Cell labeling
NALM-6 were washed with serum free media andlabeled with 5uM lipophilic tracer spDII [Invitrogen] for 10
minutes at 37°C. The cells were resuspended in media withserum and seeded into the 4 wells. The adherent cells MCF7 and SK-BR-3 were trypsinized at confluence, washed withserum free media and labeled in the same way like NALM-6
cells. The cells were left to adhere for 24 hours.
Fibronectin Coating
The imaging dish used µ-Dish 35 mm Quad [iBIDI] wascoated with 100 uL of 15 ug/mL Fibronectin [Invitrogen] ineach of the four wells and left for 1 hour in the hood. After 1
hour the solution was removed and the dish was left to dry
for another 30 minutes before adding the NALM-6 cells.

Fig. 1. Morphology of NK-92 cells during contact with target cells
labeled with spDII. Green arrows indicate the conjugate formation.
A. NK-92 cell is conjugated with NALM-6 cell (orange) B. NK-92
cell is conjugated with SK-BR-3 cell (orange) C. NK-92 cell isconjugated with MCF-7 cell (orange)Morphology of NK-92 cells in contact with target cells
was followed during the 8 hour time-lapse. Initially NK-92
cells spread lamellipodia in search for the target cell. Also,
the lamellipodia are present for a period of time after thecontact was established. The activated NK-92 cells have asymmetrical spreading across target cells surface. The large
immunological synapse is followed by a contraction phaseof NK-92 cell which determines shrinkage of adherent cells.
Visible changes occur in morphology of adherent cells, thetarget cells became more granulated and rounder, indicating
the apoptosis of cells.
Size of NK-92 clumps is influenced by the interaction
with target cells (Fig. 2).

Cytotoxicity assay
The NK-92 cells were centrifugated, resuspended in XVivo media supplemented with 5% HP, 500 U/mL IL-2,
counted and added at the E:T ratio of 1:1 in each well. Livecell imaging of cells was performed using Nikon Biostation
IM-Q at 37°C in a 5% CO2 atmosphere with image
acquisition every 5 minutes for 8 hours.
Data analysis
The size of the NK-92 clumps was measured using NISElement Software [Nikon] and the data is represented as
mean+-SD.
Fiziologia - Physiology • 2018.28.2(96)Fig. 2. Measurement of NK-92 cell clumps during the 8-hour time
lapse imaging

5

To determine if the NK-92 cells have a different
clumping behavior depending on the type of target cells wemeasured the diameter of formed clumps every hour during
the 8 h time-lapse. After 2-hour incubation with target cells,
the NK-92 cells started to form bigger clumps when incontact with NALM-6 cells. A drop in the size of clumps isregistered after 5 hours in case of NK-92 incubated with
NALM-6 cells. The increase in size of clumps continue after
6 hours and reach a plateau size after 7 hours. The
aggregation size of NK-92 cells in contact with adherent
cells was comparably smaller with NALM-6 cells, butfollowed an increasing path during the incubation time.
Differences in NK-92 target cell attachment to target
cells (Fig. 3.)

surface of the cell and drag the target cell to a close
multicellular cluster (Fig.3 C). Moreover, we observed NK-92
cells that form conjugates with the target cells drag them into
the NK-92 cell clumps, which results in an accumulation of
NALM-6 cells inside the NK-92 clumps.
Only at the encounter with NALM-6 cells, the NK-92
cells accumulate target cells at the site of NK-92 cellclusters. The tendency of smaller clumps to fuse with othersmaller clumps determine the aggregation into much bigger
clusters formed of NK-92 cells and NALM-6 cells (Fig.3 A).
Some of the NALM-6 cells brought together inside an NK-92
clumps were shaded away (Fig.3 B).
The engagement of NK-92 clumps towards MCF-7 and
SK-BR-3 cells was different, without the integration of target
cells in the NK-92 clumps. Some of the times the killed target
cells remain attached to the NK-92 cells for a short period, but
the NK-92 cells release them before engaging in another kill.

DISCUSSION

Fig. 3. NK-92 cell contact dynamics with NALM-6 target cells labeledwith spDII. A. Smaller NK-92 clumps fuse with other NK-92 clumps
to form a bigger cluster. The NK-92 cells are strongly attached to
NALM-6 cells (orange) and transport them inside the NK-92
clumps; B. After the binding and close interaction of NK-92 cells to
NALM-6 cells the NK-92 clumps shed away the NALM-6
aggregates (orange) from the clump formation; C. Motility of a NK92 cells which binds to NALM-6 cell (orange) and drag the cell.

During the time-lapse imagining we observed a different
interaction of NK-92 cells with NALM-6 cells adherent on
fibronectin compared to NK-92 cells co-incubated withadherent target cells MCF-7 and SK-BR-3. The NK-92 cells inclose contact with a NALM-6 target cells spread across the6

Using time-lapse imaging we have compared the
behavior of NK-92 cells co-incubated with different types oftarget cells. Leukemia tumor NALM-6 cells are single small,round cells cultivated in suspension and MCF-7, SK-BR-3
cells are adherent tumor breast cancer cells. NK-92 cells
were added at the same E:T concentration of 1:1 for all typeof cells.
The cytotoxic activity of NK-92 cells is establishedduring the immunological synapse when the NK-92 cell
forms a firmer contact with the target cell. The duration of
the contact is important, because shorter contacts indicate
an inhibitory synapse where the NK-92 cells does not kill thetarget cell. Prolonged contact lead to stronger conjugate
effector-target cells with efficient cytotoxicity [14]. NK-92
cells activation is known to be determined by theengagement of activating receptors like NKG2D, NK 1.1,
2B4, DNAM-1 and NCRs [15]. The activation status enables
the rearrangement of actin filaments inside the cells totransport the lytic enzymes at the interface of theimmunological synapse [7,16]. The release of the enzymes
such as perforin determine the pore formation on the target
cells in which granzyme B can enter and induce apoptosisof the cell [17].
Each of the immunological synapse stages determine
modification in the morphology of the effector cell. Changes
in membrane morphology are characterized by spreadingover the target cells which increases the contact of ligandreceptors at the level of immunological synapse [14,18]. We
have noticed that individual NK-92 cells form lamellipodia
during their surveillance for target cells and also at the firstcontact with the target cell. After the contact was establishedthe NK-92 cells become bigger and spread over the target
cell. Olofsson et al. [14] showed that IL-2 activated cells arelarger and spread across target cells during conjugation.
Results from our imaging method are in line with otherFiziologia - Physiology • 2018.28.2(96)studies where during synapse formation activated NK cells
display elongated and irregular shape compared to resting
NK cells which are smaller and rounder [14,17,19].
We identified a different behavior of NK-92 cells towards
NALM-6 cells. The NK-92 cell form conjugates with target
cells and remain in contact with them, migrating towards theclosest cluster of cells. Thus, the formation of clumps has
integrated NALM-6 target cells with-in, NK-92 cells scanningover the target cells. The motile scanning of NK cells was
also described by [14], but only for isolated conjugates.
Netter P et al. [20] demonstrated that initiation of new
contact with a target cells is followed by a rapid detachmentof NK cells from the last target. In our study, the NK-92 cellclusters were observed to contain smaller aggregates of
NALM-6 cells, which were shed together after a period of 4hours. The process through which NK cells could dissemble
the immunological synapse and engage in sequential killing
has not been identified. The termination of theimmunological synapse was studied more using NK cells,
which have different array of receptors compared to NK-92
cell line [21]. Is possible that the integration of NALM-6
target cells in the NK-92 clumps is necessary for the cells tomaintain the activation status for a longer period of time.
LFA-1 integrin plays a role in NK cell binding to target celland is regulated by the balance of activating signals from
2B4 and NKG2D receptors [6].

CONCLUSION
Imaging techniques allows for a better understanding of
the biological processes through real-time cell tracking.
Moreover, the experiments will enable us to define theinteractions between cells that can have further
consequences in the function of the cell.
Acknowledgement: This work was supported by the grant“Chimeric Antigen Receptor Targeted Oncoimmunotherapy with
Natural Killer Cells (CAR-NK)”, POC 92/09/09/2016, ID: P_37_786,
MySMIS code: 103662.

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IMAGISTICĂ TEMPORIZATĂ PENTRU
EVIDENȚIEREA INTERACȚIUNILOR DINTRE
CELULELE NK-92 ȘI LINII CELULARE TUMORALEREZUMATO prioritate în dezvoltarea imunoterapiei mai eficiente este folosirea celulelor natural killer pentru tratarea difertelor tipuri
de cancer. Citotoxicitatea celulelor natural killer față de celulele tumorale depinde de tipul de sinapsă imunologică formată.
Prin stabilirea unei sinapse imunologice litice, celula efectoare este activitată și eliberează enzime ce distrug celula țintă.
Terminarea interacțiunii dintre cele două celule țintă este la fel de importantă ca și crearea unei sinapse imunologice
deoarece detașarea celulei țintă poate influența citotoxicitatea continuă a celulelor natural killer. În acest studiu am urmărit
comportamentul celulelor natural killer în prezența a 3 tipuri diferite de linii celulare tumorale. Diferențele observate îninteracțiunea celulelor NK-92 cu celulele tumorale dă o perspectivă asupra factorilor implicați în citotoxicitate.
Cuvinte cheie: sinapsă imunologică, citotoxicitate, celule natural killer, celule tumorale, time-lapse

8

Fiziologia - Physiology • 2018.28.2(96)MECHANISMS OF BONE LOSS IN
GLUCOCORTICOID INDUCED OSTEOPOROSISANDREEA LILI BARBULESCU1, RALUCA ELENA SANDU2, FLORENTIN
ANANU VREJU3, SINETA CRISTINA FIRULESCU4, CRISTINA CRIVEANU3,
BEATRICE ANDREEA CHISALAU4, CRISTINA DORINA PARVANESCU4,STEFAN CRISTIAN DINESCU3, DANIELA ALEXANDRA CIOBANU4,OANA TAISESCU5, ELENA ANCA TARTEA6, CARMEN VALERIA ALBU6
1Department of Pharmacology, University of Medicine and Pharmacy of Craiova, Romania
2Department of Biochemistry, University of Medicine and Pharmacy of Craiova, Romania
3Department of Rheumatology, University of Medicine and Pharmacy of Craiova, Romania
4PhD Student, University of Medicine and Pharmacy of Craiova, Romania
5Department of Anatomy, University of Medicine and Pharmacy of Craiova, Romania
6Department of Neurology, University of Medicine and Pharmacy of Craiova, Romania

ABSTRACT
Osteoporosis (OP), defined by decreased bone mass and altered bone microarchitecture, is a generalized skeletal diseaseassociated with a potentially high fracture risk and consequent severe complications; it has become a major health problem,with over 200 million people diagnosed and over 9 million new fractures annually. Glucocorticoid (GC) therapy is used withhigh efficiency in a wide variety of inflammatory and autoimmune diseases and in long term administration constitutes themost common cause of osteoporosis. Glucocorticoid induced osteoporosis (GIO) is recognized to be associated with asecondary increased prevalence of vertebral fractures due to a main effect on trabecular bone. The fracture risk is directly
related to dose, appears even at small doses, and it has been published to be increased, after the first 6 months oftreatment, both for vertebral and non vertebral fractures. It is reported that over 10% of the patients that have chronic GC
treatment are diagnosed with a fracture.
GC effects on bone mass are visible as two types: first, a rapid phase, with a percentage between 6 and 12 reduction in
the first year, due to an enhanced regulation of osteoclastogenesis and increased bone resorption, and a second phase,with a maximum 3% loss per year, explained by an altered bone formation. The mechanisms concurring to GIO
pathogenesis are complex and imply both systemic effects, as well as local consequences on every bone tissue cell type.
Keywords: glucocorticoids, osteoporosis, fracture risk

INTRODUCTION
Osteoporosis (OP), defined by decreased bone mass
and altered bone microarchitecture, is a generalized skeletal
disease associated with a potentially high fracture risk andconsequent severe complications [1]; it has become a major
health problem, with over 200 million people diagnosed andover 9 million new fractures annually [2]. Usually fractures
occur in spine, hip, distal forearm and proximal humerus;
vertebral fractures are known to be associated with further
vertebral fractures, with a reported incidence of 19.2% in thefirst year; more than 60% of them aren’t diagnosed andrepresent one of the major causes of death among elderly[3, 4].
Glucocorticoid (GC) therapy is used with high efficiency
in a wide variety of inflammatory and autoimmune diseases
and in long term administration constitutes the mostcommon cause of secondary osteoporosis. GC remain
major anti-inflammatory drugs in many rheumatic diseases,
both at the time of diagnosis, as well during flares, observed
both by clinical examination, as well as using imagistic
techniques, like high resolution ultrasonography, which has

Received September 16th 2018. Accepted October 20th 2018. Address for correspondence: Raluca Sandu, Department of Biochemistry,
University of Medicine and Pharmacy of Craiova, Petru Rares Street No. 2, Craiova, Romania, RO-200349; Phone: +40740017164; e-mail:ralucasandu80@gmail.com

Fiziologia - Physiology • 2018.28.2(96)9become an important diagnostic and evaluation tool; it ispublished that about 0.5-2% of the general population
receives GC therapy, with a percentage up to 90 among
patients with rheumatoid arthritis [5-9].
Glucocorticoid induced osteoporosis (GIO) is
recognized to be associated with an increased prevalenceof vertebral fractures due to a main effect on trabecularbone. The fracture risk is directly related to dose, appears
even at small doses, and it has been published to be
increased, after the first 6 months of treatment, both forvertebral and non vertebral fractures. It is reported that over
10% of the patients that have chronic GC treatment are
diagnosed with a fracture [10]. GC determine a more evidentbone loss mostly in the first 6 months of treatment, due toan increased bone resorption but also associated to the
concurrent inflammation of the underlying disease, with aslower decrease during long term administration [11, 12].
GIO has been intensively studied, both regarding
pathogenesis and improving therapeutic measures, adapted
to each category of patients and it should receive greaterawareness, with early intervention, in order to prevent futurecomplications.
We aimed to make a brief review of the most recent aspects
regarding GIO pathogenesis, assessment and treatment.

MECHANISMS OF BONE LOSS
GC effects on bone mass are visible as two types: first,
a rapid phase, with a percentage between 6 and 12
reduction in the first year, due to an enhanced regulation of
osteoclastogenesis and increased bone resorption, and asecond phase, with a maximum 3% loss per year, explainedby an altered bone formation [13]. The mechanisms
concurring to GIO pathogenesis are complex and imply both
systemic effects, as well as local consequences on everybone tissue cell type [14].
Osteoblasts. In patients with GC therapy appear
intricated actions on osteoblast progenitors, osteoblasts andosteocytes, which contribute to bone loss and GC induced
osteoporosis [10]. Several experimental studies
demonstrated that GIO is characterized by an important
decrease of osteoblast cells [15].
MSCs (mesenchymal stem cells), a subpopulation ofbone marrow cells capable of bone formation, that candifferentiate into multiple cellular lines, are altered by GC
therapy, by stimulating the expression of different adipocytespecific transcription factors, like upregulation of PPARγ2(peroxisome proliferation-activated receptor gamma
receptor 2) and CCAAT (cytosine-cytosine-adenosineadenosine-thymidine)-enhancer-binding proteins (C/EBPs)
α, β and γ; consequently, the balance is shifted towards
adipogenesis and limited capacity of osteogenesis andosteoblast production [16-21].
Osteoblasts proliferation is inhibited by GC antianabolic
effects, as suppression on the synthesis of different growth
10

factors, such as insulin growth factors (IGF), hepatocyte
growth factor (HGF), or inhibiting the activity ofkinases/phosphatases [22, 23]. Another mechanism
responsible for osteoblasts’ impaired proliferation isrepresented by attenuation of cell cycle progression; several
studies reported that cell cycle regulators CDK2, 4,6 cyclin
D, c-Myc and E2F are downregulated and cyclin-dependent
inhibitors, such as p21 and p27, and the tumor suppressor
gene, p53, is stimulated [24-26].
High doses of GC inhibit osteoblasts differentiation byinterfering with pathways essential for this process:downregulation of Wnt/β-catetin and BMP (bonemorphogenetic protein) signaling pathway or upregulationof Notch inhibitory pathway. Physiologically, Wnt signaling
prevents osteoblasts apoptosis and stimulate their
proliferation. Wnt/ β-catetin signaling pathway is activatedby Wnt binding to frizzled (Fz) receptors and co-receptors
for low density lipoproteins receptor related protein (LRP)
5 and 6, which determines a reduced osteoclasts
generation and osteoblasts differentiation and maturation[15, 27, 28]. High GC doses reduce Wnt expression andincrease sclerostin and Dkk1 expression, thus inhibiting
osteoblasts differentiation; by inducing Notch1 and Notch2
mRNA in osteoblasts, their differentiation is also inhibited[2, 29, 30].
Osteoblasts apoptosis. It is extensively reported thatan important contributor to bone loss is represented by GCinduced apoptosis in osteoblasts and osteocytes [15, 28,31]. There are several mechanisms that potentiallycontribute to apoptosis: excessive GC determine anincrease of pro-apoptotic factors, Bim and Bak, anddecrease the expression of BclXL, a pro-survival factor;also, high GC doses upregulate p53 protein levels, activate
proline-rich tyrosine kinase 2 (Pyk2), by Ca entrance fromextracellular space, and consequently JNK (c-Jun Nterminal protein kinase) activation, cell detachment andapoptosis [32]. Experimental studies showed that in humanosteoblasts dexamethasone reduces β1 integrin expression
and prevents cell-matrix adhesion, with cell death [33].
Therefore, osteogenic cellular line is prone to apoptosis due
to GC administration through inhibiting anti-apoptotic factors
and enhancing different pro-apoptotic pathways.
Osteocytes. Osteocytes, the most abundant bone cell
type, with a tight connection that allows them tocommunicate, sense mechanical alterations, repair andmaintain bone integrity, are differentiated osteoblasts withcharacteristic dendrites. GC dramatically influence
osteocytes function and number, as they upregulate thehormonal factor FGF23, event that decreases serum 1,25
(OH)2-vitamin D3 levels and phosphorus [34, 35]; also,
osteocytes are a major source of RANKL, and GC stimulate
the expression of RANKL and of inhibitors of Wnt signaling,as Dkk1 (dickkopf signaling pathway inhibitor 1) and Sost
(sclerostin) [36, 37]. Several studies have proposed toestablish the dose and time of GC exposure that determines
a decrease of osteocyte number; it was reported that long
Fiziologia - Physiology • 2018.28.2(96)term high GC doses (2.8-5.6mg/kgc/day) induce theirapoptosis and low doses, under 2.8mg/kgc/day, determine
autophagy [38, 39]. Weinstein el al reported that bone aging
is directly related to an increased activity 11-beta-HSD1, a
GC-activating enzyme, sustaining an increased sensitivity ofosteocyte response to GC in older subjects [40].
Autophagy. Autophagy, a fundamental process for cell
survival, can become insufficient or extensive and produce
apoptosis. Bone homeostasis is maintained by autophagy inall cellular lines (osteoblasts, osteocytes and osteoclasts),
and is relevant to several pathologies, including GIO [41].
GC interfere with osteoblast autophagy, the results ofseveral experimental studies indicating that they may inhibit
the process in a dose and time dependent manner [42, 43].
In a similar way, apparently autophagy is a protective
mechanism for osteocytes in response to GC administration[32].
Osteoclasts. GC induced bone lost begins with a fast
process of bone resorption due to an enhanced osteoclasts’
number and activity [44]. GC increase the production ofreceptor activator of nuclear factor-κB ligand (RANKL) anddecrease osteoprotegerin (OPG) production by osteoblastic
cells and osteocytes, with a consequent enhancing ofosteoclasts’ proliferation and differentiation [45, 46].
RANKL, receptor activator of nuclear factor-kappa B(RANK) and OPG, members of TNF family, have a majorrole in bone remodeling and disorders of mineralmetabolism. The differentiation and activation of osteoclasts
is promoted by RANKL binding to its receptor, RANK [47].
OPG functions as a decoy receptor for RANKL, which
prevents RANKL binding to RANK and constitutes aprotective factor against bone loss [48]. The reports ofseveral studies that investigated the RANKL/RANK/OPG
system, concluded that is represents a major regulator ofbone resorption [49]. Experimental studies have reported
that GC administration induces the production of RANKL inbone marrow adipocytes and consequently supports
osteoclasts differentiation in vitro [37, 50-52]. Proinflammatory cytokines, as TNF-α, IL-1, IL-6 and IL-17 canactivate osteoclasts directly; production of RANKL and MCSF is stimulated by TNF α and IL-17, produced by Th17
cells, that can release directly RANKL; these proves suggestthat Th17 cells can be considered a major factor involved inosteoclastogenesis [53-58].

CONCLUSIONSGC therapy is used with high efficiency in a wide variety
of inflammatory and autoimmune diseases and in long term
administration constitutes the most common cause of
osteoporosis. GIO is an important pathology with potentiallysevere complications and identifying risk categories andinitiating proper therapeutic measures is essential for
osteoporosis and fracture prevention in chronic GC users.

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MECANISMELE PIERDERII OSOASE ÎN
OSTEOPOROZA GLUCOCORTICOID INDUSĂREZUMAT
Osteoporoza, reprezentată de scăderea densității și masei osoase, alături de alterarea microarhitecturii osoase, este oafecțiune generalizată a scheletului osos, asociată cu un risc fracturar înalt și complicații potențial severe; este recunoscută
ca o problemă majoră de sănătate publică, cu peste 200 de milioane de cazuri diagnosticate și peste 9 milioane de fracturi
anuale. Terapia glucocorticoidă este utilizată cu eficiență înaltă în afecțiuni inflamatorii și autoimmune, iar administrarea petermen lung constituie cea mai importantă cauză de osteoporoză secundară. Osteoporoza glucocorticoid indusă este
recunoscută a fi asociată cu o prevalență crescută a fracturilor vertebrale, în special datorită efectelor asupra osuluitrabecular. Riscul fracturar este direct asociat cu doza, este prezent chiar și la doze mici, în special în primele 6 luni detratament, atât pentru fracturile vertebrale, cât și pentru cele non-vertebrale; peste 10% dintre pacienții cu terapie cronică
cu glucocorticoizi sunt diagnosticați cu o fractură.
Efectele glucocorticoizilor asupra masei osoase apar în două faze: prima, rapidă, determină reducerea masei osoase cu
6-12% în primul an, consecutive osteoclasogenezei și intensificării resorbției osoase, și a doua, datorată alterării formării
oasoase, ce determină o pierdere a masei osoase de aproximativ 3% pe an. Mecanismele ce determină apariția
osteoporozei glucocorticoid induse sunt complexe și asociază efecte sistemice și locale, asupra fiecărui tip de celulă
osoasă.
Cuvinte cheie: glucocorticoizi, osteoporoză, risc fracturar

Fiziologia - Physiology • 2018.28.2(96)13SIMULTANEOUS CANCERS OF THE HEAD
AND NECK – CASE PRESENTATIONIOAN CRISTIAN MOT, MARIOARA POENARU, NICOLAE CONSTANTINBALICA, DELIA IOANA HORHATENT Department, “Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACT
We present a 76 years old patient with a Laryngeal tumor stage III, T3N0Mx with a s simultaneous neoplasm localized at
the oropharyngeal level stage IIA (left palatine tonsil T2aN1Mx). The patient underwent a complex oncologic surgicalprocedure: total laryngectomy, left palatine tonsillectomy with bilateral lymph node dissection, level I-IV, followed byradiotherapy. Histopathologic exam revealed a squamous cell carcinoma in both tumors. Patient was followed-up for 5
years, being free of disease. Multiple head and neck cancers are not rare entities and according to studies in the literature,
their incidence is increasing. A patient who suffered a head and neck cancer is exposed to a lifetime risk of developing asecond malignancy.
Keywords: simultaneous cancers, synchronous cancers, laryngeal cancer, oropharyngeal cancer, head and neck cancers

INTRODUCTION
The head and neck represents the most complex parts of
the human body, and cancers with this localization presents a
tremendous importance. Head and neck cancer includes
mucosal and tissues malignant tumors. Head and necktumors implies tumors occurring in oral cavity, oropharynx,
rhinopharynx, hypopharynx, larynx, nasal cavity, paranasal
sinuses and salivary glands, etc. [1,2]. Simultaneous,synchronous and methachronous cancers are explained by
the phenomenon of “field cancerization”, not being rare
entities; according to studies in the literature, patients with ahead and neck cancer are at risk of a concurrent,
synchronous and methachronous cancers appearance.
A patient who suffered a head and neck cancer is
exposed to a lifetime risk of developing a second
malignancy [3]. The real incidence of simultaneous,synchronous and metachronous cancers is not known,
but according to some studies, it appears to be
increasing [4]. Head and neck cancers presents a
incidence of approximately 4% in the United States, andup to 35-45% in less developed countries [1,5]. Annuallyare diagnosed about 650,000 new cases of head and neckcancers all over the world, with a ratio of 3:1 between men
and women [3], the majority being in advanced stages(stage III or IV) [6]. From histopathological point of view,

more than 90% of head and neck cancers are squamouscell tumors, and the oral cavity and larynx interests
approximately 60-70% of patients, followed by thepharynx, nasal cavity, salivary glands, thyroid gland, middle
ear, etc. [1,5,7]. Besides squamous cell carcinomas, there
are adenocarcinomas in approximately the rest 10% of thecases, followed by melanoma, soft tissue tumors andlymphomas [1,8].
Despite the medicine evolution regarding head and neckcancer patients’ survival rate, we did not encountered an
improvement, explained probably by the high risk ofrecurrence and the development of a second primary cancer.

CASE PRESENTATION
We present a 76 years old patient which presented in
our ENT Department with dysphonia for 6 months, mild
odinophagya, 10 kg weight loss and intermittent headache.
ENT clinic and endoscopic exam, performing 70º Rigid
Hypopharyngolaryngoscopy revealed a tumor withsupraglottic localization with glottic extension (the righthemilarynx being fixed) (Figure 1) and a simultaneous tumor
localized on the left palatine tonsil (oropharynx).

Received October 17th 2018. Accepted November 20th 2018. Address for correspondence: Nicolae Balica, MD, PhD, ”Victor Babes” Universityof Medicine and Pharmacy Timisoara, ENT Department; Eftimie Murgu Square No. 2A, RO-300041, Timisoara, Romania; phone: +40256
498205; e-mail: balica@umft.ro14

Fiziologia - Physiology • 2018.28.2(96)Fig.1. Supraglottic tumor with glottic extension (right hemilarynx)

Being incriminated several factors and affections suchas lifestyle, genetic susceptibility, immune deficiencies,
radiotherapy, chemoradiotherapy etc. a complete
anamnesis was performed, revealing that the patient
smokes 30 cigarettes / day, drinks approximately 100 ml
pure alcohol/day and his father died with a pharyngeal tumor(without following any treatment modality).
A double biopsy and panendoscopy followed clinic
examination. The biopsies revealed carcinomas of thelarynx and oropharynx (squamous cell carcinoma).
The following diagnostic was established: Laryngeal
cancer staged III, T3N0Mx (T3 supraglottic carcinoma withglottis extension (right hemilarynx) with a simultaneous
neoplasm localized at the oropharyngeal level stage IIA (leftpalatine tonsil T2aN1Mx). The patient underwent a complex
oncologic surgical procedure: total laryngectomy, leftpalatine tonsillectomy with bilateral lymph node dissection,level I-IV. At 14 days after the surgical procedure thenasogastric tube was removed and the patient wasaddressed to radiotherapy. The follow-up continued for 5
years as follows: in the first year at 2 months, in the second
year at 3-4 months, in the 3rd year at 6 months and in the 4th
and 5th year annually. Patient was free of disease.
Simultaneous, synchronous and methachronous cancers
are not a rare entities. In our ENT Department, evaluating aperiod of 10 years, we encountered a number of 23 patientswith head and neck simultaneous, synchronous andmethachronous cancers (5 patients presented simultaneouscancers, 2 patients presented synchronous tumors and 16
patients presented metachronous tumors). The most frequently
involved organ was the larynx, followed by the hypopharynx,
oropharynx and oral cavity.

DISCUSSIONS
Patients who presented during their life a head and neckcancer and who were treated for it, have a high risk todevelop multiple cancers. Simultaneous, synchronous andmetachronous cancers appears in 3-5% of patients,
presenting three cancers involves approximately 0.5% ofFiziologia - Physiology • 2018.28.2(96)patients and the concomitance of four cancers occur in lessthan 0.3% of patients [9].
From historical point of view Billroth reported for the firsttime in 1889 a simultaneous-synchronous cancers astomach cancer and an external ear cancer (the second
primary cancer) [10,11]. The term “field cancerization” might
explain the phenomenon that occurs in the upper aerodigestive tract when mucosa at this level is exposed to thesame types of carcinogens, if they act consistently and for along time [5,12,13]. “Field cancerization” mainly affects theoral cavity, oropharynx and larynx, lung, esophagus, vulva,
cervix, colon, breast, bladder and skin [14].
From clinical and oncological point of view it is important
to differentiate between recurrence and second primary
metachronous cancer.
The cancers might be simultaneous tumor appears inthe same time with the primary (initial) tumor; synchronoustumors, which develop within 6 months of initial cancer
diagnosis and metachronous tumors that occur at least 6months after initial primary tumor development [15,16].
From statistically point of view a second primary tumor may
appear with an incidence of 15% for synchronous tumours
and 4% of the metachronous ones [17].
In our ENT Department simultaneous, synchronous andmethachronous cancers are not a rare entity. In a period of 10
years we encountered a number of 23 patients with head andneck simultaneous, synchronous and methachronous cancers(5 patients presented simultaneous cancers, 2 patients
presented synchronous tumors and 16 patients presented
metachronous tumors). More than 20,000 patients were
evaluated, out of which 8,000 there were oncologic patients.
The most frequently involved organ was the larynx, followed by
the hypopharynx, oropharynx and oral cavity.
Mortality risk is higher in patients with second primary headand neck cancer, having the primary tumor in an early stage,compared with mortality risk from the primary tumor [7,18,19].
The prognosis for simultaneous and synchronous
cancers is worsten than the prognosis of and methachronous
cancers [16].
There are incriminated several factors and affections
such as lifestyle, genetic susceptibility, immune deficiencies,
radiotherapy, chemoradiotherapy etc. Follow-up of patients
who have had a head and neck cancer and periodic control
are important for early detection of multiple cancers.

CONCLUSION
Multiple cancers of the head and neck are not as rare as
we might think. The number of patients with synchronous or
methachronous cancers in head and neck area has
increased. Due to increased life expectancy it is very
important to monitor and investigate all oncologic patients
regarding tumor evolution and the entire upper
aerodigestive tract for a possible early detection of second
primary cancers.
15

REFERENCES
1. Shah PJ, Lydiatt W. Treatment of Cancer of the Head andNeck. Ca – A Cancer Journal for Clinicians 1995;45:352- 368.
2. Shah PJ, Patel S, Singh B. Jatin Shah’s Head and Neck
Surgery and Oncology, 4th edition, Mosby, 2012.
3. Munro AJ, Stafford ND. Head and Neck Cancer, In: Price P,
Sikora Karol, T Illidge T. Treatment of Cancer, Hodder Arnold,
2008:343-345.
4. Lyons MF, Redmond R, Covelli H. Multiple Primary Neoplasiaof the Head and Neck and Lung. Cancer 1986; 57:2193-97.
5. Evans PHR, Montgomery PQ, Gullane PJ. Principles and Practiceof Head and Neck Oncology, London: Martin Dunity, 2006.
6. List MA, Siston Amy, Haraf D, et al. Quality of Life andPerformance in Advanced Head and Neck Cancer Patients on
Concomitant Chemoradiotherapy: A Prospective Examination.
J Clin Oncol 1999; 17:1020-1028.
7. Bradley PJ. Head and Neck Cancer, In: Ludman H, Bradley PJ.
ABC of Ear, Nose and Throat, Fifth Edition, Blackwell
Publishing, 2007:93-101.
8. Dwivedi RC, Chisholm E, Kanwar N, et al. Epidemiology,
aetiology and natural history of head and neck cancer, In:
Staffieri A, Sebastian P, Kapre M et al. Essentials of Head andNeck Cancer, India, 2012:1-7.
9. Mehdi I, Shah AH, Moona MS, et al. Syncrhronous andmetachronous Malignant Tumors Expect the Unexpect. J Pak
Med Assoc 2010; 60:905-909.
10. McGuirt WF, Matthews B, Koufman JA. Multiple Simultaneous
Tumors in Patients with Head and Neck Cancer. Cancer
1982;50:1195-1199.
11. Jones AS, Morar P, Phillips DE, et al. Second Primary Tumors
in Patients with Head and Neck Squamous Cell Cancer.
Cancer 1995; 75:1343-1352.

12. Bronchud MH. Selecting the Right Targets for Cancer Therapy,
In: Bronchud MH, Foote MA, Giaccone G et al. Principles of
Molecular Oncology, IIIrd New Jersey: Edition, Humana Press,
2008:1-10.
13. Hittelman WN. Head and Neck Field Carcinogenesis, In:
Ensley JF, Gutkind JS, Jacobs JR, Lippman SM. Head andNeck Cancer Emerging Perspectives, USA: Academic Press,
2003: 227-243.
14. Braakhuis B, Tabor M, Kummer A, et al. A Genetic Explanationof Slaughter’s Concept of Field Cancerization: Evidence andClinical Implications. Cancer Res 2003;63:1727-30.
15. Gallo O, Sardi I, Pepe G, el al. Multiple Primary Tumors of theUpper Aero-digestive Tract: Is There a Role for Constitutional
Mutations in the p53 Gene? Int J Cancer 1999; 82:180-186.
16. Gale N, Zidar N. Benign and Potentially Malignant Lesions of
the Squamous Epithelium and Squamous Cell Carcinoma, In.
Cardesa A, Slootweg PJ. Pathology of the Head and Neck,
Springer, Germany, 2006: 1-38.
17. Chang S, Ha P. Biology of Head and Neck, In: Flint PW,
Haughey BH, Lund VJ et al. Cummings Otolaryngology, Headand Neck Surgery, Fifth Edition, Volume One, Mosby Elsevier,2010:1015-1029.
18. Stack BC, Clarke B, et al. Head and Neck Squamous Cell
Carcinoma: Therapy with Fusaric Acid/Paclitaxel, In: Hayat
MA. Methods of Cancer Diagnosis, Therapy, and Prognosis,
Volume 7, General Overviews, Head and Neck Cancer andThyroid Cancer, USA, 2010:229-233.
19. Kelly DJ, Shah JP. Global Incidence and Etiology, In:
Close LG, Larson DL, Shah JP. Essentials of Head andNeck Oncology, New York: Thieme Medical Publisher,
1998:3-10.

CANCERE SIMULATANE DE CAP ȘI GÂT –PREZENTARE DE CAZ
REZUMATÎn această lucrare prezentăm un pacient de 76 de ani, cu tumoră laringiană stadiul III, T3N0Mx cu un neoplasm simulatan
localizat la nivel orofaringian stadiul IIA (amigdala palatină stângă T2aN1Mx). Pacientul a suferit o procedură chirugicală
oncologică complexă: laringectomie totală, tonsilectomie palatină stângă cu disecție bilaterală a ganglionilor limfatici, nivelul
I-IV, urmată de radioterapie. Examenul histopatologic a arătat carcinom cu celule scuamoase la nivelul ambelor tumori.
Pacientul a fost urmărit timp de 5 ani, neavând remisiuni. Cancerele multiple de cap și gât nu sunt entități rare și, conform
datelor din literatură, incidența acestora este în creștere. Un pacient care a dezvoltat un cancer la nivelul capului sau gâtuluiprezintă un risc pentru tot restul vieții de a dezvolta o afecțiune malignă.
Cuvinte cheie: cancer simultan, cancer sincron, cancer laringian, cancer orofaringian, cancere de cap și gât

16

Fiziologia - Physiology • 2018.28.2(96)ALERGIC RHINNITIS – METHODS OF PREVENTIONCRISTINA M. GOANŢĂ1,2, MIHAIL TUȘALIU1,3, ANDREEA STANCIULESCU4
1University of Medicine and Pharmacy “Carol Davila”, Bucharest, Romania
2Clinical Emergency Hospital “Sf. Pantelimon”, Bucharest, Romania
3Institute of Phonoaudiology and Functional ENT Surgery “Prof. Dr. D. Hociotă”, Bucharest, Romania
4 Centre Hospitalier de Gonesse, Service d’Explorations Fonctionnelles Neurosensorielles

ABSTRACT
Allergic rhinitis is a frequent pathology affecting about 20% of the population. The risk is higher if the patient associates
asthma, different types of eczema or has a history of allergic diseases. You don’t have an age limit for allergic rhinitis – itcan develop also in newborns or in elder people. Symptoms have remission periods – meaning they can disappear
completely for a long period of time in which the patient can feel he has been completely cured. Allergic rhinitis is causedby the reaction of the nasal mucosa to small particles called allergens. Most commonly we classify the type of allergic
rhinitis by its duration. We can have seasonal allergic rhinitis (rhinitis that occurs in allergy-specific seasons) and perennial
allergic rhinitis (which is present anytime during the year.)
Key words: allergic rhinitis, nasal obstruction, allergens

INTRODUCTION
Allergic rhinitis is a frequent pathology affecting about
20% of the population. Allergic rhinitis is defined as a
symptomatic affection of the nose induced by allergen
exposure, its inflammation being mediated by IgE.
The risk is higher if the patient associates asthma,different types of eczema or has a history of allergic
diseases. It is important to remember you don’t have an age
limit for allergic rhinitis – it can develop also in newborns orin elder people. Usually the symptoms have remission
periods – meaning they can disappear completely for a long
period of time in which the patient can feel he has been
completely cured [1].

Main causes for the development of alergic rhinitis
Allergic rhinitis is caused by the reaction of the nasal
mucosa to small particles called allergens.
This allergic reaction is characterized by activation of
two types of inflammatory cells: mast cells and basophils.
These cells produce pro-inflammatory substances such as
histamine and these substances cause congestion andstimulate fluids to gather in the nasal mucosa leading toitching and sneezing. After this cascade is activated, othermechanisms continue to activate, and the symptoms
become persistent and increasingly unpleasant [2].
Most commonly we classify the type of allergic rhinitis
by its duration. We can have seasonal allergic rhinitis(rhinitis that occurs in allergy-specific seasons) andperennial allergic rhinitis (which is present anytime during
the year.)
Seasonal allergic rhinitis is most often caused byallergens coming from tree pollen, grass, various plants(the most common being ambrosia) or molds. Seasonalallergies generally do not occur in children under 2 yearsof age [3].
Allergens that produce perennial rhinitis are the ones
that surround us throughout the year and are usually foundright inside our house. The most commonly encountered are
dust, mites, hair or puff of certain animals, kitchen beetles.
This type of allergy is the most difficult to treat because it isvery difficult to remove the allergen from the patient's
environment [4].
ARIA (Allergic Rhinitis and its Impact on Asthma)classifies allergic rhinitis in intermittent : less than 4 days aweek or less than 4 weeks or persistent: more than 4 daysa week or more than 4 weeks.

Received July 14th 2018. Accepted September 16th 2018. Address for correspondence: Cristina Maria Goanta, MD, PhD, Clinical Emergency
Hospital “Sf. Pantelimon”, Pantelimon Street no. 340-342, RO-021623 Bucharest, Romania; phone: +40723648919; e-mail:cristina.goanta@gmail.com

Fiziologia - Physiology • 2018.28.2(96)17Main symptoms of allergic rhinitis
The most common symptoms are:‐ Nose related: the nose will usually flow in the formof aqueous secretions, it will be clogged, thepatient breathes hard, sneezes, and has the
feeling of secretions in the back of the nose .
Sometimes they can associate a lack of taste or asense of pressure or pain in the sinus area.
‐ Eyes related: itching may occur and the eyes may
be red. Sometimes the patient can describe afeeling of sand in their eyes.
‐ Throat related - there is a feeling of numb, dry
throat, the voice can be hoarse and a chronic
cough that does not respond to classic coughtreatment can appear.
‐ In addition, the patient can relate that he breathes
more than the mouth, sleeps poorly and wakes up
often, during the day is tired and focuses hard on
work.
Allergic rhinitis is diagnosed by detailed history,
including questions about possible asthma, and nasal
examination, together with inspection of throat, ears andchest where possible, backed up by specific allergy tests,
either skin prick or blood tests, for specific IgE to allergens
suggested by the history.

Clinical history: symptom type, duration and frequency
and exacerbating factors are important for diagnosing andclassifying rhinitis which is characterised by two or more nasal
symptoms: itching, sneezing, obstruction and rhinorrhoea.
The timing of these in relation to exposure to allergen (i.e.,
specific season or animal) is highly relevant. Upon such
exposure,symptoms occur in minutes and last for hours.
Late-phase symptoms can include nasal obstruction,
hyposmia, postnasal mucous discharge and nasal hyperreactivity [5].
What can we do to prevent allergic rhinitis?
The best advice for the patient is to eliminate theallergen from the environment. For that the first step is to
identify the allergen. There are four major categories oftrigger:‐ Pollen - spring and summer - trees, grass, autumn- hay‐ Insects - kitchen beetles, ladybugs‐ Allergens of animal origin - skin, fur, fluff, saliva.
‐ Molds
The most common indoor allergens are dust and dog or
cat hair. It is good to inform your patient with regard to indoor
allergens, that a period of 3 to 6 months is required until thesymptoms are resolved after the allergen is removed [6].
Mites - these are a very small type of insect microscopic
living in mattresses, sofas or other types of material. They
do not bite or produce any harm other than allergies. They
18

absorb moisture from the atmosphere and feed on organic
matter (human or animal skin). They can be removed byusing special shells against the mites. The patient should be
advised to cover the bed with special anti-mucus covers thatare very tight on the bed. Improvise plastic bedding glued
with adhesive tape as described by various patients should
not be improvised. They are inefficient and put the patient's
life in danger by choking. Lingerie should be washed weekly
in warm water and dry in the dryer if this is possible [7].
Exposure may be reduced if the patient uses a vacuumwith a HEPA filter, wipes the dust frequently, and does not
sleep on upholstered furniture (for sofas).
Air humidity must be maintained at a level between 30
and 50%. Humidifiers are not recommended because there
are some studies that claim their use amplifies the problem.
When possible unnecessary items should be removed,excess furniture, curtains, drapery rugs, especially in rooms
where the patient spends a lot of time (bedroom, office)because they are sources of dust. Plush toys are to be
avoided [8].
Puff / animal fur -any animal can cause allergies. In cats,allergies are produced by a protein found in its saliva, theskin, the urinary and reproductive tracts, and not the hair as
it is popular believe. So a cat with short fur is as allergenic
for a patient with allergic terrain as a long-haired cat. If thepatient really wants a pet, he can try a reptile, turtle or fish although fish are not recommended because they live under
the pool they live in is a perfect environment for allergy
development.
If the patient is allergic to an animal then it must be
removed from the house. Limiting it to an area of the house
is not effective because allergens are frequently carried by
the air and the clothes of the people who come in contact
with the animal. After the animal has been removed from thehome, the carpets, sofas, curtains, drapes and linen shouldbe thoroughly cleaned. You should recommend a thoroughcleaning of the home especially after cats because theallergens produced by it are very adherent and theirelimination from the environment lasts months after the cat
has been removed [9].
Other advices for allergic patients [10]:‐ Discard garbage every night to prevent the bugs
from appearing‐ Wash dishes and cutlery immediately after use‐ Do not leave water overnight in the sink or bathtub.
‐ Molds live in wet environments, such as theshower wall, sink pipe area, basements, drain
trays. It is very important to remove the mold every
time you view it. Mold grows well on the soap filmthat covers the sink, the shower or the faience.
‐ Change the air filters frequently‐ If the patient is allergic to a factor outside thehouse, try to avoid exits in the allergic season. If
he should expose to a known allergen, it isadvisable to use a specially designed mask.
‐ The shower before bedtime is essential
Fiziologia - Physiology • 2018.28.2(96)‐Nasal lavage twice a day is very important toeliminate allergens.

bed and use a HEPA filter vacuum cleaner to keep the air in
the room as clean as possible.

There are a few medications that can be helpful:
-

-

-

-

Sea water sprays - helps treat sneezing, dry noseand throat.
Spray with glucocorticoids (steroids) - these are
the first line in the treatment of allergies. They
have few side effects and are very effective insolving aergic rhinitis. Studies show that in somecases they are even more effective thanantihistamine pills. These are usually applied as
one or two puffs twice a day. Although the effectcan be noticed from the first day, treatment should
be used for at least a few weeks, and in somepatients it may be necessary to use it continuallyeven when the patient is well [11].
People with severe allergic rhinitis may need touse a decongestant nose spray in the first few
days toghether with the steroid .
Antihistamines - are antiallergic drugs that reduceitching, sneezing but do not reduce the feeling of a
stuffy nose. They must therefore be taken in
combination with the nasal spray. Some drugs of thistype can cause drowsiness, although those of the
new generation no longer produce this symptom.
In some selected cases, vaccins can be made to
reduce the body's sensitivity to a specific allergen.
They can only be given for certain allergens suchas pollen, dog or cat hair, dust, mites or mold. The
most important role of immunization is thepossible prevention of asthma. Studies show that
this treatment method is more effective if
administered over a period of 3-5 years [12].

CONCLUSION
If we know the allergen we will avoid exposing thepatient to this factor as much as possible. For example, if
the patient is allergic to pollen, he should not go out in thehigh-grade pollen days, keep the doors and windows of thehouse and car closed, wash himself every evening before

Fiziologia - Physiology • 2018.28.2(96)REFERENCES
1. Chipps B, Spector S, Farrar J, Carr W, Meltzer E, Storms W,
Kaliner M, et al. Differences in recommendations between
the Allergic Rhinitis and its Impact on Asthma Update 2010
and US Rhinitis Practice Parameters. Journal of Allergy andClinical Immunology, 2011; 127(6): 1640-41.
2. Masuda S, Fujisawa T, Katsumata H, et al. High prevalence
and young onset of allergic rhinitis in children with bronchialasthma. Pediatr Allergy Immunol 2008; 19:517-22.
3. Jorksten B, Clayton T, Ellwood P, et al. Worldwide time trends
for symptoms of rhinitis and conjunctivitis: Phase III of theInternational Study of Asthma and Allergies in Childhood.
Pediatr Allergy Immunol 2008; 19:110-24.
4. Brożek JL, Bousquet J, Baena-Cagnani CE, Bonini S,
Canonica GW, Casale TB, etal. Allergic Rhinitis and its Impact
on Asthma (ARIA) guidelines: 2010 Revision: Journal ofAllergy and Clinical Immunology, 2010; 126(3): 466-476.
5. Scadding GK. Optimal management of allergic rhinitis. ArchDis Child 2015; 100:576-582.
6. Canonica GW, Compalati E. Minimal persistent inflammationin allergic rhinitis:implications for current treatment strategies.
Clin Exp Immunol., 2009; 158:260-71.
7. Jacobsen L, Niggemann B, Dreborg S, et al. Specificimmunotherapy has long-term preventive effect of seasonal
and perennial asthma: 10-year follow-up on the PAT study.
Allergy, 2007; 62:943-8.
8. Roberts G, Mylonopoulou M, Hurley C, et al. Impairment inquality of life is directly related to the level of allergen exposureand allergic airway inflammation. Clin Exp Allergy 2005;
35:1295-300.
9. Vuurman EF, van Veggel LM, Uiterwijk MM, et al. Seasonalallergic rhinitis and antihistamine effects on children’s learning.
Ann Allergy 1993; 71:121-6.
10. Santos CB, Pratt EL, Hanks C, et al. Allergic rhinitis and its
effect on sleep, fatigue, and daytime somnolence. Ann Allergy
Asthma Immunol. 2006; 97:579-86.
11. Stelmach R, do Patrocinio TN, Ribeiro M, et al. Effect oftreating allergic rhinitis with corticosteroids in patients with
mild-to-moderate persistent asthma. Chest 2005;
128:3140-7.
12. Radulovic S, Calderon MA, Wilson D, et al. Sublingual
immunotherapy for allergic rhinitis. Cochrane Database Syst
Rev. 2010; (12):CD002893

19

INVESTIGATION OF NEUTRAL POLYSACCHARIDESFROM FOMES FOMENTARIUS: A PRELIMINARY STUDY
1BOJIN L.*, 2COJOCARIU C.*, 1GEORGESCU M.*, 1PUIU M.,
2,3PASCARIU M.C.#, 1SERB A#, 1PUSCASIU D, 1TATU C, 1SISU E
1“Victor Babes” University of Medicine and Pharmacy Timisoara, Faculty of Medicine, 2 Eftimie Murgu Sq., RO-300041 Timisoara,

Romania
2“Vasile Goldis” Western University of Arad, Faculty of Pharmacy, 86 Liviu Rebreanu, RO-310414, Arad, Romania
3National Institute of Research & Development for Electrochemistry and Condensed Matter, 144 Aurel Păunescu-Podeanu, RO-300569
Timisoara, Romania* these authors have equal contribution# corresponding authors: aserb@umft.ro; mihai.cosmin.pascariu@gmail.com

ABSTRACTThe combined chemical and spectral analysis of the polysaccharide content from the Fomes fomentarius mushroom,
harvested from the spontaneous flora, highlights the presence of a neutral polysaccharide. This glycan is a linear or
branched galactomannan which does not contain any fucose residues, in contrast to what was found in previous research
regarding neutral polysaccharides isolated from this species.
Key words: Fomes fomentarius, MALDI-TOF-MS, Glycan

INTRODUCTION
The total number of mushroom species on the planet isestimated to be around 140 000, of which only 10% (~ 14000 named species) are known. They comprise a vast andyet largely unexplored resource of promising new
pharmaceutical products and have been valued as food andmedical resource for thousands of years [1]. Several
bioactive molecules, which include antitumor compounds,
have been identified in mushrooms. The fruit bodies,cultured mycelium and culture broth of many
Basidiomycetes mushrooms contain biologically active
polysaccharides [1]. Most polysaccharides have uniquestructures in different species, and even different strainsof one species can produce polysaccharides withdifferent properties. They are the best known and mostpotent mushroom derived substances which possess
antitumor and immunomodulating properties [2-10]. Suchpolysaccharides do not attack cancer cells directly, butinstead they activate various immune responses in the host,thus producing their antitumor effects. This requires anintact T-cell component; their activity is mediated through athymus-dependent immune mechanism. However, besides
the biological properties, the biotechnological availability isalso important for practical applications [1].
Fomes fomentarius, which is a member ofBasidiomycetes, has been used in traditional oriental
medicine for treating various diseases, i.e. gastroentericdisorder, hepatocirrhosis, oral ulcer, inflammation, andvarious cancers. Previous research revealed various
appealing biological activities for this fungus, including
antidiabetic, anti-inflammatory, antioxidant and anticancer
properties [11-14]. The polysaccharides from this species
were investigated in the 1970s by Lindberg and Kubala.
They determined the composition and structure for two
types of polysaccharides in the aqueous extract, i.e. neutral
and acidic [15-17].
Lindberg examined the fruit bodies of Fomes
fomentarius for polysaccharide content. The materials,
which were extracted with hot water, were further
investigated. Neutral and acidic polysaccharides wereseparated by treatment with DEAE cellulose. Neutral

Received September 12th 2018. Accepted December 10th 2018. Address for correspondence: Assistant Professor Dr. Alina Florina Serb, Facultyof Medicine, “Victor Babes” University of Medicine and Pharmacy Timisoara, 2 Eftimie Murgu Sq., RO-300041 Timisoara, Romania; phone:
0740 176 484; e-mail: aserb@umft.ro; Mihai-Cosmin Pascariu, Assistant Prof. Dr., Faculty of Pharmacy, “Vasile Goldiş” Western Universityof Arad, 86 Liviu Rebreanu Street, RO-310414 Arad, Romania; phone: 0720 647 622; e-mail: mihai.cosmin.pascariu@gmail.com

20

Fiziologia - Physiology • 2018.28.2(96)polysaccharides were subjected to stepwise acid hydrolysis,
and the fragments of low molecular weight were isolated.
The monosaccharides were separated and identified as
D-mannose, D-galactose, and L-fucose. From the structural
studies, it was found that the mannofucogalactans from
Fomes fomentarius and Fomes igniarius have similar
structures. They consist of a backbone of α(1→6) linked
α-D-galactopyranose residues, of which about 30%(F. fomentarius) and 40% (F. igniarius) are substituted in the
2-position. Most of the substituents are either 3-O-α-Dmannopyranosyl-L-fucopyranosyl or fucopyranosyl residues.
[15,16]At the same time, Kubala determined the structure ofneutral polysaccharides, extracted with water from Fomes
fomentarius. It was found that the backbone of thepolysaccharide consists of D-galactose units, linked together
with (1→6)-glycosidic linkages. Some of the galactose units
are found as branch points substituted in the 3-position byside chains. It can be concluded that the predominant
amount of L-fucose is found in the side chains. On the other
hand, some of the side chains are formed by single L-fucose
units, while others consist of mannosyl-fucose [17].
Bellow we present the results obtained during theextraction, isolation, purification and deciphering of thestructure of neutral polysaccharides from the fruiting body ofFomes fomentarius, harvested from the spontaneous floraof the Vâlcea County in Romania.

EXPERIMENTAL
Equipment
Rotary Evaporator RII (Buchi); Alpha 1-2 Freeze Dryer(Christ); 200 Benchtop Centrifuge (Labofuge); pH meter
inoLab 7110; Glass Oven B-580 (BUCHI); CAMAG Model3 TLC scanner equipped with CAMAG CATS 4 software;
CAMAG Horizontal Developing Chambers (for plate10x10); Bruker Vertex 70 (Bruker Daltonik GmbH,Germany) ultrafleXtreme MALDI-TOF workstation (Bruker
Daltonik).
Reagents
TRIS hydrochloride (ROTH), sodium hydroxide,
phenol (>99%), sulfuric acid 95-97%, 2,5-DHB (2,5dihydroxybenzoic acid) (all from Merck); sodium chloride
99.8%, ethyl alcohol (abs) (from Chimopar S.A., Bucharest);
2,5-dihydroxybenzoic acid (DHB) >99%, α-cyano-4hydroxycinnamic acid (HCCA) 99%, trans-2-[3-(4-tertbutylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB)≥99.0%, 2’,4’,6’-trihidroxyacetophenone monohydrate(THAP) ≥ 99.5% (all from Sigma-Aldrich, Spruce Street,
St. Louis, USA); Sepharose Fast Flow (GE Healthcare
Sweden); dialysis membranes, molecular weight cutoff(MW/CO) 1000 were purchased from Spectrum Europe
B.V. (Breda, The Netherlands).

Fiziologia - Physiology • 2018.28.2(96)SampleThe main steps of extraction, isolation, purification andfractionation of the polysaccharide blend obtained from thefruiting body of Fomes fomentarius are presented below:a) Isolations and control: The fungus was harvested
from Vâlcea County (Romania) in October 2015 and stored
until used at -78 ºC. The analysis and taxonomic
classification of the collected material was carried out by
experts in systematic botany from the Faculty of Chemistry,
Biology, Geography within the West University of Timisoara.
The fungus was treated with liquid nitrogen and grounded
into a powder.
b) Extraction: An aliquot of 300 g from the powder was
extracted by refluxing with:- hexane (3 x 0.5 L);- acetone (3 x 0.5 L);- water (3 x 0.5 L).
The aqueous volumes were combined and brought tominimum volume by evaporating the solvent (in a rotary
evaporator) at low pressure.
c) Purification: The minimum aqueous extract volume(50 mL) was diluted with EtOH (abs) – ten times theminimum volume (500 mL) – precipitating thepolysaccharide fraction, followed by cold (0–4 ºC) filtration.
The obtained precipitate was washed repeatedly withmethanol and acetone (0–4 ºC) and dialyzed (MW/CO =1000) against water for 48 hours. The dialysate was thenlyophilized, yielding 118 mg of yellow-brownish powder.
d) Column chromatography: The column (20 x 2.5 cm),
was filled with DEAE Sepharose Fast Flow and eluted with
a 0.01 M Tris solution adjusted to pH 7 with 0.1 M NaOH.
A sample of 30 mg of raw mixture of polysaccharide in 3 mL
eluent was loaded into the column and washed with 750 mL
of 0.01 M Tris solution adjusted to pH 7, collecting a seriesof 3 mL fractions. The presence of polysaccharide in thefractions was checked by using a spectrophotometricmethod [18]. The column was then eluted with 0.1 M, 0.4 M
and, respectively, 1 M NaCl in 0.01 M Tris solution (3 x 750mL). The representation of the absorbance as a function of
the number of tubes gives us the elution profiles (Figure 1).
Mass Spectrometry
MALDI-TOF mass spectra of the samples wererecorded by using a Bruker ultrafleXtreme MALDI-TOF
workstation controlled by FlexControl (Bruker Daltonics,
Bremen, Germany) equipped with a Smartbeam II laser(Bruker Daltonics) of 355 nm and operating in positivemode. The following settings were used: laser frequency,
2,000 Hz; smartbeam, ‘4_large’; sample rate and digitizer
settings, 1.25 GS/sec; accelerator voltage, 20.07 kW;
extraction voltage, 18.87 kW; lens voltage, 5.58 kW; anddelayed extraction, 250 nsec. A 1,000 laser shots were used
for each individual spectrum and a minimum of ten individual
spectra were cumulated and saved. External calibration was
21

performed using maltodextrin (Paselli MD-6, AVEBE,
Veendam, The Netherlands). FlexAnalysis version 3.3(Bruker Daltonics) was used for data processing. The
analyzed sample was spotted on MTP 384 polished steel
BC targets (Bruker Daltonik). Four types of matrices, withthree different concentrations, known to be used in the
MALDI-TOF determination of oligosaccharides, glycans
and glycoconjugates, were used in combination with threedifferent sample concentrations to find the optimal
ionization conditions. The trials as well as the evaluationof the quality of the spectra by using various settings are
given in Table I.
Preparation of the matrix solution:
DHB matrix solution: 50 mg DHB in 0.5 mL H2O + 0.5 mL
ACN (C1); for C2 and C3 (dilution 1:3 and, respectively, 1:5)
DCTB matrix solution: 50 mg DHB in 1 mL ACN (or THF)
with 5µL TFA (C1); for C2 and C3 (dilution 1:3 and,
respectively, 1:5)
HCCA matrix solution: 50 mg HCCA in 0.5 mL ACN + 0.5
mL MeOH (C1); for C2 and C3 (dilution 1:3 and,
respectively, 1:5)
THAP matrix solution: 50 mg THAP in 0.5 mL H2O + 0.5 mL
ACN (C1); for C2 and C3 (dilution 1:3 and, respectively, 1:5)
Infrared spectroscopy (IR)The FT-IR spectra were obtained in attenuated totalreflectance (ATR) mode by using a Bruker Vertex 70
instrument (Bruker Daltonik GmbH, Germany) equipped with
Platinium ATR, Bruker Diamond Tip A225/ Q.I. 128 of coadded scans were collected in the range of 4000-400 cm-1,
with a resolution of 4 cm-1.
UV spectrophotometry
Carbohydrate determinations using the PhOH/H2SO4
method [18] were performed on a Biochrom Libra S12
UV/VIS Spectrophotometer in 1 cm cuvettes at λ=490 nm.
TL-Chromatography
The linear ascending development was carried out in a
CAMAG glass twin through chamber (10 cm × 10 cm), which
was previously saturated with 10 mL of mobile phase, i.e.
ethyl acetate:1-propanol:anhydrous acetic acid:water =4:2:2:1 (v/v). Plates were developed to a distance of 80 mm,
after which the TLC plates were dried in an air current witha hair dryer. The dried plate was visualized by spraying withCe(SO4)2:(NH4)6Mo7O24·4H2O:H2SO4:H2O = 1:5:10:90 (w)and then dried 5 min to 120 °C.
HPTL–chromatography
Samples (20 μL) were deposited at 10 mm from thebottom of the plate, after which the plate was developed in
the planar HPTLC chamber (CAMAG). After elution (9 cmfrom bottom) the plate was dried and visualized using
Ce(SO4)2:(NH4)6Mo7O24·4H2O:H2SO4 and 5 min heating at
120 °C. The color is stable for 75 minute.
22

Densitometry
The CAMAG densitometry (CAMAG Model-3 TLC
scanner equipped with CAMAG CATS 3 software) used areflectance spectrometer with 190–700 nm monitoring
domain. The slit was set to 8x0.4 mm and data acquisitionand processing were performed using the WinCATS
software. The measurements were performed at λ=580 nm
wavelength.
Total hydrolysis of NP2
3 mg of NP2 was pre-hydrolyzed with 72% (w/w) sulfuric
acid for 1 h at 25 °C, followed by hydrolysis with 1 M sulfuric
acid for 3 h at 100 °C. The reaction mixture was neutralizedwith BaCO3, filtered and then lyophilized [19]. The obtained
product, dissolved in the minimum quantity of water, wasanalyzed by TLC.

Fig. 1. Elution profiles of raw polysaccharides on DEAE-Sepharose
FF column ( 20 × 2.5 cm). The column was equilibrated with 10 mM
Tris-HCl buffer (pH 7.0), then eluted as follows: a) 1-90 tubes 10
mM Tris-HCl buffer; b) 91-170 tubes with 0.1 M NaCl in 10 mM
Tris-HCl c) 171-220 tubes with 0.4 M NaCl in 10 mM TrisHCl d) 221-250 tubes with 1.0 M NaCl in 10 mM Tris-HCl; elution
speed: 1mL/min; volume =3 mL/tube; PhOH/H2SO4 colorimetricmethod (Aλ=490 nm); NP = neutral polysaccharides, AP = acidic
polysaccharides
Table I. The quality of the spectra strongly depends on the sample
application protocol.

M/P first 1 L matrix solution, then 1 L sample solution;
P/M first 1 L sample solution then 1 L matrix solution; MP1 L matrix solution + 1 L matrix solution, vortex andapplication of 1 μL to the target. Quality of spectra: m missing; p - poor; g - good, e - excellent; P-sample; P1-5 mg
sample in 1 mL H2O; P2-1.7 mg sample in 1 mL H2O; P31.7 mg sample in 1 mL H2O; M1=2,5-DHB; M2=4-HCCA;
M3=THAP; M4=DCTB; C1=50 mg matrix; C2=17 mg matrix;
C3=10 mg matrix; # add 0.5 L TFA; * add 0.5 L
HCOOH

Fiziologia - Physiology • 2018.28.2(96)SPOTTED
M/P1
P1/M
MP1
M/P2
P2/M
MP2
M/P3
P3/M
MP3
M/P1 *
P1/M *MP1 *M/P2 *
P2/M *MP2 *M/P3 *
P3/M *
MP3*

C1
g
p
#p
m
m
#p
g
p
g
e
g
g
m
p
g
e
g
e

M1
C2
M
M
P
P
#P
G
P
M
P
G
G
E
M
P
E
G
p
g

C3
m
p
g
m
m
m
p
m
#p
p
p
#p
p
g
p
g
g
p

C1
P
m
#m
m
m
P
#m
P
P
G
P
G
m
m
m
P
m
m

M2
C2
m
p
#m
p
p
m
m
m
#m
p
m
m
p
p
p
m
m
p

RESULTS AND DISCUSSIONS
Starting from the information provided by Park’s work[20] (who extracts, purifies and separates polysaccharides
from Fomes fomentarius), we extracted,
purified and separately collected
polysaccharides from Fomes fomentarius
harvested from the native Romanianflora. Some changes were made as
follows: after the fruiting bodies of Fomes
fomentarius were converted into powder,
300 g were extracted with hexane (toremove lipids) and then with acetone (toremove some of the colored compounds)
and finally with water. After purificationand isolation, chromatography is
performed on Sepharose. In the firststage, during elution with a 0.01 M Tris solution adjusted
to pH 7, the migration of neutral polysaccharides takes
place, after which, during elution with NaCl (0.1 M, 0.4 M
and 1 M, respectively), the elution of acid polysaccharides
occurs. The eluates were tested for the presence ofcarbohydrates using the PhOH/H 2SO4 method according
to a well-established protocol [18]. Graphical representationof the absorbance measured at λ=490 nm as a functionof the number of fractions leads to an elution profile,which is shown in Figure 1 (see Experimental: UV
spectrophotometry).
In contrast to the previous works, our chromatogram
reveals three fractions at neutral/low alkaline pH elution andtwo fractions at acidic pH elution. The three fractions wereisolated, lyophilized, dialyzed (against water) and finallylyophilized. FT-IR analysis of each fraction did not reveal the
carbonyl valence vibration (1700-1800 cm-1), so it was
Fiziologia - Physiology • 2018.28.2(96)

C3
P
P
P
M
p
p
m
m
m
m
p
g
m
m
p
m
p
m

C1
M
m
p
m
p
p
p
m
p
g
p
p
m
m
m
p
m
p

M3
C2
P
M
P
P
M
M
P
P
M
P
P
M
M
M
P
M
M
M

C3
m
m
m
p
p
m
m
m
p
m
m
p
p
m
p
m
m
p

C1
#m
#p
#m
#m
#m
#p
#m
#m
#m
m
m
p
p
m
g
p
p
p

M4
C2
#m
#p
#p
#m
#m
#p
#m
#m
#p
m
m
m
m
m
p
p
m
m

C3
#p
#m
#p
#p
#p
#m
#m
#m
#m
p
m
m
g
p
p
m
m
p

concluded that it does not contain uronic acids or acetyl
residues. The three fractions were named NP1, NP2 andNP3 (from neutral polysaccharides). The FT-IR spectrum of
NP2 fraction is shown in Figure 2.

Fig. 2. FT-IR spectra in attenuated total reflectance (ATR) modeof the NP2 fraction in the 4000–400 cm-1 domain

Using the mass spectrometry to investigate the structureof natural and chemically modified polysaccharides is always
a challenge, which is rewarded when good quality spectra
offer a large number of details regarding the composition ofglycans, the type of connections, and the alternation mode of
the constituent monosaccharides [21-26]. The mass
spectrometric analysis of the NP2 fraction required several
attempts to find the proper conditions (matrix type, matrix
concentration, ionization promoters, sample concentration,
application order on targets etc.) that would generate a good
ionization and would give a high quality spectrum. These
attempts, as well as the related explanations, are given in
Table I. The mass spectrum of NP2, under the specified
conditions, is shown in Figure 3 (A-C).

23

Table II Assignment of the ions observed in (+)MALDI TOF
spectrum of NP2

Fig. 3. Positive MALDI-TOF-MS of the NP2-FF sample in reflectronmode A) m/z 600- 4500; B) m/z 600-1900; C) m/z 1900-4200;$ = [Hexn + Na+]+ ;∆ =[Hexn + Na+-H2O]+; *= [Hexn + K+]+The spectrum analysis immediately reveals the 162 Da“step”, which indicates the existence of a polysaccharide,more specifically of a polyhexose. The absence, on the wholemeasuring range (m/z 600-5000) of a weight loss of 146 Da,
suggests that neither rhamnose nor fucose are present. The
assignment of all peaks from the mass spectrum presented in
Figure 3 (A-C) is given in Table II.

24

Nr.

m/z=

ChargeAssignments

Nr.

m/z=

ChargeAssignments

1.

3933.4+1

Hex24 +Na+

32

2164.3+1

Hex13 +K+2

3787.4+1

Hex23 +K+

33

2148.3+1

Hex13 +Na+

3

3771.3+1

Hex23 +Na

34

2130.3+1

Hex13 + Na+H2O

43753.2+1

Hex23 +
Na+-H2O

35

2002.1+1

Hex12 +K+

53625.2+1

Hex22 +K+

36

1986.1+1

Hex12 +Na+6

3609.1+1

Hex22 +Na+

37

1968.1+1

Hex12 + Na+H2O

73591.1+1

Hex22 +
Na+-H2O

38

1840.1+1

Hex11 +K+

8

3462.8+1

Hex21 +K+

39

1824.0+1

Hex11 +Na+

93446.9+1

Hex21 +Na+40

1806.0+1

Hex11 + Na+H2O

103428.7+1

Hex21 +
Na+-H2O

41

1677.8+1

Hex10 +K+

113300.6+1

Hex20 +K+

42

1661.8+1

Hex10 +Na+

123284.6+1

Hex20 +Na

43

1643.7+1

Hex10 + Na+H2O

13

3266.5+1

Hex20 +
Na+-H2O

44

1515.9+1

Hex9 +K+

143138.2+1

Hex19 +K+

451499.9+1

Hex9 +Na+15

3122.2+1

Hex19 +Na+

46

1481.8+1

Hex9 + Na+-H2O

16

3104.3+1

Hex19 +
Na+-H2O

47

1355.8+1

Hex8 +K+

172974.9+1

Hex18 +K+

48

1337.8+1

Hex8 +Na+

18

2958.9+1

Hex18 +Na+49

1319.7+1

Hex8 + Na+-H2O

192940.9+1

Hex18 +
Na+-H2O

50

1191.6+1

Hex7 +K+

202812.7+1

Hex17 +K+

51

1175.6+1

Hex7 +Na+

212796.7+1

Hex17 +Na+

52

1157.7+1

Hex7 + Na+-H2O

222778.8+1

Hex17 +
Na+-H2O

53

1029.7+1

Hex6 +K+

232650.5+1

Hex16 +K+

54

1013.7+1

Hex6 +Na+

24

2634.5+1

Hex16 +Na+

55

995.6

+1

Hex6 + Na+-H2O

252616.5+1

Hex16 +
Na+-H2O

56

867.7

+1

Hex5 +K+

26

2488.4+1

Hex15 +K+

57

851.6

+1

Hex5 +Na+

27

2472.3+1

Hex15 +Na+

58

833.6

+1

Hex5 + Na+-H2O

282454.2+1

Hex15 +
Na+-H2O

59

705.4

+1

Hex4 +K+292326.6+1

Hex14 +K+

60

689.4

+1

Hex4 +Na+30

2310.6+1

Hex14 +Na+

61

671.3

+1

Hex4 + Na+-H2O

312292.5+1

Hex14++++1

Na+-H2O

Fiziologia - Physiology • 2018.28.2(96)It can be clearly seen in the spectrum that only three ion
series, i.e. [Hexn + Na+], [Hexn +K+] and [Hexn+Na+-H2O], arepresent. It remains to be investigated whether we are
dealing with a homoglycan or a heteroglycan. The total acidhydrolysis of an NP2 sample was carried out, which, after
processing, was analyzed by TLC using galactose,mannose and fucose as references. The analysis ofchromatograms from Figure 4 (A-C) clearly shows that NP2
is a (Gal)x(Man)y heteropolysaccharide. In order to be able
to estimate the relationship between the two components,
the NP2 hydrolysate separation was performed on the
HTPLC plate, which was followed by visualization anddensitometry analysis of the chromatogram.

The densitogram taken at λ=580 nm is shown in Figure
5. From the analysis of the ratios of the areas of the twocomponents, galactose and respectively mannose, we
can conclude that the linear or branched heteropolymer
is a galactomannan with the presumptive formula of[(Gal)18(Man)5 ]nFig. 5. Densitometric chromatogram of hydrolyzed NP2 fraction
after development and vizualisation. Scanned at 580 nm. The
HPTLC plate coated with Silica gel 60 F254 was developed using
the mobile phase ethyl acetate: 1-propanol:anhydrous acetic
acid:H2O = 4:2:2:1 (v/v) and visualized with Ce(SO4)2:(NH4)6Mo7O24.4H2O : H2SO4 : H2O = 1:5:10:90 (w) then 5 min to
120 °C.

CONCLUSIONS

Fig. 4. TLC chromatograms of acidic hydrolisated sample NP2-FF.
A) Identification of galactose: Line1-acidic hydrolysate of NP2, Line 2:
standard galactose; Line 3: standard mannose; Line 4: acidichydrolysate of NP2 + Gal; B) Identification of mannose Line1-acidichydrolysate of NP2, Line 2: standard galactose; Line 3: standard
mannose; Line 4: acidic hydrolysate of NP2 + Man; C) Absence of
fucose: Line1-acidic hydrolysate of NP2, Line 2: standard galactose;Line 3: standard mannose; Line 4: acidic hydrolysate of NP2 + Fuc;Line 5: standard Fucose; Elution: AcOEt:1-PrOH;AcOH:H2O= 4:2:2:1(v/v); visualization:
Ce(SO4)2:(NH4)6Mo7O24.4H2O:H2SO4:H2O =1:5:10:90 (w) then 5 min to 120 oC.

Fiziologia - Physiology • 2018.28.2(96)The analysis of the polysaccharide content from Fomes
fomentarius, harvested from the Romanian spontaneous
flora, by combined methods (chemical and spectral), reveals
the existence of a neutral polysaccharide, which is a
galactomannan and which does not contain any rhamnoseor fucose residues, in contrast to what was found in otherprevious research regarding polysaccharides from thismushroom. A judicious choice of sample content, matrix
type, ionization promoters and application mode allowed forthe best ionization method to be established, which allowed
for high accuracy and quality MALDI-TOF spectra.
ACKNOWLEDGEMENT
This work was supported by the Romanian National Authority for
Scientific Research (CNCS-UEFISCDI) through project PN-IIPCCA-2011-142. Part of the research was done at the Center ofGenomic Medicine from the “Victor Babes” University of Medicine
and Pharmacy of Timisoara, POSCCE 185/48749, contract25

677/09.04.2015. The research collective wishes to express the fullgratitude towards prof. Sinitescu from West University of Timisoara
for his selfless support.

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26. Sisu E., Bosker W. T. E., Norde W., Slaghek T. M. ,Timmermans J. W., Katalinic J. P., Cohen-Stuart M. A., ZamfirA., Electrospray ionization quadrupole time-of-flight tandem
mass spectromteric analysis of hexamethylenediaminemodified maltodextrin and dextran, Rapid Commun.Mass
Spectrom. 2006;20(2):209-218

Fiziologia - Physiology • 2018.28.2(96)INVESTIGAREA POLIZAHARIDELOR NEUTREPROVENITE DIN FOMES FOMENTARIUS:
UN STUDIU PRELIMINARREZUMATPrin combinarea analizei chimice și spectrale a conținutului de polizaharide din ciuperca Fomes fomentarius, recoltată din
flora spontană, se pune in evidenta prezența unei noi polizaharide neutre. Acest glican este un galactomanan liniar sau
ramificat, care nu conține resturi de fucoză, spre deosebire de cele descoperite în cercetările anterioare privind
polizaharidele neutre izolate din această specie.
Cuvinte cheie: Fomes fomentarius, MALDI-TOF-MS, Glican

Fiziologia - Physiology • 2018.28.2(96)27

MANAGEMENT OF EPISTAXIS IN NASAL TUMORSAND HEART DISEASES AT THE UNIVERSITYHOSPITAL OF TIMISOARA
ALINA MARIA MARIN, AUREL MARIN, MARIOARA POENARU
ENT Department, “Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACT
Background and aim. Epistaxis is a frequently disease, affecting up to 60% of the population and is the common reason
for healthcare utilization.
Methods. The study includes patients presenting to the E.N.T department of the University Hospital of Timisoara fromJanuary 2014 to October 2018 with anterior and / or posterior epistaxis. Demographic factors (age, rase, gender),medication use (anticoagulants and anti-aggregant medication) and several comorbidities were analyzed as potential
predictors of episodes.
Results. A total of 346 patients were identified with a total of 456 individual epistaxis episodes over 4 years. Multivariate
analysis identified nasal tumors, hypertension, overdose of anticoagulant medication and seasonal variations as predictorsof higher number of cases.
Conclusions. Epistaxis occurred more frequently in older patients with arterial hypertension. Allergic rhinitis, chronicsinusitis, nasal tumors and hypertension are associated with increased epistaxis incidence.
Keywords: epistaxis, nasal tumor, hypertension, nasal packing, cauterization, anticoagulants.

INTRODUCTION
Epistaxis is an otolaryngologic affection known since
antiquity and the term is derived from the Greek, epistazein(epi – above, over; stazein – to drip)[1]. Epistaxis limited tonasal bleeding was introduced by the Englishman Cullen in
1785 and the Frenchman Pinel in 1818 and later became
widespread in medical language [2].
This is a frequently disease and it is estimated from a
Scandinavian survey in 1974 of 410 people that up to 60%of the population will one episode of epistaxis in their lifetimeand 6% will seek medical attention[2]. This is a frequently
disease and the Scandinavian study in 1974 of 410 people
is evaluated that up to 60% of the population will have atleast one episode of epistaxis in their lifetime and 6% willneed health care utilization [2].
A US health examination survey from 1972 of 6672
adults revealed a 7% to 14% incidence of epistaxis [2].
Incidence from most reports from Europe and America is
about 10%-15% of the population [3-5]. Incidence of
epistaxis in a US health investigation study from 1972 of

6672 people is estimated between 7% and 14% [2]. The
Europa and America reports revealed a 10%-15% incidenceof the epistaxis.
Although epistaxis may occur at any age or at any timeand in any season, it is a common complaint in the pediatricage group and the winter months but has been shown tohave bimodal age range presentation in reports from North
America and Europe [3-6]. Reports from North America andEuropa demonstrated that epistaxis may occur at any age
or at any time and in any season, it is a common disease in
the pediatric age group and the winter months but has beenshown to have bimodal age range presentation [3-6].
It is a frequent otolaryngologic emergency, which inserious cases will need a full complement of resuscitative
measures to stabilize the patient and prevent or address
hypovolemic shock [3-7]. Knowledge of history of epistaxis
in a patient will assist the physician or surgeon in taking
precautions while planning any treatment modality be it
medical or surgical. Failure to take these precautions maylead to unpleasant consequences [3-8]. In serious cases of
epistaxis will need resuscitative measures to stabilize the

Received September 17th 2018. Accepted November 20th 2018. Address for correspondence: Nicolae Balica, MD, PhD, ”Victor Babes”
University of Medicine and Pharmacy Timisoara, ENT Department; Eftimie Murgu Square No. 2A, RO-300041, Timisoara, Romania; phone:+40256 498205; e-mail: balica@umft.ro28

Fiziologia - Physiology • 2018.28.2(96)patient and prevent hypovolemic shock [3-7]. Previous
medical history of epistaxis in a patient will direct thephysician or surgeon to take the precautions and to choose
medical or surgical treatment. Failure to take theseprecautions may lead to unpleasant consequences on thehealth of the patient [3-8].
The etiologies includes local factors, such as trauma or
direct irritation and systemic factors. Yüksel et al. [4] foundthat the evidence available was insufficient to prove asignificant association between hypertension and epistaxis.
Many cases of epistaxis are prevented with the moisturizing
nasal mucosa, avoidance of direct irritation or trauma, andnasal spray with oxymetazoline and epinephrine. The
severity of epistaxis ranges from a minor symptoms tohemodynamically significant events, including death in rarecases. When prevention methods fail, recourse to thecompression, vasoconstrictor nasal sprays, chemical orelectrical cauterization, hemostatic agents and nasal
packing. The primary aim of this study is to assess thefactors favoring the occurrence of epistaxis according to age
and associated pathologies within the patients who
presented to the Emergency Room at the E.N.T.
Department of the University Hospital of Timisoara.
Secondary aims include assessing the therapeutic options
depending on the severity of the bleeding.

METHODS
We conducted a retrospective study on sample which
was obtained by consecutive sampling of all admissions for
epistaxis at the Ear, Nose and Throat (ENT) Department of
the University Hospital of Timisoara, in the period between
January 2014 and October 2018. Our centre it is a tertiarycare hospital which has 47 beds for ENT department. The
study contain 346 cases that were analyzed in terms of
demographics, clinical aspects and treatment. In thestatistical analysis, we used measurements of central
tendency and of dispersion for quantitative variables (age,blood pressure values, and length of hospital stay andseverity of epistaxis) and absolute and relative frequencies
for qualitative variables (gender, society, location of theepistaxis, plugging, antiaggregation, anticoagulation,
hypertension and sphenopalatine artery cauterization). The
study was accomplished with the approval of the ethics
committee of the University of Medicine and Pharmacy“Victor Babes” Timisoara.
Patient selection
346 patients were selected for the study. The study
inclusion criteria were: male and female patients of any age– patients admitted to the ENT department with a diagnosisof serious spontaneous epistaxis requiring at least one nasal
pack; patients with minor epistaxis easily managed by ﬁrst
aid measures and/or immediately successful local treatment
Fiziologia - Physiology • 2018.28.2(96)(cauterization), posttraumatic epistaxis (including iatrogenic
epistaxis after nasal surgery) and patients with nasal
tumors.
Management of epistaxis
Management of epistaxis ranged from unilateral nasal
packing to endoscopic surgery depending on the severity ofepistaxis. Patients with systolic blood pressure (BP) higher
than 160 mmHg also received Enalapril LPH 10 mg tocontrol BP. Anterior nasal packing was performed with
Polyvinyl Alcohol sponge (MEROCEL-Standard nasal
dressing) or compression belts with antihemostatic(Adrenostazin or Etamsilat); anterior and posterior nasal
packing was performed with a double balloon nasal catheteror buffer nasopharynx compression. Nasal packs were
removed after 48-72 h if the patient did not re-bleed. In
cases of re-bleeding after removal of nasal packs, nasal
packing was repeated. Sphenopalatine artery cauterization
was indicated in the case of anemia, persistent bleeding
after 2 or 3 nasal packs and after the tumor excision.
Study protocol
A history of hypertension was deﬁned as patientstreated with antihypertensive drugs. Patients with a selfreported history of epistaxis or who had been alreadyhospitalized for at least one episode of epistaxis wereconsidered to have a history of epistaxis. The mean of all
BP measurements during hospitalization was determined,
and BP that was difﬁcult to control was deﬁned as BP
higher than 140/80 mmHg. Epistaxis was classiﬁed as
grade 1 (serious) or grade 2 (severe). Serious epistaxis
was deﬁned by the need for medical management
requiring hospitalization of the patient for epistaxis,
anterior nasal packing, under 5 days of hospitalization.
Severe epistaxis (grade 2) was deﬁned as follows: lengthof hospital stay more than 5 days, nasal packing using
double balloon nasal catheter or buffer nasopharynx
compression, two or more nasal packs, presence ofhematologic consequences: anemia (hemoglobin\10 g/dl)
and/or blood transfusion, patients treated by cauterizationof sphenopalatine artery.
Statistical Analysis
Data are presented as absolute numbers, percentages
and means where appropriate. A descriptive analysis was
performed with calculation of mean and medians forcontinuous variables and percentages for categoricalvariables. Multiple ordered logistic regression models andlinear regression models were constructed to evaluate theassociation between covariates and epistaxis severity.
Values were considered statistically significant for P < 0.5.
All statistical calculations were performed using CHISQUARE test.
29

RESULTS
Of the study subjects, 58.84% were men and 41.16%
were women, with a male/female ratio of 1.42/1 (oddsratio=1.4296), although this ratio varies by age group. The
median age for the patients with epistaxis was 75 (71-80)
years with a range of 11-98 years (Figure 1) and 60 (50-70)
years in nasal tumor cases. Of the total, 83.53% were aged
over 50 years. According to the anatomical location, thecases of epistaxis were distributed into posterior (8.09%;
28/346) and anterior (91.91%; 318/346).

Also, we observed a predominance of hospitalized
epistaxis in the months of March and January, with adecrease in summer periods (Figure 3).

Fig. 3. Correlation between the season and the appearance ofnasal bleeding.

Fig. 1. The median age for the patients with epistaxis was 75(71-80) years with a range of 11-98 years.

Associated local factors were much less frequent thansystemic factors. Arterial hypertension was the mostprevalent and was present in 84.39% (292/346) of cases,
while 43.64% of patients (151/292) were aged over 71
years. Anti-aggregation accounted for 19.08% (66/346) andanticoagulation for 17.92% (62/346). Nasal bleeding
occurred in 8.41% (29/346) of patients with nasal tumors
which was performed the surgery (Figure 4).

Pathologies incriminated in nosebleeds were: hypertension(84.39%, 292/346), ischemic heart disease (21.68%, 75/346),
atrial fibrillation (12.72%, 44/346), diabetes (12.43%, 43/346),
nasal tumors (8.38%, 29/346), viral (5.49%, 19/346), trauma(1.73%, 6/346) and post-surgery (0.87, 3/346). 0.87% of all thepatients showed no pathology (Figure 2).

Fig. 4. Number of patients with nasal bleeding in the nasal tumor
cases.

In this cases the median age was 60 (50-70) years andthe hospitalization days of these patients was on average 8days (Figures 5 and 6).

Fig. 2. Pathologies incriminated in nosebleeding.
30

Fig. 5. Correlation between the age and tumors.
Fiziologia - Physiology • 2018.28.2(96)At the anterior and/or posterior nasal packing refractory
we have used arterial cauterization. Sphenopalatine arterycauterization was performed at a rate of 59% to stop thenasal bleeding in patients with nasal tumors (Figure 9).

Fig. 6. Days of hospitalization at the tumors pathology.

According to the severity of epistaxis, nasal tumors wereclassified in grade 2 of severity and the rest of pathologies
in grad 1. In nasal tumor cases we observed that posterior
epistaxis (59%, 17/29) was more frequently than anterior
epistaxis (41%, 12/29) (Figure 7). Management of epistaxis
in our patients included 4 methods, starting with first aid,
than anterior nasal packing with compresion belts or
MEROCEL, posterior nasal packing and nasal cauterization.

Fig. 7. The posterior epistaxis (59%, 17/29) was more frecvently
that anterior localization (41%, 12/29).

In 82.66% of cases we practiced anterior nasal packing
and 7.80% of cases were treated by posterior nasal packing.
We have identified spontaneously stopped bleedings in
9.54% of total cases (Figure 8).

Fig. 8. Anterior nasal packing was more used than posterior packing
and in 9.54% of cases the bleedings were spontaneously stopped.
Fiziologia - Physiology • 2018.28.2(96)Fig. 9. Correlation between tumors and arterial cauterization.

DISCUSSION
Epistaxis is one of the most common emergencies of our
specialty. The male predominance of epistaxis has been
well documented by most authors. It is a constant feature in
the literature, which in our series was reflected across all
age groups until the eighth decade of life and the result isstatistically significant (p=0.0010, p<0.5). Epistaxis
incidence by gender and age distribution was described by
Tomkinson et al. [9] in 1997, with a male predominancebetween the ages of 20 and 49 years. However, thisdifference by gender was not present after the age of 50
years in his study. From this age, the incidence in men was
similar to that in women, a fact that the author relates to thepossible protective effect of estrogens in pre-menopausal
women. It is a condition that can affect all age groups, butthere is an increased incidence in the population above age
50. In 1974, Juselius reported that up to 71.4% of hispatients exceeded this age. This finding was later confirmed
by other authors. Walker et al.[10], in their extensive seriesof 21,770 admitted epistaxis cases, reported a median ageof 70 years, in which approximately 75% of patients wereover 40. The classification by decades in our series showed
an ascending distribution, and the vast majority of cases
were over 50. Another epidemiological datum in theliterature has been the environmental and seasonal
incidence of nasal hemorrhages. We recorded a nonuniform distribution with a higher incidence during themonths of January and March, thus supporting the seasonalvariability described in classic references. Danielides et al.
[11] showed a significant association between weatherchanges and the incidence of epistaxis, with the maximum31

and minimum daily temperatures and the pressure of water
vapour being the most influential environmental factors. A
greater influence of the environment during the coldest anddriest periods of the year has also been highlighted.
However, there is no full agreement in this respect and otherauthors such as Bray et al. [12] have failed to demonstrate
this relationship.
In our series, epistaxis with anterior local source
accounted for a high percentage at the patients with heart
pathologies. The posterior epistaxis predominated over
the anterior in the nasal tumors cases. The highpercentage of epistaxis with a posterior location is evident
in the case of a hospitalized population, which is eight days
in most of the cases and which is also associated withnasal tumors (p=0.35, p<0.5). In the work of Pino V. et al.
[13], which evaluated the etiopathogenesis and treatmentof cases of epistaxis admitted between 1990 and 2000, thepercentage of posterior epistaxis slightly exceeded 70%.
Local factors associated were present in 11% of cases,
with a predominance of nasal tumors, followed by local
trauma and infections of the upper airway [13]. The vast
majority of patients presented an associated systemic
disease, primarily in the form of hypertension, ischemic
heart disease, atrial fibrillation and diabetes. Despite this,the descriptive design of the study prevents us from statingthat these were the direct causes of bleeding. There aremany publications that have attempted to explain theinfluence of arterial hypertension in epistaxis. Contrary towhat is classically considered, there is, to date, insufficientevidence to say that arterial hypertension behaves as arisk factor. Most studies do not permit an assessment of
this association as they are case series without a control
group. Moreover, the impossibility of knowing the figuresof blood pressure prior to bleeding and the inability to knowwhether these are a cause or an effect in patients withnasal hemorrhage greatly impedes their study. Knopfholz
et al. [14] studied a series of 121 patients with epistaxis
and a prior history of hypertension. They investigated theblood pressure values during episodes of bleeding and theincidence of epistaxis according to the severity ofhypertension, classifying their sample into three groups.
They found no differences between baseline bloodpressure figures in routine checks and the figures recorded
during active bleeding. Neither were the cases of epistaxis
more frequent in patients with higher levels ofhypertension. Neto et al. [15] failed to confirm a definite
association between these two parameters. They argue
that the real factors contributing to bleeding is sustained
hypertension (for more than 5 years), because it produces
a series of arteriosclerotic degenerative changes in thevessel walls, weakening them and thus making it easier forthem to break. There are very few publications that havecontinued this avenue of research. A high proportion of ourpatients presented abnormalities of hemostasis by antiaggregation or anticoagulation due to drugs, which could
32

indicate that drugs altering hemostasis influenced patients
hospitalized for epistaxis. The relationship between
anticoagulant drugs and epistaxis has been poorly
documented in the literature. Its range of therapeutic hypocoagulability is measured by its international normalized
ratio (INR); its normality index values typically range
between 2.5 and 3.5, depending on the pathology being
treated. The classic belief considering that poor
anticoagulation control carried an increased risk ofepistaxis was challenged by Garcia Callejo et al. [16] in a
1990-1995 study. In their extensive series of 1410
anticoagulated patients, nasal hemorrhages were moreprevalent in patients with correct anticoagulation control,and only 20% of patients with nasal bleeding presented
altered INR levels [16]. In our series, the anticoagulants
were not involved in the occurrence of epistaxis (17.92%,
62/346). Most authors agree that, in these patients, theaverage hospital stay is longer than that for patients not
requiring anticoagulation, but this is a fact that Denholm etal. [17] could not confirm. The association between
consumption of non-steroidal anti-inflammatory drugs(NSAIDs) and epistaxis is better documented [17]. In 1990,
Watson et al. [18] had already confirmed this association,
showing that 32% of patients admitted for epistaxis in theirstudy were anti-aggregated, compared to 6% who required
hospitalization for other causes. In our study, antiaggregated use were associated with the nasal bleeding in
19.08% of cases. Aspirin was the most used and its one of
the most potent platelet anti-aggregation agents. It
interferes with the metabolism of arachidonic acid,producing an irreversible inhibition of the synthesis of
cyclooxygenase. It thus promotes a permanent platelet
dysfunction for 5-7 days, which is the average life of aplatelet. This is one of the main aspects that differentiate
it from other NSAIDs, as the latter have shorter durationand potency because the inhibition they generate on
cyclooxygenase is variable in level and reversible.
Spenopalatine artery cauterization was performed to stop
the nasal bleeding only in patients with nasal tumors.

CONCLUSIONSThis first Romanian report on the predictors of epistaxis
disorders showed that the typical pattern of patients
admitted for nasal bleeding is a preferably male patient, of
middle or advanced age with an anterior location and who
displays some associated comorbidity or treatment. In ourstudy, the only variable that behaved as a risk factor for
bleeding was the posterior location of the epistaxis which
occurred more in patients with nasal tumors. In these cases,
patients require several days of hospitalization and severityof epistaxis is higher than in patients with other associatedpathologies.

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The influence of meteorological factors on the frequency of
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Mafessoni R, Gleissner AL. Is epistaxis evidence of end-organ
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MANAGEMENTUL EPISTAXISULUI ÎN TUMORINAZALE ȘI AFECȚIUNI CARDIACE LA SPITALULUNIVERSITAR DIN TIMIȘOARAREZUMAT
Background și scop. Epistaxisul este o boală frecventă, care afectează până la 60% din populație și este un motiv uzualde adresabilitate la sistemele de sănătate.
Meode. Studiul include pacienți care s-au prezentat la departamentul ORL al Spitalului Universitar Timișoare în perioada
ianuarie 2014 – octombrie 2018, prezentând epistaxis anterior și/sau posterior. Factorii demografici (vârstă, rasă, gen),medicamentele utilizate (anticoagulante sau medicație anti-agregantă) și alte comorbidități au fost analizate ca potențiali
predictori ai noilor episoade.
Rezultate. Au fost incluși 346 pacienți în studiu, din totalul de 456 pacienți cu episoade de epistaxis în ultimii 4 ani. Analiza
multi-variant a identificat predictorii pentru un mare număr de cazuri: tumori nazale, hipertensiune, supradoză demedicamente anticoagulante și variațiile sezoniere.
Concluzii. Epistaxisul apare mai frecvent la pacienții vârstnici cu hipertensiune arterială. Rinita alergică, sinuzita cronică,tumorile nazale și hipertensiunea sunt asociate cu o creștere a incidenței epistaxisului
Cuvinte cheie: epistaxis, tumoră nazală, hipertensiune, packing nazal, cauterizare, anticoagulant.

Fiziologia - Physiology • 2018.28.2(96)33