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Fiziologia - Physiology  2010.20.1 (65)CONTENTS
1. REVIEW: The Race to Cure Diabetes: How Far Are We From a Breakthrough?........................................................................................................................................................... 4
Oana I. Gavriliuc, Calin A. Tatu, Virgil Paunescu
2. REVIEW: Interleukin 24......................................................................................................................................................................................................................................... 9
Laura Marusciac, Carmen Bunu Panaitescu, Carmen Tatu, Gabriela Tanasie, Virgil Paunescu
3. Health, Longevity and Ecology - An Integrated Paradigm......................................................................................................................................................................................13
Dan Riga, Sorin Riga, Aurel Ardelean, Francisc Schneider
4. Insulin Growth Factor 1 Involvement in the Etiology of Pectus Excavatum in Children ...........................................................................................................................................17
David VL, Izvernariu DA, Bojin Florina, Gavriliuc Oana, Popoiu MC, Tănăsie Gabriela, Puiu Maria, Boia ES
5. Comparison between Differentiation Potential of Dog Periosteum-Derived Cells and Bone Marrow-Derived Cells to Osteogenic, Chondrogenic and Adipogenic Lineages................21
Anghel Simona, Bojin Florina, Igna Cornel, Tanasie Gabriela, Tatu Calin, Seres Monica, Tatu Carmen, Luca Calin, Ordodi Valentin, Gruia Alexandra, Rosca Adriana, Gurban Camelia, Georgescu Remus, Paunescu
Virgil6. Aristolochia Clematitis in Medicine: the Good and the Bad....................................................................................................................................................................................26
Mirabela I. Cristea, Alexandra T. Gruia, Simona Anghel, Elena Gai, Calin A. Tatu, Virgil Paunescu
7. Beta-Adrenergic Stimulation and Pulmonary Function Modulation in Asthma and Chronic Obstructive Pulmonary Disease....................................................................................29
Camelia Pescaru, Voicu Tudorache, Cristian Oancea
8. Mathematical Models of Breast Cancer Risk Calculation and Its Applications..........................................................................................................................................................33
Dana Carmen Zaha, Liana Antal, Ioan Magyar, Elena Lazar
9. Stromal Cells - Tumor Microenvironment Interactions – Part I...............................................................................................................................................................................37
Florina Bojin, Calin Tatu, Oana Gavriliuc, Alexandra Gruia, Gabriela Tanasie, Carmen Tatu, Mirabela Cristea, Felicia Ciuculescu, Alexandru Tocut, Daniela Crisnic, Adrian Carabineanu, Daciana Nistor, Carmen
Bunu, Virgil Paunescu
10. From Inactive Carrier to Reactivation of Hepatitis B Virus....................................................................................................................................................................................42
Diana Nicoliţă, Ioan Sporea, Roxana Şirli, Isabel Dan, Corina Vernic
11. Electrodiagnostic of the Neuromuscular Junction in Myasthenia Gravis...............................................................................................................................................................46
Bogdan Istrate, Daniel Dumitru Banciu, Crisanda Vîlciu, Ana Câmpeanu, Maria-Luisa Flonta
12. Lipoperoxides – Markers of Oxidative Stress in the Newborn with Perinatal Asphyxia..........................................................................................................................................51
Gabriela Zaharie, Melinda Matyas, Adina Mureşan, Monica Popa

CUPRINS
1. REVIEW: Inovatii biomedicale pentru diabetul zaharat: vindecare sau tratament?.................................................................................................................................................. 4
Oana I. Gavriliuc, Calin A. Tatu, Virgil Paunescu
2. REVIEW: Interleukina 24....................................................................................................................................................................................................................................... 9
Laura Marusciac, Carmen Bunu Panaitescu, Carmen Tatu, Gabriela Tanasie, Virgil Paunescu
3. Sanatate, longevitate si ecologie - o paradigma integrata....................................................................................................................................................................................13
Dan Riga, Sorin Riga, Aurel Ardelean, Francisc Schneider
4. Implicarea factorului de crestere insulinic 1 în etiologia pectus excavatum la copii................................................................................................................................................17
David VL, Izvernariu DA, Bojin Florina, Gavriliuc Oana, Popoiu MC, Tănăsie Gabriela, Puiu Maria, Boia ES
5. Analiza comparativa a potentialului de diferentiere a celulelor derivate din periostul si maduva osoasa canina spre liniile osteogenica, condrogenica si adipogenica.....................21
Anghel Simona, Bojin Florina, Igna Cornel, Tanasie Gabriela, Tatu Calin, Seres Monica, Tatu Carmen, Luca Calin, Ordodi Valentin, Gruia Alexandra, Rosca Adriana, Gurban Camelia, Georgescu Remus, Paunescu
Virgil6. Aristolochia clematitis in medicina: beneficii si efecte adverse..............................................................................................................................................................................26
Mirabela I. Cristea, Alexandra T. Gruia, Simona Anghel, Elena Gai, Calin A. Tatu, Virgil Paunescu
7. Stimularea beta-adrenergica si modularea functiei pulmonare in astm si in bronhopneumopatia cronica obstructiva............................................................................................29
Camelia Pescaru, Voicu Tudorache, Cristian Oancea
8. Modele matematice de evaluare a riscului de cancer de sân şi aplicaţiile lor..........................................................................................................................................................33
Dana Carmen Zaha, Liana Antal, Ioan Magyar, Elena Lazar
9. Interactiunea dintre micromediul tumoral si compartimentul stromal – partea I..................................................................................................................................................37
Florina Bojin, Calin Tatu, Oana Gavriliuc, Alexandra Gruia, Gabriela Tanasie, Carmen Tatu, Mirabela Cristea, Felicia Ciuculescu, Alexandru Tocut, Daniela Crisnic, Adrian Carabineanu, Daciana Nistor, Carmen
Bunu, Virgil Paunescu
10. De la starea de purtător la reactivarea virusului hepatic B...................................................................................................................................................................................42
Diana Nicoliţă, Ioan Sporea, Roxana Şirli, Isabel Dan, Corina Vernic
11. Electrodiagnosticul jonctiunii neuromusculare in miastenia gravis .....................................................................................................................................................................46
Bogdan Istrate, Daniel Dumitru Banciu, Crisanda Vîlciu, Ana Câmpeanu, Maria-Luisa Flonta
12. Lipoperoxizii – markeri ai stresului oxidativ la nou-nascutii cu asfixie perinatala.................................................................................................................................................51
Gabriela Zaharie, Melinda Matyas, Adina Mureşan, Monica Popa

2010.20.1(65)  Fiziologia - Physiology

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REVIEWTHE RACE TO CURE DIABETES: HOW FAR ARE WE FROM A
BREAKTHROUGH?
OANA I. GAVRILIUC1, CALIN A. TATU2,3, VIRGIL PAUNESCU1
Physiology-Immunology Department, “Victor Babes” University of Medicine and Pharmacy Timisoara
2
Biology Department “Victor Babes” University of Medicine and Pharmacy, Timisoara
3
Regional Center for Transplant Immunology, Emergency Clinical County Hospital Timisoara

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ABSTRACT
Currently recognized as a major public health concern of both highly industrialized and developing countries, diabetes, a metabolic disorder affecting more than 220
million people worldwide, warrants a definitive cure. Exogenous insulin supply, the current treatment, is not fully capable of achieving tight control of glucose regulation,
while pancreatic islet transplantation is not a viable solution due to the shortage of donors. This mini-review discusses progressive therapeutic initiatives launched in thequest for the cure of diabetes, from gene therapy to cellular replacement using embryonic or mesenchymal stem cells converted to β-pancreatic cells, immunotherapy andcombinations of the above. Although the article addresses mainly autoimmune-derived diabetes, type 2 diabetes mellitus will equally benefit from β-cell replacement
and regenerative therapies.
Key words: diabetes, embryonic stem cells, mesenchymal stem cells, gene therapy, autoimmune, immunotherapy, transplantation

BACKGROUND
Frederick Banting, one of the co-discoverers of insulin, recognized in his Nobel
Prize lecture in 1923 that ‘Insulin is not a cure for diabetes; it is a treatment’. Afteralmost a century since the revolutionary discovery, exogenous insulin supply still
represents the main therapeutic option for Type 1 diabetes (T1D) patients and isbeneficial for some severe Type 2 diabetes (T2D) cases, but it is far from being
perfect and it may seem primitive from a current medical perspective. Insulin
therapy is not fully capable of achieving a tight control of glucose metabolism,
being often associated with an increased risk of hypo- or hyperglycemic episodes(“the yo-yo effect”). Even the latest insulin analogues fail to prevent long-term
complications of high blood glucose levels that include severe damage to themicrovasculature and nerves (1) and ensuing diseases, like diabetic nephropathy
or diabetic retinopathy.
Transplantation of pancreatic islets under the Edmonton protocol (2) offers abetter approach than simply administering insulin. However, it is obvious that onlya small number of patients worldwide will be able to benefit from this therapeutic
approach that would still require long-term immunosupression regimens andpatient monitoring (3). Islet allotransplantation can provide insulin independence
for a limited period of time (4), but it represents proof-of-concept that cell-based
treatments for T1D can be effective.
In Type 1 diabetes mellitus, the insulin-secreting β-cells in the pancreatic islets
of Langerhans are selectively and irreversibly destroyed by an autoimmune attack.
As diabetes is caused by the destruction of only one type of highly specialized cell,
it follows logically that therapeutic approaches should focus on replacing thisparticular cell type. Overall, two distinctive pathways can be considered as meansof obtaining functional β-cells: gene-therapy or factor-based differentiation ofstem or progenitor cells. Gene-therapy refers to the in vivo or in vitro transfer of aforeign gene into any type of cell, allowing it to produce insulin. The foreign genecan be insulin or another gene (e.g. transcription factors) that can in turn activate or
repress on demand the insulin gene. The factor-based approach involves exposing
stem cells to a cocktail of growth factors and cytokines over an extended period ofin vitro culture, followed by transplantation of the differentiated insulin-producing
cells into the receiving diabetic patient. In the present review, we wish to highlightthe successes attained in each of these fields and also the hurdles still needed to
be overcome before a safe, effective, and widespread therapeutic application ispossible. Although the long road from treatment to cure is still unwinding, thenew developments in gene and cell-based therapy could remove in the near future
diabetes from the list of incurable, chronic diseases. In the same time, personalized
genomics that would allow identification of certain risk genes predisposing todiabetes mellitus could dramatically improve disease prevention.

NEW CONCEPTS, OLD PROBLEMS
Embryonic and adult stem cells for diabetes
The minimum properties sought for in a surrogate β-cells have been considered
before (5, 6). One requirement is that a significant number of replacement cells will
be needed as current transplantation protocols replace approximately 2-4x 109 β-cells per recipient and a significant fraction is lost immediately post-transplant
due to procedure or host factors. Mature β-cells, although long-lived, have very
limited proliferative capacity, unlike progenitor or stem cells, which represent a
more suitable candidate for expansion and directed in vitro differentiation due totheir intrinsic plasticity. However, an essential prerequisite of any potential stem celltherapy and its translation into clinics is to ensure that differentiation was efficientand no residual stem cells are implanted into patients. These uncommitted stem
cells might prove detrimental on account of spontaneous differentiation to divergent
lines or unchecked proliferation resulting in the formation of tumors.

Received January 2010. Accepted February 2010. Address for correspondence: Oana Gavriliuc, Physiology-Immunology Department, “Victor Babes” University of Medicine and Pharmacy
Timisoara, 2A Eftimie Murgu Square, RO-300041, Phone/Fax: +40256490507, e-mail: oana_gav@yahoo.com

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Fiziologia - Physiology  2010.20.1 (65)The most important question is which stem cells would represent an appropriate starting material: embryonic or adult stem cells? The use of allogeneic or
xenogeneic embryonic stem cells will most likely generate immune rejection thathas to be controlled in addition to underlying autoimmunity, while the patient’s
own tissue stem cells will be less likely to pose this problem (7) although the issueof autoimmune response may still pose difficulties.
In recent years, many research groups have reported the creation of insulinproducing cells from tissue progenitor or stem cells by means of transdifferentiation(i.e. the switching of a committed cell’s fate towards another distinct phenotype);
however, earlier, promising studies have so far failed to translate into reliable protocols for generating functional β-cells (8, 9). Bone-marrow derived mesenchymal
stem cells (MSCs) have been a favorite candidate for differentiation studies due totheir availability, ease of isolation, apparent plasticity and immunosuppressive
properties (10, 11). However, recent studies demonstrated that MSCs enhance
the regeneration of endogenous β-cells in experimental diabetes mouse models
rather than repopulate the pancreas by transdifferentiation (12). Microenvironment
manipulation differentiation studies on other types of adult stem cells, including
neural progenitor cells (13), umbilical cord-blood stem cells (14), multipotent
acinar pancreatic stem cells (15), liver progenitor cells (16), in the absence of any
genetic manipulation, resulted in only partial successes.
The first notable progress in the field of factor-based differentiation towards
β-cells came from studies that applied developmental principles to stem cell biology,
recapitulating embryonic differentiation in vitro (17, 18). The protocol required firsta definitive endoderm specification of the embryonic stem cells, then the step-wise
generation of pancreatic progenitors, endocrine progenitors and mature β-cells.
Human embryonic stem cells were exposed initially to activin A, a member of the
TGFβ family, in the absence of serum, resulting in the efficient formation of definitive
endoderm. Later, TGFβ signaling was eliminated and the cell cultures were exposed
to retinoic acid, which initiated the endocrine lineage commitment. The Baetge
group (19) arrived at this model by quantitatively monitoring gene expression in the
differentiating human embryonic stem cells after manipulation of culture environment using a combination of growth factors and signaling molecules. The samegroup reported more recently the obtaining of glucose-responsive mature endocrine
cells in mice after a three months period of in vivo incubation (20).
It is not surprising that the embryonic stem cells were the first to be differentiated to β-cells, given their well-documented plasticity. Additionally, these cells canproliferate indefinitely in vitro, in an undifferentiated state, thus being capable ofsupplying the high number of cells required for transplantation. However, long-term
immunosuppression would be a prerequisite in this case, the embryonic stem cells
and their insulin-producing derivatives behaving essentially as an allograft upontransplantation.
An alternative that could circumvent the ethical considerations and immune
rejection issues associated with embryonic stem cells seem to be the induced
pluripotent stem cells (iPS), a sort of surrogate ESC pioneered by Yamanaka’slaboratory in 2007 (21, 22). The discovery that human somatic cells, including
those from patients with T1D (23), can be reprogrammed to a state of pluripotency
highly similar to that of embryonic stem cells, marked a turning point in regenerative
medicine. These autologous iPS cells, can be differentiated into insulin producing
cells by a protocol similar to that employed for hESC (24) and used as an autograftthat would obviously not require immunosupression. Original methods for obtaining
iPS cells involved the viral transduction of four transcription factors associated withpluripotency (Oct4, Sox2, Klf4 and c-Myc) in the target cells, but subsequent studies
reported the generation of iPS cells using nonintegrative transfer vectors better suitedfor future clinical applications, and even membrane-soluble recombinant proteins(25). Although a promising alternative to ESC, the iPS cells still need to be more2010.20.1(65)  Fiziologia - Physiology

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thoroughly characterized and their features and potential limitations understoodcorrectly before any clinical studies are to be performed.
The functional architecture of the β-cell
The pancreatic β-cell is a highly differentiated and complex cell that harbors
several mechanisms critical for the regulated transcription and translation ofproinsulin, a controlled secretory pathway and a stimulus-secretion coupling,which may not be individually unique to β-cells, but together they sustain the
homeostatic purpose of β-cells. The β Langerhans cell is able to monitor circulating
glucose levels because it expresses the high capacity glucose transporter GLUT2 andglucokinase. These elements would be necessary characteristics of any candidate
cell, however they are not sufficient to reconstruct the glucose-responsive systemof the actual β-cells, as it was demonstrated by the attempt to express GLUT2 andGK in intermediate pituitary cells, which resulted in a defective glucose metabolism
and induction of apoptosis (26). In vivo, insulin secretion is also influenced by otherfuel secretagogues (27), such as leucine, and can be regulated by incretin hormones(GIP, GLP-1) (28) or as a result of neuronal input. The response of engineered cells toall of the stimuli above, in a tightly coordinated fashion, does not seem very likely
to be achieved with current gene therapy tools.

Fig. 1 A Signal transduction in β-cells. Glucose is transported into the β-cells on thehigh-capacity GLUT2 transporter and metabolized within the cell, generating ATP, which binds toATP-sensitive K+-channels, leading to the closure of those channels. The inhibition of K+ efflux leads
to plasma membrane depolarization and opening of voltage-dependent Ca2+-channels. Extracellular
Ca2+ enters β-cells resulting in elevated concentrations of intracellular Ca2+ and initiation of insulin
secretion. B Insulin translation and post-translational processing. After insulin is released from the
β-cell by exocytosis, specific transcription factors bind to the insulin gene promoter and upregulate
insulin transcription into preproinsulin mRNA, leading to proinsulin biosynthesis in the ER. Proinsulin
is exported to the Golgi vesicles where it will be packed into secretory granules. Only here willproinsulin be cleaved by PC2 and PC3 endoproteases and carboxypeptidase H to yield mature insulin
and C-peptide.

Insulin secretion can be stimulated within minutes after exposing the β-cellto a high glucose concentration. Glucose is transported and metabolized within
β-cells, generating ATP, which binds ATP-sensitive K+-channels, leading to theclosure of channels. The inhibition of K+ efflux leads to β-cell plasma membrane
depolarization and opening of voltage-operated Ca2+-channels. Extracellular Ca2+
enters β-cells resulting in elevated intracellular Ca2+, activation of Ca2+-sensitive
downstream signaling and rapid discharge of insulin stored in secretory granules
by exocytosis (29) (Figure 1). This first phase of insulin secretion is followed by agradually increasing second phase that lasts as long as glucose stimulation is applied (30). Normal glucose homeostasis absolutely depends on rapid and biphasic
kinetics of insulin release.
5In order for insulin to be released from mature insulin secretory granules asa biologically active protein, it must be first cleaved from proinsulin by β-cellsspecific endoproteases PC2 and PC3 and carboxypeptidase-H. In turn, proinsulin
production in the β-cell is predominantly regulated at the level of translation (31,32) by heteronuclear mRNA processing and control of preproinsulin stability. In
the long-term, translational control is supplemented by transcriptional regulation
involving a variety of transcription factors, and among these Pdx-1, NeuroD1 andMafA play a crucial role.
Should we make a β-cell?
If we consider all the fine-tuning mechanisms we need to engineer and put
into a candidate β-cell, the answer is a definite ‘No’. It is also negative from thepoint of view of Type 1 diabetes as an autoimmune chronic condition associatedwith the perpetual generation and activation of autoreactive T cells recognizing
pancreatic β-cell autoantigens. Presently, there is still no complete list of these
β-cell autoantigens, but the closer we will come to β-cell identity, the more likely
the newly engineered transplanted cell will be subject to autoimmune destruction.
A very important question is how close do we have to come to imitating a β-cell inorder to achieve a normal metabolic control of glycemia and still somehow avoid(self) destruction by the diabetic patient’s immune system.
Gene-therapy for diabetes
The possibility of ectopic insulin expression by gene transfer in a non-β-cellhas been explored since 1983 and further experiments have shown that simple
replacement of insulin expression is not likely to be useful as a therapy for T1D
unless it contains a system to regulate insulin levels in response to minute variations of blood glucose. Considering the complex machinery of stimulus-secretion
coupling in β-cells, a successful gene therapy for diabetes should deliver in the target
cells, separately or in combination, at least two key elements: a regulatory system
responsive to glucose concentrations, and appropriate processing of proinsulin into
mature, biologically active insulin.
One method to achieve glucose-regulated insulin release involves
glucose-responsive promoters linked to the insulin gene that ensure activation oftranscription of the insulin gene under hyperglycemic conditions and inhibition
under hypoglycemic conditions. Several research groups have experimented withliver-specific LPK (L-type pyruvate kinase) and glucose-6-phosphatase promotersin liver cells (33). Still, transcriptional stimulation of gene expression takes no lessthan 2 hours, while the ‘off’ response depends on the half-life of preproinsulin
mRNA, which is at least of 6 hours (34), not even comparable to the few minutes
response time necessary in vivo.
Another hurdle of insulin gene therapy - correct proinsulin processing in non-β
cells - has been overcome by engineering a mutated version of the proinsulin gene,in which the aminoacid cleavage sites recognized by β-cell-specific proteases havebeen replaced with the sites recognized by the more ubiquitary protease furin (35).
A different approach focused on expressing a modified single-chain insulin (36),lacking C-peptide, which has a higher biological activity than proinsulin, but still
only about 30% of that of the normally-occurring insulin, and does not require
any enzymatic cleaving.
Obviously, such somatic insulin gene therapy that attempts to replace every
element involved in the physiological function of β-cells, has very small chancesof being successful. Non-β-cells possessing some of the characteristics of ourtarget are better suited in the experimental design. For instance, hepatocytes havea glucose-sensing system, involving GLUT2 and glucokinase, but do not containproinsulin processing enzymes and secretory granules, while neuroendocrine
cells on the other hand contain components of the regulated secretory pathway,
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including PC2 and PC3 and secretory granules. Another type of cells has received
some attention – the K cells (37), an endocrine cell type located in the gut thatcan secrete the incretin hormone GIP. These cells seem to be a very good target
for insulin gene therapy as they show glucose responsiveness and exocytosis andcontain the necessary endoproteases.
Taking their cue from recent work on iPS cell formation (21, 22, 38), thegroup of Douglas Melton pointed to the possibility of converting one type of cell
into another directly, without reverting to an undifferentiated pluripotent state
first and exemplified this concept by reprogramming, in vivo, adult pancreatic
exocrine cells to β-cells (39). The key point of this strategy, similar to that used
for iPS cells, is the delivery of a combination of β-cell specific transcription factors singled out by mutational studies on pancreatic embryogenesis (40). Threetranscription factors (Ngn3, Pdx1 and MafA) were transduced into the murine
pancreas by means of adenoviral vectors, resulting in extra-insular secretion of
insulin from cells that closely resemble β-cells in size, shape and gene expression. It is still unclear whether the same method can be successful in vitro, in
the absence of signals from the pancreatic microenvironment, however, it mightprove to be a more logical alternative to the laborious genetic reconstruction of
β-cell mechanisms of metabolic control.
Combined therapy for type 1 diabetes
Cell replacement therapeutic options, as presented above, are purposefully
designed to restore the diminishing β-cell mass in T1D subjects; unfortunately,
the newly transplanted β-cells will require protection from the recurrence of
islet-specific autoimmune attack. Ideally, we should address both the cause andthe consequence of type 1 diabetes (41) in a combined therapy pairing β-cell
replacement or regeneration with immunomodulation. Self-reactive effector T
lymphocytes responsible for the selective destruction of insulin-producing cells
could be tempered by enhancing local suppressor lymphocyte subpopulations,
explicitly CD4+CD25+FoxP3+ regulatory T cells (Tregs) (42), as means of immunotherapy. The loss of frequency or function of Tregs has already been proposed as one
mechanism that contributes to the development of type 1 diabetes (43), therefore
a method of controlled induction of functional Tregs without causing generalized
immune suppression would likely prove beneficial, if not necessary, as an adjuvant
therapy for diabetes. Nevertheless, other pathways of immune intervention havebeen explored besides the expansion of Tregs: encapsulated islets as synthetic
immune barriers, induction of apoptosis in the effector T cells, overexpression ofanti-apoptotic genes in the pre-diabetic islets (44), co-infusion of mesenchymal
stem cells with transplanted β-cells (11), use of anti-inflammatory agents, suchas IL-1R antagonists, for improving β-cell survival (45).
Mesenchymal stem cells (MSCs), which generate minimal immune reactivity
and have anti-inflammatory (46) and immunomodulatory (47-49) effects, havebecome favourites in recently initiated or on-going clinical trials involving the useof stem cells for the treatment of diabetes mellitus, matching the more comprehensive interest in the potential of MSCs to alleviate abnormal immune responses
in graft-versus-host-disease (50), solid organ transplantation (51) and Crohn’sdisease, among others. The precise therapeutic mechanism through which MSCs
would be able to treat T1D, either by release of regulatory cytokines such as TGF-βor IL-10 or by cell-cycle inhibition of lymphocyte proliferation (52), has yet to beconclusively determined. Furthermore, should we be able to unlock the transdifferention capacities of MSCs in the direction of β-cell replacement, this cell typemight become one of the most useful therapeutic tools, linking immunomodulation
with pancreatic repopulation in type 1 diabetes.
Figure 2 is depicting the most current therapeutic tools that could be employedin the treatment of type 1 diabetes in the near future.
Fiziologia - Physiology  2010.20.1 (65)Fig. 2. Therapeutic strategies for type 1 diabetes. Insulin producing cells can be generated
from various sources, such as embryonic stem cells (hESC) or autologous adult stem cells andreprogrammed somatic cells, or regenerated from existing β-cells. For a successful ‘cure’ of diabetes,
cell-replacement approaches should be combined with immune intervention that would address theunderlying cause of diabetes by blocking the islet-directed autoimmune attack.

CONCLUSIONSThe standards we set for a ‘cure’ in type 1 diabetes are proportionally highwith respect to the complex immunologic and metabolic disruption elicited by
the disease. While we do expect to obey the first principle of medicine, that is thetreatment should not aggravate the disease, a life-long regimen of immunosuppression following transplantation is barely acceptable. Strategies focused on replacing
β-cells either by a milieu-based differentiation or genetic engineering of adult orembryonic stem cells stem cells portend a new therapeutic era for type 1 diabetes
management should we overcome the two most stringent requirements for a
successful clinical application: that the surrogate β-cells are able to secrete insulin
in response to metabolic stimuli and not constitutively, and that they circumvent
acute rejection, chronic rejection and the reactivation of pre-existent autoimmune
diabetes following transfer into the receiving diabetic patient.
Considering a pre-emptive approach to arrest the development of full-onset
diabetes by determining people at risk of manifesting the disease in a preclinicalphase and treating them to prevent islet damage, we are still far from reconstructing
the etiology of type 1 diabetes. Genome-wide association studies (GWAS) aimed
at identifying single nucleotide polymorphisms responsible for susceptibility tocertain autoimmune conditions uncovered 41 distinct loci that increase the risk fortype 1 diabetes. Environmental factors, such as infectious agents, chemicals andstress are also known to play an important part leading to the accelerated destruction of Langerhans islets, while the exact molecular mechanisms and causative
autoantigens are yet to be detailed.
As for future prospects, we expect a steep improvement in the current knowledge of the predisposing factors and progression of type 1 diabetes, before any
breakthrough definitive treatment is possible. Nevertheless, we are confident thatthe solution is not beyond our current or near-future scientific endeavors.

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10. Ianus, A., et al., In vivo derivation of glucose-competent pancreatic endocrine
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12. Urban, V.S., et al., Mesenchymal stem cells cooperate with bone marrow cells
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13. Hori, Y., et al., Differentiation of insulin-producing cells from human neuralprogenitor cells. PLoS Med, 2005. 2(4): p. e103.
14. Denner, L., et al., Directed engineering of umbilical cord blood stem cells to
produce C-peptide and insulin. Cell Prolif, 2007. 40(3): p. 367-80.
15. Trucco, M., Regeneration of the pancreatic beta cell. J Clin Invest, 2005. 115(1):p. 5-12.
16. Li, W.C., et al., In vitro transdifferentiation of hepatoma cells into functional
pancreatic cells. Mech Dev, 2005. 122(6): p. 835-47.
17. D'Amour, K.A., et al., Efficient differentiation of human embryonic stem cells todefinitive endoderm. Nat Biotechnol, 2005. 23(12): p. 1534-41.
18. D'Amour, K.A., et al., Production of pancreatic hormone-expressing endocrine
cells from human embryonic stem cells. Nat Biotechnol, 2006. 24(11): p. 1392401.
19. Baetge, E.E., Production of beta-cells from human embryonic stem cells. Diabetes
Obes Metab, 2008. 10 Suppl 4: p. 186-94.
20. Kroon, E., et al., Pancreatic endoderm derived from human embryonic stem
cells generates glucose-responsive insulin-secreting cells in vivo. Nat Biotechnol,
2008. 26(4): p. 443-52.
21. Takahashi, K., et al., Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007. 131(5): p. 861-72.
22. Yamanaka, S., Strategies and new developments in the generation of patientspecific pluripotent stem cells. Cell Stem Cell, 2007. 1(1): p. 39-49.
23. Maehr, R., et al., Generation of pluripotent stem cells from patients with type 1
diabetes. Proc Natl Acad Sci U S A, 2009. 106(37): p. 15768-73.
24. Tateishi, K., et al., Generation of insulin-secreting islet-like clusters from humanskin fibroblasts. J Biol Chem, 2008. 283(46): p. 31601-7.
25. Zhou, H., et al., Generation of induced pluripotent stem cells using recombinant
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Semin Immunopathol, 2006. 28(1): p. 63-76.
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46. Ortiz, L.A., et al., Interleukin 1 receptor antagonist mediates the antiinflammatory
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48. McTaggart, S.J. and K. Atkinson, Mesenchymal stem cells: immunobiology
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49. Potian, J.A., et al., Veto-like activity of mesenchymal stem cells: functional
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Immunol, 2003. 171(7): p. 3426-34.
50. Cohen, J.L. and M. Sudres, A role for mesenchymal stem cells in the control ofgraft-versus-host disease. Transplantation, 2009. 87(9 Suppl): p. S53-4.
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Transplantation, 2009. 87(9 Suppl): p. S57-62.

INOVATII BIOMEDICALE PENTRU DIABETUL ZAHARAT:
VINDECARE SAU TRATAMENT?
REZUMAT
Considerat in prezent o problema majora de sanatate publica atat in statele industrializate cat si in tarile in curs de dezvoltare, diabetul zaharat, defect metabolic ce afecteaza
global mai mult de 220 de milioane de persoane, necesita urgent o solutie terapeutica. Tratamentul curent, ce presupune administrarea exogena de insulina, nu asigura uncontrol eficient al glicemiei, in timp ce transplantul de insule pancreatice este limitat de numarul redus de donatori. Scopul prezentului articol este de a compara initiative
terapeutice inovatoare explorate ca posibile solutii pentru diabet: terapie genica si terapie cu celule stem embrionare sau celule stem mezenchimale diferentiate catre celule
β-pancreatice, imunoterapie si combinatii ale acestora. Desi articolul discuta in principal diabetul de tip autoimun, optiunile de regenerare a masei de celule β-pancreaticesecretoare de insulina pot fi aplicate cu succes comparabil in tratamentul diabetului de tip 2.
Cuvinte cheie: diabet, celule stem embrionare, celule stem mezenchimale, terapie genica, boala autoimuna, imunoterapie, transplant.

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Fiziologia - Physiology  2010.20.1 (65)REVIEW
INTERLEUKIN-24
LAURA MARUSCIAC, CARMEN BUNU, CARMEN TATU, GABRIELA TANASIE, VIRGIL
PAUNESCU
Physiology-Immunology Department, “Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACT
Gene therapy could prove to be an alternative strategy for use in cancers resistant to conventional therapies. One of the choices is to use a therapeutic gene which codes
for a protein with antitumor properties. Such a gene has been shown to be the interleukin-24 (IL-24) gene. In this review we discuss the genomic profile, localization,
expression and functions of interleukin-24.
Key words: interleukin-24, mda7, il-10 cytokine family, cancer, apoptosis

INTRODUCTION
IL-24 has been identified in 1993 by substration hybridization techniques,using human melanoma cells, and after inhibiting their growth and inducing
differentiation with IFN-β and mezerein (MEZ)(1). Immunohistochemistry assays showed protein expression in the melanoma cells, but the expression was
significantly decreased in the later, invasive phases of the melanoma (2). It wastherefore suggested that the loss of MDA-7 expression is critical in the progressionof the melanoma and that this protein is a viable candidate for melanoma genetherapy (3).
The protein was originally named mda-7 (melanoma differentiation associated gene), but following the discovery of its gene’s chromosomal localization andits homology with IL-10, mda-7 was classified as a citokine and renamed IL-24.

THE IL-10 FAMILY OF CYTOKINES
Nowadays IL-24 is considered to be part of the IL-10-like family, together with
IL-10, IL-19, IL-20, IL-22, IL-26, IL-28. IL-10 is known to have immunomodulatory
effects on many different cell types; it acts as inhibitor to a number of cytokines,
such as IFN-γ, TNF, IL-2, GM-CSF (4). The other related interleukins have various
functions: IL-19 production by B lymphocytes and monocytes is up-regulated bytheir stimulation with GM-CSF and LPS (5), IL-20 regulates the proliferation anddifferentiation of keratinocytes (6), while IL-22 is responsible for the productionof acute-phase proteins in inflammation (7). IL-20 over expression in transgenic
mice is lethal during the neonatal period and it produces abnormalities of theepidermal differentiation6. IL-28 is involved in the acquired immune response.
The amino acid sequence for IL-24 across species is highly homologous,
unlike the lower homology (20-30%) which exists between the different
members of the IL-10 family (8).

THE STRUCTURE AND LOCATION OF THE
IL-24 GENE
The human IL-24 gene is located on chromosome 1q32-33 (Fig 1), in aregion spanning 195 kb and it is part of a cluster of genes that also contains
genes for other members of the IL-10 family: IL-10, IL-19, IL-20 (9). The ARNm
for IL-24 is approximately 2 kb, and the protein has a molecular weight of 23.8

kDa (10). The gene consists of 7 exons and 6 introns (9). The reading frame lies
between untranslated regions of 274, and 823 bp. The untranslated region fromthe antisense strand contains 3 polyadenylation signals which are essential forthe posttranscriptional stability of the ARNm. By using sequencing techniques, asignal peptide of 49 amino acids was identified, which allows the splicing and thesecretion of the protein. Sequencing also revealed 3 positions where glycosilationis possible – amino acids 95, 109, and 126. The different glycosilation possibilities
allow for different molecular weights for IL-24 (11).

Fig. 1. Chromosome 1

HOMOLOGOUS MOLECULES
IL-24 is highly conserved across species, as proven by the fact that murine
and rat equivalents for IL-24 share a structural homology of 69 (12) and 82% (13),respectively. Both rat and murine IL-24 have a molecular weight of 23 KDa11, while
human glycosilated IL-24 has a molecular weight of 33 KDa. This glycosilation has
no effect on the binding properties, nor on the activity of the protein, as shownby the fact the murine IL-24 can bind to and activate human IL-24 receptors (14).
The gene’s localization in the IL-10 gene cluster is identical for the human, rat andmouse counterparts (9). The human IL-24 gene is located on chromosome 1, amonga gene sequence that codes for IL-10, IL-19, IL-20, and IL-24, while murine IL-24 lies
on chromosome 13q13, in the same gene cluster. The organisation and the size ofthe exons are similar for humans and mice, with the exception of the human exon
2, which is the signal peptide. Therefore, murine exons 3-5 and the coding region
form exon 6 are identical with human exons 4-6 and the coding region from exon
7, coding the corresponding amino acids (14). The rat homologous gene for IL-24
has been identified in fibroblasts during tissue repair processes and named c49a
(13). The murine equivalent for IL-24 is expressed by Th2 lymphocytes stimulated
by IL-4 and it was named FISP (12) (IL-4-induced secreted protein).

Received December 2010. Accepted February 2010. Address for correspondence: Marusciac Laura, MD, Physiology-Immunology Department, “Victor Babes” University of Medicine andPharmacy Timisoara, 2A Eftimie Murgu Square, RO-300041, Phone/Fax: +40256490507, e-mail: laura.marusciac@gmail.com
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c49a/mob-5
There seem to be some differences between the human and rat orthologous.
Different sets of up or downregulated genes have been found in rat fibroblasts
during tissue repair processes (13). By comparing amino acid sequences from C49A
and IL-24, a 58,7% homology has been found, which seems to suggest that thesemolecules are related, and not true homologues (11). C49A is expressed in injured
dermal cells, which led to the conclusion that it is involved it proliferation processes.
Another IL-24-like protein, MOB-5, has been isolated from embryonic rat fibroblasts(15). This is identical with C49A, with the exception of two amino acids. The mob-5
expression is induced by the H-ras and K-ras oncogenes, and it seems to also have arole in proliferation (16). In conclusion, even though C49A and MOB-5 are similar
to IL-24, they stimulate proliferation, and don’t inhibit it.

FISP
FISP is expressed in lymphocytes which have been induced to differentiate
towards Th2 cells, and the expression is regulated by T cell receptors and IL-4. The
protein is 93% homologous with the rat c49a/mob-5 and 69% homologous withthe human IL-24. Even though FISP functions are not fully known, there is someevidence that they are similar to those of IL-24. For example, inhibition of the growthof liver tumor cells has been induced by intramuscular introduction of FISP in mice.
This seems to suggest that IL-24 functions are more similar to those of FISP, than
to those of c49a/mob-5 (17).

IL-24 EXPRESSION
Human IL-24 has been detected in various cell types, such as cultivatedmelanocytes (13), and cells which are part of the immune system – periferal bloodmononuclear cells (PBMC) activated with either concanavalin A (15) and phytohemagglutinin (18), or IL-4 and LPS (19), splenocytes (13), thymocytes (10), butalso in tumor cells. Like other members of the IL-10 family, IL-24 can be expressedby Th2 cells stimulated with protein kinase C and IL-4 (20). IL-24 expression canbe detected by using RT-PCR (Reverse Transcription Polymerase Chain Reaction)in activated T cells and monocytes stimulated with LPS (21). Also, an elevation of
IL-24 (8) expression has been detected in vivo, in skin injuries, and the high level
was maintained for several days, returning to base level after approximately 14
days (13). Considering the fact that IL-24 expression was also elevated before andduring the proliferation phase of the tissue repair process, it is likely that IL-24 is
involved in cell proliferation.

IL-24 RECEPTORSThe IL-24 receptors belong to the class II cytokine receptors family, alongwith other receptors for IL-10-like interleukins (22). They consist of two chains –R1 and R2 – of different intracytoplasmic lengths (23). There are 3 subtypes of R1
chains (IL-10R1, IL-20R1, and IL-22R1), and 2 subtypes of R2 chains (IL-10R2 andIL-20R2) (24). The R1 chain contains a long cytoplasmic domain and represents
the main signalling component, while the R2 chain has only a short cytoplasmic
domain (23). The IL-20 specific receptor consists of two subunits – IL-20R1 andIL-20R2, which connect on the surface of the keratinocytes, in order to form thefunctional receptor for IL-20. It has recently been observed that IL-20 can also bindto a receptor which contains IL-22R1. The IL-22 receptor is made up from the IL-22R1
and IL-10R2 (25). IL-24 can bind to either IL-20R1/IL-20R2, or IL-22R1/IL-20R2. The
most common type of receptor is IL-20R1/IL-20R2, which is common in most tissues
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throughout the body, while IL-22R1/IL-20R2 can only be found in the liver, colon,
small intestine, and pancreas. After binding with either of the two receptors, IL-24
activates the STAT3 pathway (26). However, it has been shown that the activationof the JAK/STAT pathway is not indispensible for inducing apoptosis, and cell death
is induced by intracellular IL-24 through a mechanism independent from receptors.
Intracellular IL-24 cand be found at the endoplasmic reticulum, where it induces astress response, which leads to the apoptosis of the tumor cells through different
pathways, which depend or not on mitochondria (26).
Unlike the IL-10 receptor (IL-10R1/IL-10R2), which is expressed by mosthematopoietic cells, the expression of IL-24 receptors seems to be restricted by
the presence of the IL-20R2 in some non-hematopoietic tissues, such as skin,lungs, testicles, and ovaries, which suggests a pleiotropic role for IL-24. The overexpression of IL-24 can be found in psoriatic skin, which suggests a connection
between overactivation of IL-24 and IL-24 (which share the same receptors) anddisease (27).

THE BIOLOGICAL FUNCTIONS OF IL-24
IL-24 can perform its functions in two different ways: like a normal cytokine,
through the specific receptors, or independent of its receptors. The latter manner
only applies to certain types of cancer cells.
Receptor Dependent Functions
One of the most important physiological functions of IL-24 is to be involvedin tissue repair processes. The target cells for this function are keratinocytes. In
certain skin pathologies, such as psoriazis, monocytes migrate directly beyond thedermic layer of the skin and secrete IL-24 which leads to persistent proliferationof keratinocytes (28).
In vitro, IL-3-dependent murine proB cells proliferate in the presence of IL24. IL-24 can also function as growth factor for B lymphocytes (14) and stimulate
the expression of other cytokines, such as TNF-α, IL-6, and IFN-c by PMBC (15),which suggests an involvement in inflammation. Ras oncogenes can induce not
only IL-24 expression, but also the expression of their receptors, which suggests
a paracrine or autocrine function for IL-24, which contributes to the survival andproliferation of tumor cells (29).
It has also been shown that it has antiangiogenic properties through a
receptor-mediated mechanism (30), although no specific receptors have beenfound on the endothelial cells.
Receptor Independent Functions
Several studies have shown an antitumor effect of IL-24 which is not mediatedby receptors. Most of these studies have produced IL-24 overexpression with the aidof an adenoviral vector, this construct being named Ad.mda-7.
Immunohistochemistry and RNA studies on melanocytes have shown themessenger RNA expression for IL-24, as well as the protein levels. IL-24 expression
decreases with the progression of the melanoma, from the radial growth phase, tothe vertical phase (2). This suggests the fact that IL-24 has a tumor suppression role,and the loss of expression is a critical step in melanoma progression from noninvasive
tumor to invasive malignity with metastatic potential (31).
It has been shown that IL-24 overexpression induces apoptosis in various
types of tumor cells – melanomas (3), malignant gliomas (4), prostatic (32),ovarian (33), and lung carcinomas (34), and on the other hand it does not inhibit
the growth of normal cells – for example, fibroblasts (32), breast (35) and prostatic
epithelium (32), astrocytes (31). The antitumor activity does not seem correlated
Fiziologia - Physiology  2010.20.1 (65)with the status of tumor suppresor genes (p53, p21) (11).
IL-24 performs its antitumor effects by different mechanisms. One suchmechanism might be modifying the ratio between pro- and antiapoptotic intracellular proteins – IL-24 tips the balance towards apoptosis mediated by BAX
and other related proteins. Inside the cell there is a delicate balance between
the molecules which promote or inhibit apoptosis (36). Proteins such as BCL-2,
BCL-XL, MCL-1, BCL-W, and Ad-E1B protect cells from apoptosis, while proteinssuch as BAX, BAD, BAK, and BCL-XS stimulate it (37). This effect has been provenon breast cancer cells with different p53 genotypes, which suggests the fact that
it is a p53-independent mechanism. IL-24 modified the balance between these
proteins, upregulating the BAX protein, raising the BAX/BCL-2 ratio, and tipping thebalance towards apoptosis (35). Although Bax is considered to be p53-dependent,
the abillity of IL-24 to upregulate the protein levels independently from p53 seems
to suggest alternative pathways for tumor cell apoptosis. Also, ectopic BCL-2 (orits viral equivalent Ad-E1B) can prevent the inhibition of tumor cell growth, whichsuggests a possible role of mitochondria in apoptosis (33).
IL-24 can also activate tumor cell apoptosis through the MAPK (mitogenactivated protein kinase) pathway, by activating p38. This activation inhibits cell
growth in melanomas and malignant gliomas and it expresses the GADD genefamily (DNA damage inducible) (38). It has also been observed that blocking p38
or GADD expression saves the melanoma cells from apoptosis. In the case of tumor
lung cells, the Ad.mda-7-induced apoptosis correlates with the upregulation of RNAdependent protein kinase (PKR), which is a mediator for antiviral and antitumoral
responses in target cells (39).
It can also inhibit tumor angiogenesis. This has been shown in vitro, by using
human umbilical vein endothelial cells (HUVEC) (18). Other cytokines from the IL-10
family have antiangiogenic effects, as well. IL-10, besides its immunosuppressive
actions, also inhibits tumor cells growth when it is over expressed in tumor cells,
by suppressing angiogenesis.
Other studies have shown a possible bystander antitumor effect of IL-24, inthe case of pancreatic cancer (40).

CONCLUSIONS

So far, other candidate molecules for gene therapy have not met the highexpectations. It seems that IL-24’s specificity does not allow for toxic effects
on normal, healthy cells. Also, its growth suppression and apoptosis actions
maninfest themselves in a large spectre of cancers: melanoma, osteosarcoma,
glyoma, mesothelioma, breast, cervical, colorectal, kidney, liver, ovarian,pancreatic carcinomas.
These present findings suggest that IL-24 is worthy of interest as therapeuticgene for cancer and it is to be hoped that future studies will provide additional
data and enhance the benefits for patients.
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a novel human cytokine that signals through the interferon receptor-related proteinsCRF2-4 and IL-22R. J Biol Chem 2000; 275:31335-39.
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related cytokines and receptors. Annu Rev Immunol 2004; 22:929-79.
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IV, et al. Genomic structure, chromosomal localization and expression profile of anovel melanoma differentiation associated (mda-7) gene with cancer specific growth
suppressing and apoptosis inducing properties. Oncogene 2001; 20:7051-63.
10. Jiang H, Lin JJ, Su ZZ, Goldstein NI, Fisher PB Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during
human melanoma differentiation, growth and progression Oncogene 1995 Dec
21;11(12):2477-86.
11. Sauane M, Gopalkrishnan RV, Sarkar D, Su ZZ, Lebedeva IV, Dent P, et al.
MDA-7/IL-24: novel cancer growth suppressing and apoptosis inducing cytokine.
Cytokine Growth Factor Rev 2003; 14:35-51.
12. Schaefer G, Venkataraman C, Schindler U Cutting edge: FISP (IL-4-induced
secreted protein), a novel cytokine-like molecule secreted by Th2 cells. J Immunol
2001; 166:5859-63
13. Soo C, Shaw WW, Freymiller E, Longaker MT, Bertolami CN, Chiu R, et al. Cutaneous rat wounds express c49a, a novel gene with homology to the human melanoma
differentiation associated gene, mda-7. J Cell Biochem 1999; 74:1-10.
14. Wang M, Tan Z, Thomas EK, Liang P Conservation of the genomic structure
and receptor-mediated signaling between human and rat IL-24. Genes Immun
2004; 5:363-70.
15. Wang M, Tan Z, Zhang R, Kotenko SV, Liang P Interleukin 24 (MDA-7/MOB-5)
signals through two heterodimeric receptors, IL-22R1/IL-20R2 and IL-20R1/IL-20R2.
J Biol Chem 2002; 277:7341-47.
16. Zhang R, Tan Z, Liang P Identification of a novel ligand-receptor pair constitutively
activated by ras oncogenes. J Biol Chem 2000; 275:24436-43.
17. Chen W-Y, Cheng Y-T, Lei H-Y, Chang C-P, Wang C-W, Chang M-S IL-24 inhibits
the growth of hepatoma cells in vivo. Genes Immun 2005:1-7.
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SH A novel splicing variant of mouse interleukin (IL)-24 antagonizes IL-24-induced
apoptosis. J Biol Chem 2008 Oct 24; 283(43):28860-72.
21. Nagalakshmi ML, Murphy E, McClanahan T, de Waal Malefyt R Expression
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MH, et al. Design and analysis of an engineered human interleukin-10 monomer. J
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related cytokines and receptors. Annu Rev Immunol 2004; 22:929-79.
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STAT activation by IL-19, IL-20 and mda-7 through IL-20 receptor complexes of two
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distinct receptor complexes: Differences in receptor–ligand interactions mediate uniquebiological functions. J Biol Chem 2003; 277:47517-23.
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31. Su ZZ, Lebedeva IV, Sarkar D, Gopalkrishnan RV, Sauane M, Sigmon C, etal. Melanoma differentiation associated gene-7, mda-7/IL-24, selectively induces
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p53-independent manner. Oncogene 2003; 22:1164-80.
32. Jiang H, Su ZZ, Lin JJ, Goldstein NI, Young CS, Fisher PB. The melanoma differentiation associated gene mda-7 suppresses cancer cell growth. Proc Natl Acad
Sci U S A 1996; 93:9160-65.
33. Leath CA III, Kataram M, Bhagavatula P, Gopalkrishnan RV, Dent P, Fisher PB,
et al. Infectivity enhanced adenoviral-mediated mda-7/IL-24 gene therapy for ovariancarcinoma. Gynecol Oncol 2004; 94:352-362.
34. Oida Y, Gopalan B, Miyahara R, Inoue S, Branch CD, Mhashilkar AM, et al.
Sulindac enhances adenoviral vector expressing mda-7/IL-24-mediated apoptosis
in human lung cancer. Mol Cancer Ther 2005; 4:291-304.
35. Su ZZ, Madireddi MT, Lin JJ, Young CS, Kitada S, Reed JC, et al. The cancer

growth suppressor gene mda-7 selectively induces apoptosis in human breast
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38. Sarkar D, Su ZZ, Lebedeva IV, Sauane M, Gopalkrishnan RV, Valerie K, et al.
mda-7 (IL-24) mediates selective apoptosis in human melanoma cells by inducing thecoordinated overexpression of the GADD family of genes by means of p38 MAPK.
Proc Natl Acad Sci U S A 2002; 99:10054-59.
39. Pataer A, Vorburger SA, Barber GN, Chada S, Mhashilkar AM, Zou-Yang H, etal. Adenoviral transfer of the melanoma differentiation-associated gene 7 (mda7)
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RNA-dependent protein kinase (PKR). Cancer Res 2002; 62:2239-43.
40. Su ZZ, Lebedeva IV, Gopalkrishnan RV, Goldstein NI, Stein CA, Reed JC, et al.
A combinatorial approach for selectively inducing programmed cell death in humanpancreatic cancer cells. Proc Natl Acad Sci U S A 2001; 98:10332-37.

INTERLEUKINA 24
REZUMAT
Terapia genică se poate dovedi a fi o strategie alternativă utilă în cazul afecţiunilor maligne rezistente la terapii convenţionale. Una dintre posibilităţile de tratament o
reprezintă utilizarea unei gene terapeutice care codează o proteină cu proprietăţi antitumorale. O astfel de genă este cea pentru interleukina-24 (IL-24). În acest raporttrecem în revistă profilul genic, localizarea cromozomială, expresia şi funcţiile interleukinei-24.
Cuvinte cheie: interleukina-24, mda7, familia citokinica IL-10, cancer, apoptoza

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Fiziologia - Physiology  2010.20.1 (65)HEALTH, LONGEVITY AND ECOLOGY - AN INTEGRATEDPARADIGMDAN RIGA1, SORIN RIGA1, AUREL ARDELEAN2, FRANCISC SCHNEIDER3
Department of Stress Research & Prophylaxis, ”Al. Obregia” Clinical Hospital of Psychiatry, Bucharest, RO
2”V. Goldis”Western University, Arad, RO
3
Center for Applied Physiology and Molecular Biology, ”Vasile Goldis”Western University, Arad, RO

1

Abstract
Present day stage of human civilization requires new concepts for better understanding evolution mechanisms and tendencies, as well as rapid changes and reevaluations.
For this reason our paper substantiates a novel and neccesary integrated conception: global (human & Earth) health, longevity and ecology. The paradigm reunites into a new
vision three Gaia precepts: globalization, ecological strategy, and global (human & Terra) soundness. As a matter of fact, all world institutions support and supervise theseinternational programmes, i.e. Amsterdam Declaration on Global Change, July 13, 2001 and Amsterdam Declaration of the Club of Rome on Climate, Energy and Economic
Recovery, October 27, 2009. For mankind this new concept represents the reconsideration and improvement of holistic conception from Indo-Chinese and Greek-Roman
antiquity, the 2nd Renaissance of human spirit and a new beginning for humanity future.
Key words: original holistic paradigm; globalization; ecological strategy; (human & Terra) global vitality; ecological organizations, programmes and actions
A planet is passing by the Earth and asks it:- How are you, sister? I haven’t seen you for millions of years!
- Not so well… you know… I’ve got Homo Sapiens…
- Don’t worry, it’ll pass! I had it myself!
				

INTRODUCTION
Nowadays mankind needs novel prospects for rapid and better understanding evolution mechanisms and tendencies, as well as fast transformations andreevaluations (13).
For this reason, the authors have introduced a new and essential paradigm:
global (human & Earth) health, longevity and ecology.

EARTH COMMANDMENTS
At present there are three Gaia concepts-precepts, which have to be developed, implemented and supervised: globalization, ecological strategies, and global(human & Earth) soundness. In addition, they can form an integrated paradigm withimmediate positive consequences, which is more easily followed and controled.
Globalization is and will be a new stage of civilization and signifies the
local, national, regional and global integration and cooperation of all countries (1,5). Ecological strategies mean a coherent ensemble of programmes, resolutions,actions, results and effects (12, 17). Global (human & Terra) soundness represents
the unification of health (22), longevity (21) and ecology (13) concepts, concerning
both human being and Earth.

GAIA CONCEPT I - GLOBALIZATION
Globalization precept can be assimilated and represented by Atlas from Greek
mythology. Iapetus’s son, one of the Twelve Titans, Atlas (8, 10) was punished by Zeus
to sustain the vault of Heaven on his shoulders (the Earth or the Atlas mountains,in other sources). On Terra, Atlas is present everywhere: in Naples, Italy as a 2nd

A joke, but also a sad reality

century Roman copy of a Hellenistic work, in New York, NY, USA as a sculpture in
front of Rockefeller Center, in Melbourne, Australia on a building etc.
Gaia global concept was introduced by Lovelock (14), former NASA scientist,
as Gaia hypothesis and theory. This global ecological concept demonstrates thatthe bio-sphere and geo-sphere - the physical components of the Earth (atmo-,
cryo-, hydro- and litho-spheres) are closely integrated to form a complex interacting system. His role is to maintain the climatic and bio-geo-chemical conditions
on Terra in a preferred homeostasis. In this way, the Earth can be considered as aunique global organism.
Globalization, considering its general features, can be defined (2, 3, 4):- central force that presides over the restructuring of today’s world;- most extended process of socio-historical and politico-economic transformations
from humankind history;- most challenge of XXI century, involving in the same direction whole humansociery and whole geo-sphere (geo-space, geo-system);- new philosophico-political and historico-social paradigm;- power of the most important historical-cultural, scientific-military-economic,
socio-political and IT-communication factors, trends and conceptions that arereshaping and restructuring today’s world.
Globalization represents and has new values and objectives (political, economic, social, juridical, ethical, ideological), as well as restrictions and penalties, but
no negative forces against biosphere, geosphere and civilization (6, 7).

GAIA CONCEPT II - ECOLOGICAL STRATEGIES
Construction of future global ecological society, planned to be carried out

Received January 2010. Accepted February 2010. Address for correspondence: Dr. Dan Riga, Department of Stress Research & Prophylaxis, ”Al. Obregia” Clinical Hospital of Psychiatry, 10 Berceni
Road, Sector 4, RO-041914 Bucharest, Romania, e-mail: D_S_Riga@yahoo.com
2010.20.1(65)  Fiziologia - Physiology

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and consolidated in this century, will necessitate the establishment of equilibrated
relations between natural, ecological, anthropological, demographic, social andeconomic factors. Principles of sensible mankind eco-development must be
scientifically proved and afterwards placed firmly at the foundation of future ecological civilization. There are a lot of global and regional institutions, organizations,
agencies and programmes:- UNEP - United Nations Environment Programme, Stockholm, 1972;- UNCED - United Nations Conference on Environment and Development, 1992;- NAAEC - North American Agreement on Environmental Cooperation, 1994;- EEA - European Environment Agency, established by EEC Regulation
1210/1990;- IPCC - Intergovernmental Panel on Climate Change;- IGBP - International Geosphere-Biosphere Programme;- IHDP - International Human Dimensions Programme on Global Environmental
Change;- WCRP - World Climate Research Programme;- DIVERSITAS - International Biodiversity Programme.
The last four International Global Change Research Programmes presented
and introduced their conclusions in the Amsterdam Declaration on Global Change,
Amsterdam, NL, July 13, 2001 (18):- The Earth System behaves as a single, self-regulating system comprised of physical,
chemical, biological and human components;- Human activities are significantly influencing Earth’s environment in manyways;
- Global change cannot be understood in term of a simple cause-efect paradigm.
Human-driven changes cause multiple effects that cascade through the Earthsystem in complex ways;- Earth system dynamics are characterised by critical threshold and abrupt changes.
Human activities could inadvertently trigger such changes with severe consequencesfor Earth’s environment and inhabitants.
- In term of some key environmental parameters, the Earth System has moved welloutside the range of the natural variability exhibited over the last half million years
at list. The Earth is currently operating in a no-analogue state.
Recently, the Amsterdam Declaration of the Global Assembly - the Club of
Rome”Climate, Energy and Economic Recovery” Amsterdam, NL, October 27, 2009
reaffirms the actuality and acuteness of this problem (19):The Club of Rome calls for urgent action to avert the growing risk of catastrophic climate change. The most recent scientific data presented to the Assembly by
the world’s top climate scientists demonstrate the accelerating impacts of climatechange on the natural systems of the planet... Governments have directed trillionsof dollars to stabilize the financial system: we call for the required levels of financeto salvage the future of the planet...
The Club of Rome urges governments to adopt a strong climate treaty
that will be fair, that will contribute to energy and economic security, that will
respond to the growing urgency of the risks of catastrophic climate change...
We emphasize that the transition to an equitable, sustainable lowcarbon society must also engage the business and investment community, civilsociety and communities at large. There is enormous potential for business in rapidly
emerging markets for new sustainable low carbon products. Business leaders must
commit to the re-design of business models, to innovative solutions and to newenergy and resource-efficient products. They have a responsibility to regain pub-lic
trust in the ethical and sustainable basis of banking and business activities.
The goals of concerted international climate action must be (19):• To adopt, at the UN Climate Conference in Copenhagen, legally binding
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agreements that will initiate immediate action to achieve a stable climate withatmospheric concentrations of CO2 not exceeding 350 ppm.
• To establish financial mechanisms, including a carbon market, that will
enable countries, companies and communities to reduce their net carbon emissions
to the levels required.
• To accelerate support for adaptation and humanitarian assistance indeveloping countries as an integral component of national development so as toreduce the impacts of climate change (16).
• To promote the development of new models and strategies for growth,development and globalization which place a real value on natural capital andecosystems services - including the removal of CO2 from the atmosphere.
• To agree upon an international study, to be concluded within one year, which
will propose how the framework of international policies and institutions must be
adapted to meet the connected, systemic challenges of the 21st Century.

GAIA CONCEPT III - HEALTH, LONGEVITY,ECOLOGY
Construction of the Gaia IIIrd concept - Health, Longevity, Ecology - can be
exemplified by three representative goddesses in these fields (8, 9, 10):- Hygieia, goddess of health; her name is the the source of word hygiene.
- Hebe, goddess of eternal youth - longevity, the cupbearer for the gods
and goddesses of Mount
Olympus, and- Gaia, the primal Greek goddess personifying the Earth, as Gaia ecologicalconcept.

Hygieia, goddess of Health, daughterof the god of medicine, Asclepius
1st century Roman statueHebe, goddess of Eternal Youth - Longevity,
daughter of Zeus and Hera, Roman equivalent - Juventas
Antonio Canova’s (1757-1822) marble sculpture

Fiziologia - Physiology  2010.20.1 (65)CONCLUSIONSAND FUTURE ADVANCES

Gaea - Gea - Gaia, considered a Mother Goddess or Great Goddess
Pre-Olympian Greek Earth goddess, sprang from primordial Chaos,
Terra Matter or Tellus - her equivalent in the Roman pantheon

Indeed, these three notions can and must be unified, both for humanbeing and Earth as integrated conceptions, as well as between them. The result isand will be the global (human and Terra) soundness, or global (human & Earth)
health, longevity and ecology.
Surroundings (bio- and geo-spheres) are in continuous transformations(alterations or good variations), and also humans are still in evolution (bad or good
modifications). In our days the environment configuration and human evolution
depend in the greatest measure of human thinking, decisions, behaviours andactions (15). So, the human interventions on the outside (environment), and on
the inside (ourselves) are and will be decisively for global (human & Earth) health,longevity and ecology in the near future.
These can determine:l the normality of surroundings and humans with harmonious interconnections, orl unfortunately the abnormality and destruction, bringing about theaggressive environment and fragile human beings with total disagreement andantagonism between them.
By his irrational actions, man becomes an enemy of himself (dangerous andwrong life styles) and also of the natural environment (qualitative and quantitativelarge demolitions). With good reason, George Crockcroft (b. 1932, pseudonym Luke
Rhinehart) said Social consequences of a nation formed by normal people are
evident: poverty, conflicts, violence, war and a generalized gloom.
Therefore, humanity must urgently change and may act as holistic strategy
in three interconnected important directions:l remplacement of diseases of life style (pro-impairment, pro-aging andpro-pathologies), by health promotion (24), stress-related diseases prevention(20) and pro-longevity concepts (23);l substitution of surroundings destructive actions for an upright andstimulative partnership with nature;l understanding the necessity for fast implementation and combination of
global and regional strategies with national and local measures, regarding global(human & Earth) health, longevity and ecology.
The change of human behaviour is one of decisive solution. In this respect,
the famous words said in 1949 by Contantin Brâncuşi are very suitable in ourdays: If man manages to eliminate his Ego, he will be able to hear the heartbeatof nature and her whispers (11). Humans can be able to apply the Gaia concept,
mission and philosophy, namely living organisms must improve themselves andtheir environment too.
In few but cardinal words, humanity must define the question-answer, choice,pathway-solution and also the action-result To be or not to be, that is the question- a very great soliloquy on the very great topic of human being (W. Shakespeare,
Hamlet, Act 3, Scene 1).

2010.20.1(65)  Fiziologia - Physiology

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Photograph of the Earth,
Apollo 17,
taken from Appolo 17, in 1972
Mission insignia

The three Gaia philosophy and concepts (• globalization • ecological strategies • human & Earth soundness) will determine the future of minkind and of
our planet too.
For human civilization they represent:- the reconsideration and improvement of holistic conception from IndoChinese and Hellenic-Roman antiquity;- the 2nd Renaissance of human spirit; and- a new beginning for human future started with Gaia ESA (European Space
Agency) astrometry space mission (spring 2012-2017), with the obiectives tocreate the largest and most precise three dimensional chart of our Galaxy (about
one billion stars in our Galaxy and troughout the Local Group).
REFERENCES
Globalization

1. Axford B. The Global System: Economics, Politics and Culture. New York, NY,
St. Martin’s Press, 1995.
2. Huntington PS. The Clash of Civilizations. New York, NY, Simon & Schuster,1996.
3. Macridis RC. Contemporary Political Ideologies. New York, NY, Harper-Collins,
1992.
4. Robertson R. Globalization: Social Theory and Global Culture. Newbury Park,
CA, Sage, 1992.
5. Stanescu V. Stiinta globalizarii. Bucuresti, Ed. All Beck, 2005.
6. Stanescu V. Globalizarea. Spre o noua treapta de civilizatie… Cluj-Napoca, Ed.
Eikon, 2009.
7. Stiglitz J. Globalizarea. Sperante şi deziluzii. Bucuresti, Ed. Economica, 2003.

Mythology

8. Bulfinch T. Bulfinch’s Greek and Roman Mythology: The Age of Fable. New York,NY, Dover Publ., 2000.
9. Fairbanks A. The Mythology of Greece and Rome. New York, NY, D. Appleton,
1907.
10. Kernbach V. Dictionar de mitologie generala, ed. a 2-a. Ed. Stiintifica si Enciclopedica, 1989.

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Ecology
11. Andrei M, Ardelean A, Mohan G, Voica C, Anghel I. Maxime, cugetari si definitii
din biologie. Bucuresti, Ed. All, 2009.
12. Botkin DB, Keller EA. Environmental Science: Earth as a Living Planet, 7th ed.
Ed. Hoboken, NJ, John Wiley & Sons, 2009.
13. Enger E, Smith B. Environmental Science: A Study of Interrelationships, 11th ed.
New York, NY, McGraw-Hill Higher Edu., 2006.
14. Lovelock JE., Margulis L. Atmospheric homeostasis by and for the biosphere - The
Gaia hypothesis, Tellus 1974; 26(1): 2-10.
15. Nicola G, Danciu B (eds). Tratat de psihoecologie. Bucuresti, Ed. Academiei
Romane, 2008.
16. Thompson D, Metcalf J (eds). The Oxford Compact English Dictionary, Oxford
and London, UK. Oxford University Press and Dorling Kindersley, 2003.
17. Wright RT. Environmental Science: Toward a Sustainable Future, 9th ed. Upper
Saddle River, NJ, Prentice Hall-Pearson Edu., 2004.

18. * * * Amsterdam Declaration on Global Change. Amsterdam, July 13, 2001.
19. * * * Amsterdam Declaration of the Club of Rome on Climate, Energy andEconomic
Recovery. Amsterdam, October 27, 2009.
Human Health and Longevity
20. Cooper CL (ed). Stress, Medicine and Health. Boca Raton, FL, CRC Press,
1996.
21. Cutler RG, Harman SM, Heward C, Gibbons M (eds). Longevity Health Sciences.
New York, NY, New York Academy of Sciences, 2005.
22. Haber D. Health Promotion and Aging. New York, NY, Springer, 2003.
23. Klatz R, Goldman R. The New Anti-Aging Revolution. North Bergen, NJ, Basic
Health Publ., 2003.
24. Knapp M, McDaid D, Mossialos R, Thornicroft G (eds). Mental Health Policy
and Practice Across Europe. Maidenhead, UK, Open University Press, McGrawHill Edu., 2007.

SANATATE, LONGEVITATE SI ECOLOGIE - O PARADIGMAINTEGRATAREZUMATIn zilele noastre, civilizatia umana are nevoie de noi concepte pentru o mai buna intelegere a mecanismelor si tendintelor evolutive, precum si a schimbarilor si reevaluarilor
produse cu rapiditate. Din aceasta cauza, lucrarea noastra prezinta si demonstreaza o noua si necesara conceptie integrata: sanatatea, longevitatea si ecologia globala (umana
si a Pamantului). Paradigma reuneste intr-o noua viziune trei precepte Gaia: globalizare, strategie ecologica si sanatate globala (umana si a Pamantului). De altfel toate
institutiile lumii sprijina si supravegheaza aceste programe internationale, ca de ex. prin Declaratia de la Amsterdam asupra Schimbarii Globale, 13 iulie 2001 si Declaratia dela Amsterdam a Clubului de la Roma asupra Restabilirii Economice, Energetice si Climatice, 27 octombrie 2009. Acest nou concept reprezinta pentru omenire reconsiderarea
si imbunatatirea conceptiei holistice din antichitatea Indo-Chineza si Greco-Romana, a doua Renastere a spiritului uman si un nou inceput pentru viitorul umanitatii.
Cuvinte cheie: paradigma holistica originala; globalizare; strategie ecologica; vitalitate globala (umana & a Terrei); organizatii, programe si actiuni ecologice

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Fiziologia - Physiology  2010.20.1 (65)INSULIN GROWTH FACTOR 1 INVOLVEMENT IN THE
ETIOLOGY OF PECTUS EXCAVATUM IN CHILDRENDAVID VL1, IZVERNARIU DA2, BOJIN FLORINA3, GAVRILIUC OANA3, POPOIU MC4,TĂNĂSIE GABRIELA3, PUIU MARIA5, BOIA ES4
Department of Pediatric Surgery, Children's Hospital „Louis Turcanu”, Timisoara
Department of Histology, University of Medicine and Pharmacy „Victor Babes”, Timisoara
3
Department of Physiology and Immunology, University of Medicine and Pharmacy “Victor Babes”, Timisoara
4
Department of Pediatric Surgery and Orthopedics, University of Medicine and Pharmacy „Victor Babes”, Timisoara
5
Department of Human Genetics, University of Medicine and Pharmacy „Victor Babes”, Timisoara
1
2

ABSTRACT
Pectus excavatum (PE) is the most frequent anterior chest deformity occurring in approximately 1 in 1000 live births. Despite the excellent achievements in the surgicaltreatment of the disease the etiology of PE is yet to be clarified. Nowadays the most accepted etiopathogenic theory is that the posterior displacement of the sternum iscaused by the overgrowth of the disturbed costal cartilages. Insulin Growth Factor 1 (IGF1) is the main anabolic growth factor for human hyaline cartilages and plays a keyrole in cartilage homeostasis, promoting proteoglycans synthesis and cell division. In this article we present the results of our study regarding the involvement of IGF1 inthe etiology and pathogenesis of PE in children. Samples of the deformed costal cartilages from 25 children with PE were obtained during the surgical intervention for thecorrection of the disease. Cartilage samples were obtained during autopsy from 17 children in whom the cause of death was unlikely to affect the cartilage. The samples
were immunohistochemically stained with fluorescent antibody for phosphorylated IGF1 receptor (IGF1R). The majority of the samples were negative for phosphorylated
IGF1R and there were no significant differences between the two groups (p> 0.05). In both PE patients and in general populations, the majority of the costal chondrocytes
doesn’t reveal activated IGF1R. PE in children is not produced by a defect of the IGF1 axis.
Key words: pectus excavatum, IGF1, etiology, growth factor

INTRODUCTION
Pectus excavatum (PE) is the most frequent anterior chest deformity occurringin approximately 1 in 1000 live births (1). The deformity consists in the posterior
depression of the sternum and the lower costal cartilages (2). Despite the excellent
achievements in the surgical treatment of the disease the etiology of PE is yet to beclarified. Several etiopathogenic theories were proposed over time and the mostaccepted today is that the posterior displacement of the sternum is caused by theovergrowth of the disturbed costal cartilages (3). Previous histological studies
found no significant differences regarding the number, shape, area of the cell andnucleus between cartilages from PE patients and normal (4, 5). This indicates thatthe overgrowth of the cartilages has a global pattern with equal involvement of
the cells and the matrix.
Insulin Growth Factor 1 (IGF1) is the main anabolic growth factor for humanhyaline cartilages (6). IGF1 is it plays a key role in cartilage homeostasis, promoting proteoglycans synthesis and cell division (7). Compared with other growth
factors, IGF1 has the most powerful anabolic effect over chondrocytes in culture(8). Approximately 80% of the circulating of IGF1 is produced in the liver under the
direct stimulation of the pituitary growth hormone (GH) (9). Despite of that thecomplete ablation of the liver production of IGF1 has little effect on the somatic
growth indicating that alternative autocrine and paracrine mechanism are involved(10). In the serum it circulates bind to one of the 6 specific binding proteins (IGF1BP)(11). IGF1BP prolong its life time and, because their affinity for IGF1 is greater than
IGF1 receptor (IGF1R) they are the major factor influencing the biodisponibility of
the IGF1 (11). IGF1 has a specific receptor displayed by the majority of the tissues

in the human body (12).
In this article we present the results of our study regarding the involvementof IGF1 in the etiology and pathogenesis of PE in children.

MATERIAL AND METHODS
Samples of the deformed costal cartilages from 25 children with PE, age 5
to 18 years, mean 12 years, were obtained during the surgical intervention for thecorrection of the disease. Costal cartilage excision is a regular part of the Ravitch
surgical procedure for the correction of PE (13). Costal cartilage samples wereobtained during autopsy from 17 children in whom the cause of death was unlikely
to affect the cartilage, age 1 to 19 years, mean 9.5 years. The samples were cut
perpendicular to the long axis of the cartilage and apart from the costo-chondraland the chondro-sternal junction. The specimens were fixed in 10% bufferedformalin and embedded in paraffin. Three sections were cut at 3 µm thickness fromeach cartilage sample. Prior to staining, each slide was dewaxed and rehydrated
by standard protocol.
The samples were immunohistochemically stained with fluorescent antibody
for phosphorylated IGF1R (phospho Y1158) (Alexa Fluor® 488) antibody (ab20493),
Abcam®, UK using standard over night incubation technique. With this antibody
the chondrocytes membrane expressing phosphorylated IGF1R is stained in green.
The slides were examined at Nikon Eclipse E800 microscope with fluorescencefilters. Triband and green filters were used and at least one photographic image
was obtained for each sample.
Positive control was obtained using cultured fibroblast stimulated with IGF1

Received February 2010. Accepted February 2010. Address for correspondence: Dr. David Vlad, Department of Pediatric Surgery, Children's Hospital „Louis Turcanu”, Timisoara, e-mail: david.
vlad@yahoo.com
2010.20.1(65)  Fiziologia - Physiology

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and MDA-MB-231 tumoral cells (Fig. 1).

Fig. 2a. Positive cells for phosphorylated IGF1R, 10 years old patient, experimental group, triband
filter left, green filter right (400X)
Fig. 1. Immunofluorescence for IGF1R. Cultured fibroblasts stimulated with IGF1, triband filter left,
green filter right (400X)

The chondrocytes were considered positive if the cell membrane was completely stained in green. The samples were considered positive if they had large
areas of positive cells outside the subperichondral zones and in the nearby of thevascular channels.
Statistical analyzing was performed with the help of SPSS v1.7 software
Differences between groups we used chi-square test for non-numeric variables;level of significance for p was set at 0.05.

RESULTSWe found a total of 14 specimens positive for phosphorylated IGF1R, 7 for theexperimental and 7 for the control group (Fig. 2). There were no significant differences between the two groups (p> 0.05) (Table 1). The majority of the samples
were negative for phosphorylated IGF1R 72% for the experimental group and 55%
for the control group (Fig. 3). Positive cells were found nearby vascular channels onlyin 2 samples from the experimental and 3 from the control group (Fig. 4).
Table I. The results of the immunohistochemistry staining for phosphorylated IGF1R

Group
Experimental
Control

Samples25
17

Positive

7 (24%)
7 (41%)
p= 0.374
Fig. 2b. Positive cells for phosphorylated IGF1R, 1 year old patient, control group, triband filter
left, green filter right (400X)

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Fiziologia - Physiology  2010.20.1 (65)Fig. 3a. Negative cells for phosphorylated IGF1R, 10 years old patient, experimental group,
triband filter left, green filter right (400X)

Fig. 4. Positive cells for phosphorylated IGF1R in the nearby of a vascular channel, 6 years oldpatient, experimental group, triband filter left, green filter right (400X)DISCUSSIONS

Fig. 3b. Negative cells for phosphorylated IGF1R, 18 years old patient, control group, triband filter
left, green filter right (400X)

2010.20.1(65)  Fiziologia - Physiology

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PE is caused by the overgrowth of the costal cartilages (3). There are someelements that suggest that the IGF1 axis is involved in the etiology of the disease.
First of all, IGF1 is the main anabolic factor for human hyaline cartilage (7). Under
IGF1 stimulation the number of chondrocytes, the quantity of proteoglycans andcollagen II increase in superior manner to any other growth factor (8). More thanthat, IGF1 is a factor of differentiation of the mesenchymal cells towards sulfatedproteoglycans producing chondrocytes (7). Another element suggests the possibility of the IGF1 involvement is the fact that the curve of plasmatic level of the
IGF1 during childhood and puberty is following the curve of the progression of thechest wall deformity (2, 14). Serum levels of IGF1 increase progressively from birthreaching the maximum trough puberty and decreases after that (14). Parallel to thatthe deformity of the chest wall in most of the patients with PE is minor or absent
at birth, increases slowly or remain stable during childhood and aggravate rapidly
during puberty (2). After puberty the deformity usually remains stable (2).
In the same time there is no other evidence that PE patients suffer of a disturbanceof the GH - IGF1 axis. The tallness the majority of the PE patient is between the limits ofthe general population (34). But this doesn’t eliminate the possibility of involvementof the autocrin mechanism of the IGF1 axis. This was the reason why we decided tochoose to determine the phosphorylated IGF1R in the costal cartilages of children with
PE and not go for an assessment of the plasmatic level of the IGF1.
The IGF1 and his receptor are major factors influencing the proliferation,
survival and treatment resistance of tumoral cells (15). IGF1R is present in almost all
solid and blood tumors and is one of the main targets in the antitumoral treatment(16). The IGF1R blockade using monoclonal antibodies is a promising treatment
option in the treatment f cancer (17, 18). By analogy, IGF1R blockade may be used19

as a mean for stopping the overgrowth of the costal cartilages in PE and may be
used as an alternative to the surgical treatment. In order to be efficient and to avoid
systemic side effects the blockade should be made strictly local or by antibodies
specific for costal chondrocytes. These are now only speculations and more researchis to be done in this direction. We, by conducting this research have made the firststeps to finding an alternative nonsurgical treatment for PE.
The results of our study in PE patients the number of chondrocytes revealing
phosphorylated IGF1R is similar to general population. The presence of IGF1R in
human chondrocytes was already demonstrated (19).The antibody used by us
targeted the phosphorylated IGF1R. This doesn’t imply the absence of the IGF1R in
the chondrocytes but only the ones activated by IGF1. This indicates than when theexperiments were done the level of IGF1 in the cartilage was low. In the same time,
the fact that nearby the vascular channels the cells was positive for phosphorylated
IGF1R and in the rest of the cartilage was not, indicates that is happening duet ocirculating IGF1 stimulation. This means that the autocrine/ paracrine mechanism
have poor involvement in the IGF1 mediated growth of the cartilage. For PE theabsence of activated IGF1R is an indication that the overgrowth of the costal cartilages does not cur under a hyperstimulation with IGF1 by non autocrin, paracrin
or endocrine mechanism.

CONCLUSIONS

1. In both PE patients and in general populations, the majority of thecostal chondrocytes doesn’t reveal activated IGF1R. IGF1R is expressed by thecostal chondrocytes mainly in the nearby of the vascular channels and in thesubperichondral space. There are no differences between PE patients and general
population regarding the number and the distribution of the chondrocytes revelingphosphorylated IGF1R.
2. PE in children is not produced by a defect of the IGF1 axis. In the same time,
this doesn’t imply that this axis can’t be used as a therapeutic way in blocking theovergrowth of the costal cartilages in PE patients.
REFERENCES
1. Kelly RE, Lawson ML, Paidas CN et al. Pectus excavatum în a 112-years autopsy
series: anatomic findings and the effect on survival. J Pediatr Surg 2005;40:12758.
2. Kelly RE jr. Pectus excavatum: historical background, clinical picture, preoperativeevaluation and criteria for operation. Semin Pediatr Surg. 2008 Aug;17(3):181-93.
3. Haje SA, Harcke HT, Bowen JR. Growth disturbance of the sternum and pectus
deformities: imaging studies and clinical correlation. Pediatr Radiol (1999) 29:
334±341
4. Feng J, Hu T, Liu W, et al: The biomechanical, morphologic, and histochemical
properties of the costal cartilages in children with pectus excavatum. J Pediatr Surg
2001; 36: 1770-1776.
5. Geisbe H, Buddecke E, Flach A, Müller G, Stein U. Biochemical, morphologicaland physical as well as animal experimental studies on the pathogenesis of funnel
chest Langenbecks Arch Chir. 1967;319:536-41.
6. Mankin HJ, Jennings LC, Treadwell BV, Trippel SB. Growth factors and articularcartilage. J Rheumatol Suppl. 1991 Feb;27:66-7.
7. Schmidt MB, Chen EH, Lynch SE. A review of the effects of insulin-like growth
factor and platelet derived growth factor on in vivo cartilage healing and repair.
Osteoarthritis Cartilage. 2006 May;14(5):403-12. Epub 2006 Jan 18.
8. Johns DE, Athanasiou KA. Growth factor effects on costal chondrocytes fortissue engineering fibrocartilage. Cell Tissue Res. 2008 Sep;333(3):439-47. Epub
2008 Jul 3.
9. Dupont J, Holzenberger M. Biology of insulin-like growth factors in development.
Birth Defects Res C Embryo Today. 2003 Nov;69(4):257-71.
10. Yakar S, Sun H, Zhao H et al. Metabolic effects of IGF-I deficiency: lessons frommouse models. Pediatr Endocrinol Rev. 2005 Sep;3(1):11-9.
11. Hwa V, Oh Y, Rosenfeld RG. The insulin-like growth factor-binding protein (IGFBP)
superfamily. Endocr Rev. 1999 Dec;20(6):761-87.
12. Chitnis MM, Yuen JS, Protheroe AS, Pollak M, Macaulay VM. The type 1 insulinlike growth factor receptor pathway. Clin Cancer Res. 2008 Oct 15;14(20):636470.
13. Ravitch MM. The operative treatment of pectus excavatum. Ann Surg.
1949;129:429-44
14. Brabant G, von zur Mühlen A, Wüster C et al. Serum insulin-like growth factor I
reference values for an automated chemiluminescence immunoassay system: resultsfrom a multicenter study. Horm Res. 2003;60(2):53-60
15. Weroha SJ, Haluska P. IGF-1 receptor inhibitors in clinical trials--early lessons. J
Mammary Gland Biol Neoplasia. 2008 Dec;13(4):471-83. Epub 2008 Nov 21.
16. Hartog H, Wesseling J, Boezen HM, van der Graaf WT. The insulin-like growth factor 1 receptor in cancer: old focus, new future. Eur J Cancer. 2007 Sep;43(13):1895904. Epub 2007 Jul 10.
17. Párrizas M, Gazit A, Levitzki A, Wertheimer E, LeRoith D. Specific inhibition of
insulin-like growth factor-1 and insulin receptor tyrosine kinase activity and biological
function by tyrphostins. Endocrinology. 1997 Apr;138(4):1427-33.
18. Arteaga CL, Kitten LJ, Coronado EB, Jacobs S, Kull FC Jr, Allred DC et al.
Blockade of the type I somatomedin receptor inhibits growth of human breast cancer
cells in athymic mice. J Clin Invest. 1989 Nov;84(5):1418-23
19. Schmid C. Insulin-like growth factors. Cell Biol Int. 1995 1995 May;19(5):445-57.

IMPLICAREA FACTORULUI DE CRESTERE INSULINIC 1
ÎN ETIOLOGIA PECTUS EXCAVATUM LA COPIIREZUMAT

Pectus Excavatum (PE) este cea mai frecventă malformatie a peretelui anterior al toracelui şi are o incidentă de aproximativ 1 la 1000 de nou-născuţi. În ciuda realizărilor
semnificative în tratamentul chirurgical al bolii, cauza maladiei nu a fost încă stabilită. Mai multe teorii etiopatogenice au fost elaborate de-a lungul timpului, dar cea mai
acceptată astăzi este cea conform căreia înfundarea sternului se produce datorită creşterii în exces a cartilajelor costale. IGF1 este principalul factor de creştere anabolizant
pentru cartilajul hialin Si are un rol esenţial în homeostazia acestuia, stimulând secreţia de proteoglicani Si diviziunea celulară. În acest articol prezentăm rezultatele studiului
nostru privind implicare IGF1 în etiologia Si patogenia PE. Specimene de cartilaj costal au fost obţinute în timpul operaţiei pentru corecţia PE de la 25 de pacienţi. Un număr
de 17 specimene de cartilaj costal au fost obţinute de la cadavre a căror cauză de deces a fost foarte puţin probabil să afecteze cartilajul costal. Specimenele au fost colorate
prin tehnica imunohistochimică directă cu anticorp fluorescent pentru receptor IGF1fosforilat (IGF1R). Majoritatea specimenelor au fost negative pentru IGF1R fosforilat iarîntre cele două loturi nu există diferenţe semnificative (p > 0,05). Atât la pacienţii cu PE cât şi la populaţia normală, majoritatea condrocitelor costale nu exprimă IGF1R
activat. La originea PE la copil nu se află un defect al axei IGF1.
Cuvinte cheie: pectus excavatum, IGF1, etiologie, factor de creştere

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Fiziologia - Physiology  2010.20.1 (65)COMPARISON BETWEEN DIFFERENTIATION POTENTIAL
OF DOG PERIOSTEUM – DERIVED CELLS AND BONE
MARROW-DERIVED CELLS TO OSTEOGENIC,CHONDROGENIC AND ADIPOGENIC LINEAGESANGHEL SIMONA1,5, BOJIN FLORINA1, IGNA CORNEL6, TANASIE GABRIELA1,5, TATUCALIN2,5, SERES MONICA6, TATU CARMEN1,5, LUCA CALIN6, ORDODI VALENTIN2, GRUIAALEXANDRA5, ROSCA ADRIANA5, GURBAN CAMELIA4, GEORGESCU REMUS3, PAUNESCUVIRGIL1,5
Physiology and Immunology Department, University of Medicine and Pharmacy “Victor Babes”Timisoara
Biology Department, University of Medicine and Pharmacy “Victor Babes”Timisoara
3
Biophysics Department, University of Medicine and Pharmacy “Victor Babes”Timisoara
4
Biochemistry Department, University of Medicine and Pharmacy “Victor Babes”Timisoara
5
Transplant Immunology Center, County Clinical Emergency Hospital Timisoara
6
University of Agricultural Sciences and Veterinary Medicine of Banat Timisoara
1
2

ABSTRACTThe aim of the present study is to investigate the probability of dog periosteum-derived cells and bone marrow-derived cells to differentiate along the osteogenic, chondrogenic
and adipogenic lineages. Results of this experiment aim to highlight the similarities and differences between the cells obtained as a result of periosteum-derived culture
and bone marrow-derived cells cultivated in three differentiation media. While the dog bone marrow derived-cells differentiate towards all three lineages, with variable
intensity, thus confirming the results of other research studies in this field referring to other species, canine periosteum-derived cells obtained using culture of periosteal
explants, differentiate mostly towards the osteoblastic and chondrogenic lineage, and to lesser extent towards the adipogenic lineage; the cells obtained using the digestion
method of periosteal fragments positively differentiate towards all the three lineages: osteoblastic, chondrogenic and adipogenic. In addition, it was demonstrated thatcryopreserved studied cells have osteogenic, chondrogenic and adipogenic differentiation capacity.
Key words: periosteum-derived cells (explant and enzymatic isolation methods), bone marrow-derived cells, differentiation potential

INTRODUCTION

MATERIALS AND METHODS

There is a difference between bone formation during embryonic life andpostnatal bone growth, when osteoprogenitor cells directly differentiate into bone
forming osteoblasts. During the fracture healing process 2 distinct phenomena aredeveloping: osteogenesis as well as chondrogenesis. It is known that local microenvironmental conditions may be important in directing the differentiation of theperiosteum-derived cells towards either osteogenesis or chondrogenesis. As such,
periosteal cells could be used as an in vitro culture model to study the osteogenic
capacity of biomaterials developed for bone regeneration therapy. Some studies
to evaluate the osteogenic potential of biomaterials were performed which usedperiosteum-derived cells from human, rabbit, pig and bovine origin (1,2,3).
However, dog periosteum-derived cells were not yet analyzed with regard to theirsuitability to study cell/biomaterial interactions.
The strategy for obtaining cell populations with predictable tissue formation
capacity would be dependent, at least in part, on the presence of either multiple
functionally distinct cell subsets or more primitive stem cells capable of multilineage differentiation. It is important to identification and separation of the cellsubset with the desired tissue-forming capacity from cell subpopulations withunwanted differentiation potential. In the later case, cell product manufacturing,
including culture techniques, scaffold technologies, or growth factor treatments,
could be focused in the commitment to the desired tissues and in the preventionof unwanted/heterotopic tissue formation.

Isolation and cultivation of dog periosteumderived cells and bone marrow derived-cells
Biologic material was harvested from the five common-breed dogs. For harvesting the periosteal flaps the skin and underlying muscular-fibrous connective
tissue were removed to expose the periosteum. The periosteal flaps were detachedusing a periosteal elevator and processed by 2 methods: explant method andenzymatic digestion (4). The periosteal explants were placed with their osteogenic(cambium) side in direct contact with the culture plate in order to provide theadherence for explant culture, while the other periosteal flaps were exposure tocollagenase for digestion and cellular suspensions were plated into culture flasks.
Bone marrow was extracted by aspiration from the dog humerus and used in order
to isolate the mesenchymal stem cells. The Ficoll-Paque technique for the isolationof mononuclear cells, followed by the separation of mesenchymal stem cells byadherence to plastic, has been chosen.
The nutritive medium used for periosteal cells was culture medium HAM/F
12: DMEM (Dulbecco’s Modified Eagle’s Medium) (1:1) with L-glutamine + 10%
fetal bovine serum + 1% Penicillin/Streptomycin + 3ng/ml bFGF (basic Fibroblast
Growth Factor) and for bone marrow-derived cells was MEM Alpha Medium (lowglucose) + 10% fetal bovine serum + 1% Penicillin / Streptomycin + 3ng/ml
bFGF. Culture medium was replaced every 3 days. The morphology was evaluatedby phase contrast and light microscopy.

Received January 2010. Accepted March 2010. Address for correspondence: Simona Anghel, 2A Eftimie Murgu Square, 300041, Timisoara, Romania, e-mail: simoanghel@
yahoo.com
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Expression of Vimentin
Monoclonal Antibody to Vimentin (Clone V9) is use to identify qualitatively
by light microscopy tissues of mesenchymal origin using immunohistochemical
staining methods.
The cells from Nalgene Nunc Permanox chamber slides were fixed with 100%
methanol (-20oC, 10 minutes). The fixed cultures were washed twice with phosphatebuffer saline and incubated for 30 minutes with anti-vimentin primary antibody
diluted in phosphate buffer saline, at room temperature, on shaker. After washingwith phosphate buffer saline, biotinylated secondary antibody was added and thecells were incubated for another 30 minutes, at room temperature, on shaker. Afterwashing with phosphate buffer saline, the cells were incubated for 20 minutes withperoxidase-conjugated streptavidin followed by a 10 minutes incubation periodwith AEC (3-amino-9-ethylcarbazole) peroxidase substrate. After washing with tap
water and counterstaining with hematoxylin (30 seconds), the Permanox slides were
mounted in an aqueous mounting medium and the cover slip was applied.
Culture condition for differentiation to osteogenic, chondrogenic and adipogenic lineages
At passage 2 (P2), cells obtained from the two cellular sources were re-seededin culture flasks characteristic for evaluation each type of differentiation towards thethree lineages: osteoblastic, chondrogenic and adipogenic. Cell seeding required
different cellular concentrations: 15x103cells/cm2 for osteoblastic differentiation,
4x104 cells/cm2 for adipogenic differentiation, and 3x104 cells/cm2 for monolayerchondrogenic differentiation. Ready-to-use Miltenyi Biotec differentiation media
were used: NH OsteoDiff Medium, NH ChondroDiff Medium and NH AdipoDiff
Medium, supplemented with 1% antibiotic solution.
Analysis of the differentiation potential into the
osteogenic lineage
After 10 days culture in osteogenic medium, the alkaline phosphatase presence inthe cultures, the potential to form a mineralized matrix and the collagen I immunolocalization were determined histochemically as well as imunohistochemically.
a. Alkaline phosphatase histochemical staining
A ready-to-use precipitating substrate system for alkaline phosphatase is
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/ NBT) liquid
substrate system. This substrate system produces an insoluble NBT diformazan end
product that is blue to purple in color and can be observed visually.
After rinsing the cells on the Nunc Permanox chamber slides with cold (4oC)
phosphate buffer saline, the cells were fixed with acetone (-20oC) for 5 minutes.
The fixed cultures were washed with cold distilled water and allowed to dry for 30
minutes. The cultures were incubated for 10 minutes with BCIP/ NBT liquid substrate
system at room temperature. The reaction was stopped by removing the substratesolution and washing with distilled water. The Nunc Permanox slides were mounted
in an aqueous mountant.
b. The potential to form a mineralized matrix
The mineralization of the bone matrix was analyzed histochemically by Von
Kossa staining. Reveals calcium salts (phosphate, carbonate, sulfate, oxalate).
Calcium phosphate deposits can be detected by the Von Kossa technique in which
phosphate deposits are stained black.
The cultures were rinsed with cold (4oC) phosphate buffer saline and fixed with
4% formaldehyde (4oC, 10 minutes). After washing with cold distilled water, the fixed
cultures were covered with a 5% silver nitrate solution and kept for 30 min in a dark room.
Afterwards, they were rinsed and covered with distilled water and exposed to ultraviolet
light for 1 hour. The cultures were rinsed in distilled water and treated for 2 minutes with
a 5% sodium thiosulphate solution. After washing with tap water, counterstaining withhematoxylin solution for 30 seconds, followed by washing with tap water, the Nunc
Permanox slides were mounted with an aqueous mounting medium.
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c. Collagen I immunolocalization
After rinsing the cultures on the Nunc Permanox chamber slides with cold
(4oC) phosphate buffer saline, the cells were fixed with 4% formaldehyde (4oC, 8
minutes). The fixed cultures were washed with cold distilled water and let to dry.
The cultures were incubated for 30 minutes with anti-collagen primary diluted
antibody, on shaker. After washing with phosphate buffer saline, biotinylated
secondary antibody was added and the cells were incubated for another 30 minutes,
on shaker. After washing with phosphate buffer saline, the cells were incubatedfor 30 minutes with peroxidase-conjugated streptavidin followed by a 10 minutes
incubation period with AEC (3-amino-9-ethylcarbazole) peroxidase substrate. Afterwashing with tap water and counterstaining with hematoxylin, the Permanox slides
were mounted in an aqueous mountant.
Visualization is based on enzymatic conversion of a chromogenic substrate AEC
into a colored red precipitate by horseradish peroxidase (HRP) at the sites of antigen
localization, which can then be viewed using bright-field microscopy.
Analysis of the differentiation potential into thechondrogenic lineage
After 24 days under a chondrogenic environment, the dog periosteum-derived
cells and bone marrow-derived cells from monolayer cultures were processed forhistochemical staining. Safranin O and Alcian Blue were used to stain proteoglycans
in the extracellular matrix, a positive indicator of cartilaginous tissue.
Safranin O stains proteoglycans and glycosaminoglycans. The cultures on the
Nunc Permanox chamber slides were rinsed with phosphate buffer saline and fixedwith formaldehyde and then ethanol 50% for 10 minutes. After washing with distilledwater, the fixed cultures were covered with a 0.1% Light Green solution for 2 minutes.
The cultures were rinsed in distilled water and treated for 30 seconds with 1% acetic
acid and then with 0.1% Safranin O for 5 minutes, followed by washing with distilledwater. The cultures were treated with 50% ethanol for 10 seconds. The Permanox slides
were washed with distilled water and were mounted in an aqueous mountant.
For Alcian Blue stain, the cultures were fixed in the same way and treatedwith a hematoxylin solution for 3 minutes. After washing with tap water, the fixed
cultures were covered with 1% Alcian Blue solution in 3% acetic acid for 20 minutes,
followed by washing with tap water. The cultures were treated with 50% ethanolfor 10 seconds and mounted.
Analysis of the differentiation potential into theadipogenic lineage
After 21 days of adipogenic stimulation, the dog periosteum-derived cells andbone marrow-derived cells from monolayer cultures were rinsed with phosphatebuffer saline and fixed in 10% formalin for 30 minutes at room temperature. Afterwashing with sterile water, the fixed cultures were treated with 60% isopropanolfor 2 minutes and then incubated with freshly prepared Oil Red O working solution
for 5-10 minutes to stain lipid vacuoles. After washing with tap water, the Permanox
slides were mounted in an aqueous mountant.

RESULTS
Bone marrow and periosteum surrounding the femoral bone of five adult dogs
were used. Cells from the osteogenic layers of the periost were obtained by explant
cultures of periosteal fragments. Cells migrated from the tissue within 3-5 days after
harvest. In the same time interval of 3-5 days, periosteum-derived cells processedby digestion method and bone marrow-derived cells induced the occurrence offibroblast-like adherent cells within the culture flasks. Cellular confluence for studied
cellular types was reached within 10-14 days. All samples gave rise to primary cell
cultures with a fibroblast-like morphology, spindle-shape, and increased confluenceand next passage cultures showed adherent cells uniformly distributed over the tissue
Fiziologia - Physiology  2010.20.1 (65)

purple in color (Figure 3). It can be shown that within the same time interval, the osteogenesis process ismore advanced in periosteum-derived cells than in bone marrow-derived cells.

RESULTS
Bone marrow and periosteum surrounding the femoral bone of five adult dogs were used. Cells
periosteum-derived
differentiated bone marrowdifferentiated periosteum-derived
from the osteogenic layers of the periost were obtained by explant cultures of periosteal fragments. Cellsdifferentiatedcells (explant method) (x100)
derived cells (x200)
cells (digestion method) (x100)
RESULTS
migrated from the tissue within 3-5 days after harvest. In the same time interval of 3-5 days, periosteum-Fig 3. Osteogenic differentiation. a) Alkaline phosphatase activity
marrowand periosteum
the femoral boneof five adultdogs wereofused. Cells
derived cells Bone
processedby digestion
method surrounding
and bone marrow-derived
cells induced
the occurrence
from the adherent
osteogenic
layersof thethe culture
periostflasks.
were obtainedby explantfor
culturesperiosteal
Cellsto form a mineralized matrix is highlighted by the osteoblasts specialized functions
fibroblast-likecells
withinCellular confluencestudiedofcellular
typesfragments.
was The potential
to secretemineralization factors like calcium salts. Calcium phosphate deposits can be detected by the
migrated
thedays.
tissueAllwithin3-5 days
In thesame
time interval
3-5 days,periosteumreachedwithinfrom
10-14
samplesgaveafter
rise harvest.
to primarycell
cultureswith a offibroblast-like
Von
Kossa
techniquedeposits are stained black with silver (Figure 4).
derived spindle-shape,cells processed
digestionconfluence
method andcells induced
thecells
occurrence
ofin which phosphate
differentiated periosteum-derived
morphology,
andbyincreased
andbonenext marrow-derived
passage cultures showedadherent
differentiated bone marrowdifferentiated periosteum-derived
cells (explant
method)(x100)
derived cells (x200)
cells(digestion method) (x100)
fibroblast-likeadherent
cells
withinthecultureflasks.
Cellularconfluencefor
studiedcellulartypes
was
uniformly distributed
overthewithtissuedish withcells merely
having a fibroblast-likemorphology
culturedishcellsculture
merely having
a fibroblast-likemorphology(4).
Fig 3. Osteogenic differentiation. a) Alkaline phosphatase activity(4). reached within 10-14 days. All samples gave rise to primary cell cultures with a fibroblast-likemorphology, spindle-shape,and increased confluence and next passage cultures showed adherent
cellsto form a mineralized matrix is highlighted by the osteoblasts specialized functions
16
The potential
uniformly distributed over the tissue culture dish with cells merely having a fibroblast-likemorphology
to secretemineralization factors like calcium salts. Calcium phosphate deposits can be detected by the14
Von Kossa technique in which phosphate deposits are stained black with silver (Figure 4).
(4).
16

10

6

C1

14

8

C2
C3

12

Cell number x 10E6

Cell number x 10E6

12

C4
C5

10

differentiated periosteum-derived
differentiated periosteum-derived
differentiated bone marrowcells (explant method) (x100)
cells (digestion method) (x100)
derived cells (x100)
Fig.4. Osteogenic differentiation. b) Mineral deposits stained by the von Kossa techniqueFig.4. Osteogenic differentiation. b) Mineral deposits stained by the von Kossa techniqueIn the differentiated cultures from periosteum-derived cells, the mineral deposits showed much
C1 than the same marker in differentiated cultures from bone marrow-derived cells, which suggest
intensethatC2within the same time interval, the osseous mineralization process is faster in the first case, with theC3
specification
thatperiosteum-derived
cells obtained by explant
methodperiosteum-derived
seemed to induce themost intenseIn
differentiateddifferentiateddifferentiatedbone mineralization.
marrowC4 thcellsday(explant
method) (x100) towards
cells
method)(x100) collagen
derived(x100) can be observedof differentiationthe(digestion
osteoblastic
lineage,
I cellssynthesis
the C5
10
Osteogenic
differentiation.
b) Mineral deposits stained by the von Kossa technique
withinFig.4.
the cells(Figure5).

In the differentiated cultures from periosteum-derived cells, the mineraldeposits showed much intense than the same marker in differentiated cultures frombone marrow-derived cells, which suggest that within the same time interval, theIn the differentiatedculturesfromisperiosteum-derived
cells, withthe mineraldeposits showedosseousmineralization
process
faster in the first case,the specificationthat muchintense than the same marker in differentiated cultures from bone marrow-derived cells, which suggest
Fig. 1.Fig.
Comparison
between
total
cell
count
at
P2
of
periosteum-derived
cells(
explant
1. Comparison between total cell count at P2 of periosteum-derived cells ( explant andthat withinsame timeosseousmineralization
process
is fasterin themineralizafirst case, with thecellstheobtainedby interval,
explant themethodseemedto induce
the most
intenseand enzymatic digestion isolation methods) and bone marrow-derived cells
specification that cells thobtained by explant method seemed to induce the most intense mineralization. In
enzymatic digestion isolation methods) and bone marrow-derived cells
10 day oftowards
differentiation
towardslineage,
the osteoblasticI
the 10thtion.
day Inofthe
differentiation
the osteoblastic
collagen I lineage,
synthesis collagencan be observed
within the cells (Figure 5).
In order to characterize the cellular types that would be further used in differentiation processsynthesis can be observed within the cells (Figure 5).
towards osteogenic, chondrogenic
and adipogenic
lineages
we P2
analyzed
the degree cellsof Vimentin
Fig. 1. Comparison
between total
cell countof periosteum-derived( explant differentiated periosteum-derived differentiated periosteum-derived differentiated bone marrowtoandcharacterize
the cells
cellularthatatwould
bebonefurtherused in todif-theexpression. Vimentin is Ina orderproteinpresent
in theof types
mesenchymal
origin.
It belongs
enzymatic
digestion
isolation
methods) andmarrow-derived
cellsclass of cells (explant method) (x200)
cells (digestion method) (x200)
derived cells (x200)
intermediate filaments
of
the
cell
associated
with
both
the
nuclearandplasmamembranes.
Dog Fig.5. Osteogenic differentiation. c) Collagen I immunolocalization
ferentiation process towards osteogenic, chondrogenic and adipogenic lineages weperiosteum-derived
cells
and
bone
marrow-derived
cells
are
characterized
immunohistochemicalbytheir
In orderto characterize
the cellular
typesVimentinthat would
be further
analyzed
the degreeof Vimentin
expression.
is a proteinpresentusedin theincellsdifferentiation process
three weeks (24 days), the monolayer culture of dog periosteum-derived cells and bone
positive
expressionof Vimentin(Figure 2). and adipogenic lineages we analyzed the degree of After
towards
osteogenic,chondrogenic
Vimentinmarrow-derived
cells in chondrogenic medium showed that the cells were differentiated intoofmesenchymal
origin.
Itbelongs
to
the
class
of
intermediatefilaments
of
the
cell
chondrocyte-like
expression. Vimentin is a protein present in the cells of mesenchymal origin. It belongs to the classcells.
of The histochemical results of Alcian Blue and Safranin-O staining showed that thedifferentiated chondrocyte-like cells produced chondrocyte-specific extracellular matrix (Figures 6 andnuclearand plasma
membranes.
Dognuclear
periosteum-derived
intermediate associatedfilamentswith
ofboththethecell
associatedwithboth theand plasma membranes.
Dogdifferentiated bone marrowdifferentiated periosteum-derived differentiated periosteum-derived
7).
cells (digestion method) (x200)
derived cells (x200)
cells (explant
method) (x200)
periosteum-derived
bone marrow-derived
cells are characterized
immunohistochemicalby theircells andcells
boneandmarrow-derived
cells are characterized
immunohistochemical
byFig.5. Osteogenic differentiation. c) Collagen I immunolocalization
positive expression of Vimentin (Figure 2).
their positive expression of Vimentin (Figure 2).
Fig.5. Osteogenic differentiation. c) Collagen I immunolocalization
4
2
0

8
6
4

Explant

Digestion

Bone marrow

20

Explant

Digestion

Bone marrow

After three weeks (24 days), the monolayer culture of dog periosteum-derived cells and bonemarrow-derived cells in chondrogenic medium showed that the cells were differentiated into
chondrocyte-like cells. The histochemical results of Alcian Blue and Safranin-O staining showed that thedifferentiated chondrocyte-like cells produced chondrocyte-specific extracellular matrix (Figures 6 and7).

After three weeks (24 days), the monolayer culture of dog periosteum-derived
cells
and
bone marrow-derived cells in chondrogenic medium showed that the cells
periosteum-derived cells
bone marrow-derived cells
Fig.2. Expression of vimentin (x100)
were differentiated into chondrocyte-like cells. The histochemical results of Alcian
Blue and Safranin-O staining showed that the differentiated chondrocyte-like cells
Osteogenic differentiation was evaluated based on alkaline phosphatase presence in the cultures
produced chondrocyte-specific extracellular matrix (Figures 6 and 7).
(an early marker), the potential to form a mineralized matrix and the collagen I immunolocalization. The
mineralization of a collagencontainedcellsby extracellular matrix
represents
late markers.
Osteoblasts can beperiosteum-derived
bone
marrow-derived
cells
In monolayer culture, periosteal cells begin to form chondrocyte colonies andidentified morphologicallyby their cuboidal
appearance
by theirFig.2. Expressionof vimentin(x100)
Fig.2.
Expression andof vimentin(x100)association with newly synthesizedcontinued to expand forming a tightly organized mass of cells. Tendency to formbone matrix.
Committed osteogenic cells are histologically characterized by their positive expression ofcolonies is more evident in the cultures differentiated from periosteumOsteogenic differentiation was evaluated based on alkaline phosphatase presence in chondrocyte
the cultures
alkaline phosphatase, anOsteogenic
enzyme thatis involvedwas
in thebone matrix
mineralization.
BCIP/NBT liquid
differentiation
evaluatedbased andonalkaline
phosphatase(an early marker), the potential
to form a mineralized
matrixthe collagenI immunolocalization.
Thethan those differentiated from bone marrow-derived cells.
derivedcells
substrate system produces an insoluble nitro blue tetrazolium diformazan end product that is blue tomineralizationof a collagencontainedby extracellularmarkers. Osteoblasts Adipogenic
can be differentiation was evaluated based on production and accumulapresencein the cultures(an early
marker), the matrix
potentialrepresents
to form a latemineralized
identified morphologicallybytheircuboidalappearance
and
by
their
association
with
newly
synthesized
matrix and the collagen I immunolocalization. The mineralization of a collagention of neutral lipids in differentiated cells. Adipocytes are rounded and filled withbone matrix.
containedosteogenic
by extracellular
represents late characterizedmarkers. Osteoblasts
can beCommitted
cells matrix
are histologicallyby theirpositive expression
ofwhich might fuse to form vacuoles that can be stained by Oil Red O,
lipid droplets,
alkaline phosphatase,an enzyme thatinvolved
the boneandmatrixmineralization.
liquididentified morphologicallyby istheir
cuboidalinappearance
by theirassociation BCIP/NBT
a lipophilic
red dye (Figure 8).
substrate systemproducesan insoluble
nitro blue tetrazolium diformazan end product that is blue towith newly
synthesizedbone matrix.
Committed osteogenic cells are histologically characterized by their positive
expression of alkaline phosphatase, an enzyme that is involved in the bone matrix
mineralization. BCIP/NBT liquid substrate system produces an insoluble nitro blue
tetrazolium diformazan end product that is blue to purple in color (Figure 3). It
can be shown that within the same time interval, the osteogenesis process is more
purple in color (Figure 3). It can be shown that within the same time interval, the osteogenesis process isinperiosteum-derived
periosteum-derived
cells
bone marrow-derived
cells.
moreadvanced
advanced incells thaninthanbone inmarrow-derived
cells.
bbaaFig.6. Chondrogenic differentiation of periosteumFig.7. Chondrogenic
of boneFig.6. Chondrogenic
differentiation ofdifferentiation
periosteum-derived cells(explantmarrowmethod). Staining forderived cells (explant method). Staining for Alcian derived cells.
AlcianBlue (a) andAlcianStaining
Blue (a) and for
Safranin-O(b), (x200)
Blue (a) and Safranin-O (b), (x200)
Safranin-O (b), (x200)

In monolayer culture, periosteal cells begin to form chondrocyte colonies and continued to expand
forming a tightly organized
mass of cells. Tendency to form chondrocyte colonies is more evident in the
differentiated bone marrowcellsperiosteum-derived(x200)
cultures differentiatedderived
fromcells than those differentiated from bone marrow-derived
Fig 3. Osteogenic
differentiation. a) Alkaline phosphatase activity
cells.
The potential to form a mineralized matrix is highlighted by the osteoblasts specialized functions
Adipogenic differentiation was evaluated based on production and accumulation of neutral lipids
to secrete mineralization factors like calcium salts. Calcium phosphate deposits can be detected by the
Von Kossa The
techniquein which
phosphate
deposits are stainedwith silver(FigureindifferentiatedAdipocytes
are rounded and filledwith lipid droplets, bwhich might fuse to form
potential
to forma mineralized
matrixblack
iscells.
highlighted
by the4).osteoblastsaavacuoles
that
can
be
stained
OilCalcium
Red O, a lipophilic red
dyeChondrogenic(Figure 8). differentiation of periosteum- Fig.7. Chondrogenic differ
Fig.6.
specialized functions to secrete mineralization factors like calciumbysalts.
differentiated periosteum-derived
differentiated periosteum-derived
cells (explant method) (x100)
cells (digestion method) (x100)
Fig 3. Osteogenic differentiation. a) Alkaline phosphatase activity

phosphate deposits can be detected by the Von Kossa technique in which phosphate
deposits are stained black with silver (Figure 4).
2010.20.1(65) Fiziologia- Physiology
differentiated periosteum-derived
differentiated periosteum-derived
differentiated bone marrowcells (explant method) (x100)
cells (digestion method) (x100)
derived cells (x100)
Fig.4. Osteogenic differentiation. b) Mineral deposits stained by the von Kossa techniquecmyk

In the differentiated cultures from periosteum-derived cells, the mineral deposits showed muchintense than the same marker in differentiated cultures from bone marrow-derived cells, which suggestthat within the same time interval, the osseous mineralization process is faster in the first case, with thespecification that cells obtained by explant method seemed to induce the most intense mineralization. In
the 10th day of differentiation towards the osteoblasticb
a lineage, collagen I synthesis can be observed
within the cells (Figure 5).

derived differentiation
cells (explant
Staining
for for
AlcianFig.7. Chondrogenic
of bonemethod).
marrow-derived
cells. Staining
Alcian Bluederived(a) and cells. Staining fo
Blue (a) and Safranin-O(b),(b),(x200)
Safranin-O (b), (x200)
Safranin-O(x200)

In monolayer culture, periosteal cells begin to form chondrocyte colon
forming a tightly organized mass of cells. Tendency to form
23chondrocyte colcultures differentiated from periosteum-derived cells than those differentiatedcells.
Adipogenic differentiation was evaluated based on production and ac
in differentiated cells. Adipocytes are rounded and filled with lipid droplets
vacuoles that can be stained by Oil Red O, a lipophilic red dye (Figure 8).
cbaFig.6. Chondrogenic differentiation of periosteumderived cells (explant method). Staining for Alcian
Blue (a) and Safranin-O (b), (x200)baFig.7. Chondrogenic differentiation of bone marrowderived cells. Staining for Alcian Blue (a) andSafranin-O (b), (x200)

In monolayer culture, periosteal cells begin to form chondrocyte colonies and continued to expand
forming a tightly organized mass of cells. Tendency to form chondrocyte colonies is more evident in thecultures differentiated from periosteum-derived cells than those differentiated from bone marrow-derived
cells.
Adipogenic differentiation was evaluated based on production and accumulation of neutral lipids
in differentiated cells. Adipocytes are rounded and filled with lipid droplets, which might fuse to form
vacuoles that can be stained by Oil Red O, a lipophilic red dye (Figure 8).

differentiate towards all three lineages, thus confirming the results of other researchstudies in this field referring to other species, canine periosteum-derived cells
obtained using culture of periosteal explants, differentiate mostly towards theosteoblastic and chondrogenic lineage, and to lesser extent towards the adipogenic
lineage; the cells obtained using the digestion method of periosteal fragmentsbacpositively differentiate towards all the three lineages: osteoblastic, chondrogenic
Fig.8. Adipogenic differentiation of periosteum-derived cells (a. explant method – x200, b. digestion
method –Adipogenicx400) and bonemarrow-derived
cells (c) (x400). Staining
Oil RedO – x200, b. digesFig.8.
differentiationof periosteum-derived
cells (a. forexplant
methodand adipogenic. A possible explanation could be that the cells obtained from thetion method – x400) and bone marrow-derived cells (c) (x400). Staining for Oil Red O
Our findings indicate that there are special characteristic features in differentiation of studied cells osteogenic part of the periosteum (cambium) using the explant method areto adipogenic lineage according to isolation methods of dog periosteum-derived cells. The adipogenesis mainly precursors of the osteoblatic/chondrogenic lineage, while the periosteal cells
ability of bone marrow-derived cells is greater than that of periosteum-derived cells, especially than
Our findings
indicatebythat
theremethod.
are special characteristic features in differentiaperiosteum-derived
cells processed
explant
obtained using the digestion method could contain cambium cells contaminatedtion of studied cells to adipogenic lineage according to isolation methods of dog
with other cellular types derived from the fibrous upper layer. Periosteum is at theperiosteum-derived cells. The adipogenesis ability of bone marrow-derived cells is
DISCUSSION
boundary between the bone and the surrounding soft tissues and contains multiple
In bone fracture healing, it is important that restoration of the damaged area results in thegreater than that of periosteum-derived cells, especially than periosteum-derived
formation of new bone, having the same integrity as the surrounding bone tissue. To determine a suitable cell types (cells of the cambium layer, fibrous layer, and vascular pericytes) that could
cell source,
isolated dog
mesenchymal
stem cells from periosteum and bone marrow from the same
cellsweprocessedby explant
method.
potentially function as progenitor cells. This issue has important implications in thesubjects and compared their properties for expansion, multipotentiality and morphology.
The “conventional” mesenchymal stem cells are those obtained from bone marrow (5,6). preparation of cellular products for clinical applications.
However, there are increasing reports that mesenchymal stem cells can be isolated from various other
The round cuboidal cells are considered to be the osteo/chondroprogenitor
tissues as well (7,8,9). From periosteum were isolated and characterized periosteum-derived progenitor
DISCUSSION
cells by various
surface markers of mesenchymal stem cells (10). Mesenchymal stem cells have been cells (19, 20). According to Izumi et al. (19), alkaline phosphatase expression inassumed to be similar irrespective of their original tissue source since they all have self-renewal andIn bonefracturewith
healing,
it is importantthat restoration
the damaged
multidifferentiation
potentialcommonsurface epitopes(11,12). ofHowever,
the area
properties of the periosteum is not from fibroblasts or chondrocyte precursors, but from osteomesenchymal
cells
can be affectedby theirpreparations
not been properly
resultsstem
in theformationof new bone,havingthe same(13,14)integritywhich
as thehave
surrounding
blasts and osteoblast precursors. This suggests that the periosteal cells culture iscontrolled for in some studies.
bone tissue.
To determine
suitablecell source,
we isolated
dog mesenchymal
stemas a result enriched in alkaline phosphatase-positive progenitor cells or assumes that almost
Present
study aims
to reveal athesimilarities
and differences
betweenthe cells obtainedof canine periosteal-derived cells cultivated using explant/digestion method and canine bone marrowcells from periosteum and bone marrow from the same subjects and compared theirderived cells, in three different differentiation media. The expansion potential of dog periosteum-derived all the cells are able to differentiate into osteoblasts. By Hanada et al. (20), exist 2
properties for expansion, multipotentiality and morphology.
types of cells, alkaline phosphatase-positive and alkaline phosphatase-negative
The “conventional” mesenchymal stem cells are those obtained from bonecells present at early culture periods. These alkaline phosphatase-negative cells
marrow (5,6). However, there are increasing reports that mesenchymal stem cells
are not only contaminating fibroblasts from the outer layer of periosteum, but also
can be isolated from various other tissues as well (7,8,9). From periosteum were
undifferentiated mesenchymal progenitor cells capable of differentiating into theisolated and characterized periosteum-derived progenitor cells by various surface
osteogenic lineage.
markers of mesenchymal stem cells (10). Mesenchymal stem cells have been asFor the purpose of treating defective articular cartilage, mesenchymal stem
sumed to be similar irrespective of their original tissue source since they all havecells have been isolated from bone marrow, synovial membranes, periosteal tissue,
self-renewal and multidifferentiation potential with common surface epitopes
and umbilical cord blood (10,21,22,23). Periosteum has the potential to differentiate(11,12). However, the properties of mesenchymal stem cells can be affected byinto neocartilage. It contains a lot of progenitor cells that directly differentiated into
their preparations (13,14) which have not been properly controlled for in somechondrocytes and have a chondrogenic potential regardless of passage number
studies.
and donor age (15,24), therefore periosteum-derived cells can be used for hyaline
Present study aims to reveal the similarities and differences between the cells
cartilage treatment. In vitro chondrogenesis as a monolayer culture system was
obtained as a result of canine periosteal-derived cells cultivated using explant/digesperformed to evaluate the chondrogenesis potential of cells derived from periosteum
tion method and canine bone marrow-derived cells, in three different differentiation
and bone marrow. Histologically, all samples exhibited a cartilage matrix that stained
media. The expansion potential of dog periosteum-derived cells (digestion method)with Alcian Blue and Safranin O (Figures 6,7).
is no lower than that of bone marrow-derived cells cultivated in growth medium,The cryopreserved studied cells have been tested for their differentiation
with the specification that explant method provides a relatively reduced amount ofcapacity. In the present study, we demonstrated that dog periosteum-derived cells
periosteal cells than the enzymatic method, as demonstrated in our previous study
can be differentiated into mesodermal cell types, such as osteoblasts, chondrocytes(4). Cell types used in present study have a remarkable viability and proliferative
and adipocytes, just like dog bone marrow-derived cells and could be used in placecapacity. No obvious differences in morphology were noted between the studiedof bone marrow-derived cells which are widely used, as a useful source of bonecells. However, cells committed to differentiation have been found to have lower
regeneration. According to other studies, the periosteum-derived cells osteogenic
proliferative potential than cells cultivated in growth medium, thus confirming thepotential persists even after prolonged cultivation in osteogenic medium. Longer
results revealed in other similar studies (15). From the cells induced toward difculture periods (3–4 months) can be performed before final differentiation isferentiation, we concluded that explant method-obtained periosteal cells had theobtained, making these cultures suitable for long-term in vitro evaluation ofhighest proliferation rate, with an increased tendency to form cellular aggregatesbiomaterials (25,26). Moreover, sometimes harvesting periosteum-derived cells
and colonies. This tendency appears in osteogenic and chondrogenic differentiation
is easier and less invasive than yielding bone marrow-derived cells such in bone
(Figures 3,6). Thus, the use of periosteal cells may shorten the cell culture period,
reconstructive surgery, general dental medicine -regeneration of alveolar bone inthereby reducing both cost and the risk of contamination.
periodontitis and implant dentistry. Therefore, periosteum-derived cells could be
Periosteum-derived cell preparations can form cartilage and bone in vitro andcandidates for tissue engineering and cell-based therapeutic strategies.
in vivo (16,17) as well as adipocytes in vitro (18) (digestion method). The present
Acknowledgements
study focuses on characteristic features regarding the differentiation potential ofThis work was supported by PNCDI-II – Parteneriate, project number 41canine periosteum-derived cells, depending on the method chosen for processing
091/2007 and by CNCSIS –UEFISCSU, project number PNII – IDEI 130/2008of the harvested periosteal fragments. While the dog bone marrow derived-cells

24

cmyk

Fiziologia - Physiology  2010.20.1 (65)

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ANALIZA COMPARATIVA A POTENTIALULUI DE DIFERENTIEREA CELULELOR DERIVATE DIN PERIOSTUL SI MADUVA OSOASACANINA SPRE LINIILE OSTEOGENICA, CONDROGENICA SI
ADIPOGENICA
REZUMAT

Scopul acestui studiu a fost investigarea posibilitatii celulelor periostale si a celor derivate din maduva osoasa canina de a se diferentia spre liniile osteogenica, condrogenica
si adipogenica. Rezultatul acestui experiment doreste sa evidentieze asemanarile cat si deosebirile intre celulele obtinute in urma cultivarii celulelor periostale si a celulelor
derivate din maduva osoasa in trei medii variate de diferentiere. In timp ce celulele derivate din maduva osoasa canina se diferentiaza spre cele trei linii celulare, confirmand
rezultatele altor cercetari in domeniu cu aplicatii pe alte specii, celulele periostale canine se diferentiaza diferit, in functie de metoda aleasa pentru prelucrarea fragmentelorde periost recoltat. Astfel, celulele periostale obtinute prin cultivarea explantelor de periost se diferentiaza cu precadere spre linia osteoblastica si condrogenica si mai slab
spre linia adipogenica, in timp ce celulele obtinute prin metoda digestiei enzimatice a fragmentelor de periost se diferentiaza pozitiv spre toate cele trei linii celulare: osteo-,
condro- si adipogenica. In plus, s-a putut demonstra ca celulele crioprezervate prezinta capacitate de diferentiere osteogenica, condrogenica si adipogenica.
Cuvinte cheie: celule derivate din periost, celule derivate din maduva osoasa, metode de izolare, potential de diferentiere

2010.20.1(65)  Fiziologia - Physiology

cmyk

25

ARISTOLOCHIA CLEMATITIS IN MEDICINE: THE GOOD
AND THE BADMIRABELA I. CRISTEA1, ALEXANDRA T. GRUIA1, SIMONA ANGHEL1,2, ELENA GAI1,CALIN A. TATU3, VIRGIL PAUNESCU2
1
Regional Center of Immunology of Transplant, Emergency County Hospital Timisoara
2
Department of Physiology and Immunology, University of Medicine and Pharmacy Victor Babes Timisoara
3
Department of Biology, University of Medicine and Pharmacy Victor Babes Timisoara

ABSTRACT
Aristolochia clematitis, member of the Aristolochiaceae family is a medicinal plant used from time immemorial as a natural remedy, in obstetrics, in the treatment of snake
bites, therapy of arthritis, gout, rheumatism, and festering wounds. Only recently Aristolochia plant use as a natural or pharmacologic remedy was banned by the Food andDrug Administration (FDA), because it contains aristolochic acids, carcinogenic and nephrotoxic compounds, considered to be responsible for the development of Aristolochic
Acid Nephropathy (AAN) (formerly known as the Chinese Herbs Nephropathy (CHN)) and induction of upper urinary tract tumors. An interest correlation has also been madebetween Aristolochia clematitis and the etiology of Balkan Endemic Nephropathy (BEN).
The purpose of this review is to provide more insight into the role of Aristolochia clematitis as factor or cofactor in the above-mentioned diseases and to explain why theplant has been blacklisted by the current scientific community after centuries of being considered a powerful remedy in the natural pharmacopoeia.
Key words: Aristolochia clematitis, Balkan Endemic Nephropathy, carcinogenesis, nephrotoxicity

INTRODUCTION
Aristolochia clematitis (Figure 1a, b), member of the Aristolochiaceae family,
popularly named in Romania „Mărul Lupului”, „Curcubetica”, „Buruiana de remf”,
is a medicinal plant that has a very long history of medicinal use, though it israrely used by herbalists nowadays. It is an aromatic tonic herb that stimulates theuterus, reduces inflammation, controls bacterial infections, and promotes woundhealing (1).

Fig. 1b. Aristolochia clematitis fruit

Herbal drugs derived from Aristolochia species have been known since
antiquity and were used in the treatment of skin infections, and in the treatmentof snake bites in ancient Egypt and India. Contemporary traditional medicine has
used Aristolochia plant extracts for the therapy of arthritis, gout, and rheumatism(2). Externally, various parts of the plant are used in the treatment of slow-healing
cuts, eczema, infected toe and finger nails, but internal consumption can cause
uterine bleeding and especially damage to the kidneys (1).
The plant contains a complex of acids so called “aristolochic acids”, which
stimulate white blood cell activity and speeds the healing of wounds if it is usedin the right concentration (1). Aristolochic acids (AA) are a mixture of structurally
related nitrophenanthrene carboxylic acid derivatives (Figure 2) found primarily
in the genus Aristolochia and Asarum (3).
Fig. 1a. Aristolochia clematitis leaves and flowers

Received February 2010. Accepted March 2010. Address for correspondence: Mirabela Cristea, Regional Center of Immunology of Transplant, Emergency County Hospital Timisoara, Iosif
Bulbuca No. 10 Street, e-mail: bunceanum@yahoo.com

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Fiziologia - Physiology  2010.20.1 (65)

O

O
O

OH
NO2

O

O

OH
NO2

O

OCH3

AAI

AA II
Fig. 2. AA I and AAII chemical structure

Consumption of products containing aristolochic acid has been associated withpermanent kidney damage, sometimes resulting in irreversible kidney failure that has
required kidney dialysis or kidney transplantation. In addition, some patients havedeveloped certain types of cancers, most often occurring in the urinary tract.
AAN, a rapidly progressive interstitial nephropathy leading to end-stage renal
disease and urothelial malignancy, was initially reported in a Belgian group in 1992
of more than 100 patients after the intake of slimming pills containing a Chinese herb,
Aristolochia fangchi (4). This was an accidental exposure due to a mistake that occured
during the preparation of the pills, when Aristolochia roots and leaves were inadvertenlyused to replace a rather inocuous plant (Stephania tetrandra) that was supposed to be
included in the pills. Subsequently, the disease has been described in other European
countries as well, also in patients that have taken similar pills.
BEN is a chronic tubulointerstitial disease occuring almost exclusively infarming villages situated in valleys of tributaries of the Danube River, in several
countries of the Balkan Peninsula (Bosnia, Bulgaria, Croatia, Romania and Serbia).
This geographic distribution has been constant since the disease was first describedin the late 1950s. The most significant epidemiologic features of BEN are its focal
occurrence in certain villages, a familial but not inherited pattern of disease, initial
manifestation after residence in an endemic village for 15 years or more, and strong
association with urothelial cancers, similar to those encountered in AAN (5).
Aristolochic Acid Nephropathy (AAN)
Initially called Chinese-herbs nephropathy, AAN appears to be the dramaticconsequence of the substitution in the slimming pills originating in China of Stephania tetrandra with Aristolochia fangchi, the latter rich in nephrotoxic aristolochic
acids; the substitution occurred because both herbs have very similar common
names in Pin Yin (Han Fang Ji and Guang Fang Ji) (4). This two plants belong to
the same therapeutic ”Fang Ji” family in traditional Chinese medicine and generally
their common Pin Yin name is being used (4).
Nephrotoxicity manifested by progressive atrophy of renal proximal tubules
and development of a characteristic form of interstitial fibrosis involving the outerrenal cortex and progressing toward the medulla. The process tends to spare glomeruli and is associated with less inflammation than most other types of interstitialnephritides. Importantly, the syndrome was associated with a high prevalence ofurothelial cell carcinomas, which often occurred years after the onset of chronic
renal disease. Moreover, in contrast with most other urothelial cell tumors, cancers
associated with AAN tend to develop in the upper urinary tract (6).
Because aristolochic acids were identified in the slimming pills taken by thepatients with AAN, their were thought to be the main culprit of both the renal failure
and the cancers. In order to demonstrate their toxicity several in vitro and in vivo experiments ensued. Some in vitro experiments assesed the cytotoxicity of Aristolochia plants’
extracts on different cell types, like human renal epithelial HK-2 cell line, mesenchymal
stem cells, with different concentrations of aristolochic acids and also extracts from2010.20.1(65)  Fiziologia - Physiology

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different parts of the plant (7). The AA toxicity was evaluated also in vivo, on rabbits,
rats and mice, reproducing to a large extent the kidney pathology and tumorigenesis
encountered in patients with AAN. These studies have demontrated beyond any doubt
the central role of AAs in the pathogenesis of AAN/CHN (8).
Balkan endemic nephropathy (BEN)
BEN, a chronic tubulointerstitial fibrosis with slow progression to end-stage
renal disease and urothelial malignancy (4), is so named because it is presently
known to affect only rural inhabitants of Romania, Bulgaria, Bosnia, Serbia, Croatia,
in villages located along rivers of the Danube River basin (Figure 3) (5,8,9).

Fig. 3. Map showing the geographical distribution of the known Balkan nephropathy areas (9)About 25,000 people suffer or are suspected of having BEN, whereas the total
number of people at risk in this countries may exceed 100,000 (4). The adults intheir fourth or fifth decade of life are usually affected by the disease, with end-stage
renal failure developing by their sixth decade (8). The most important epidemiologic
features of BEN include its focal occurrence in certain farming villages, a familial but
not inherited pattern of disease, and strong association with upper urinary tract
urothelial malignancies (5).
This disease is relatively asymptomatic and the patient becomes symptomatic
only when the advanced renal failure becomes manifest. The clinical and paraclinicalfeatures are those of chronic renal failure, including severe anemia, polyuria, polydipsia,
nicturia, abnormalities on urinalysis, small and shrunken kidneys, and rarely hypertension (8). Upper urinary tract cancers are also frequently diagnosed in BEN.
The etiology of BEN remains obscure despite all the research and studies
conducted over the past 50 years (5). However, based on the evidence accumulated
during the last several decades, it can be stated that BEN is an environmentallyinduced disease. There are three current theories that attempt to explain the environmental cause of BEN: the aristolochic acid hypothesis, which assumes that thedisease is produced by chronic intoxication with Aristolochia clematitis seeds; themycotoxin hypothesis, which claims ochratoxin A to be the main culprit of BEN; thePliocene lignite hypothesis, which proposes that the disease is caused by long-term
exposure to polycyclic aromatic hydrocarbons and other toxic organic compounds
leaching into the well and spring drinking water from low-rank coals found in thevicinity of the endemic settlements. As not all the people living in the endemic
villages develop BEN it is logically assumed that a genetic susceptibility is also
involved in disease emergence (8). The gene-environment interaction is translated
into peculiar xenobiotic substance metabolism (controlled by cytochrome P450 andother enzymes) that increases the risk to develop BEN only in those people that bearcertain gene variants coding for those detoxification enzymes.
The „AA hypothesis”in BEN was formulated for the first time in 1969 by Ivic(14), based on the observation that Aristolochia clematitis plant occurs with higher
frequency in BEN endemic compared to nonendemic regions, and it often grows27

as a weed in fields cultivated with wheat. Ivic formulated the idea that during theannual harvest the wheat grain is mixed with Aristolochia seeds (which contain atenfold amount of Aas compared to the leaves) and as a consequence the breadmade from the contaminated flour is tainted with small amounts of AAs. Thus,
residents of the endemic region who ingest this kind of bread may be exposed,
over time, to cummulative toxic amounts of AAs (5).
Although the flour contamination hypothesis could be standing for BEN
countries like Croatia or Serbia, it is contradicted by the hilly topography of theendemic, as well as nonendemic, villages in Romania, where wheat cultivation
has been very limited. Other crop plants, like staple corn, are much more frequently
cultivated here but to what extent AA can contaminate the corn seeds and enter thehuman food chain are open questions. Another topic of discontent is the medicinaluse of A. clematitis, which has been consistently reported only for Romania although
without any significant difference between endemic and nonendemic households,
but not for the other BEN afflicted countries.
Analyzing the description of the clinical, morphological and biological featuresof AAN and BEN striking similarities can be observed. Another argument for therole of AAs as etiologic factors/cofactors in both types of nephropaties is the recent
detection of AA-DNA adducts in kidney and urinary tract tissues from patients with
AAN, and also in the renal tissues of some patients with BEN; such adducts wereclaimed to be absent in patients with chronic renal diseases or with upper urinary
tract malignancies from non-endemic areas (7-12)
Based on this kind of information, in 2001, the Food and Drug Administration(FDA) issued warnings and an import alert that herbal products are unsafe if they
contain or are suspected to contain aristolochic acids. Despite the action of the
FDA and the effort of many countries, 19 products containing AAs and 95 products
suspected to contain AAs were for sale on the Web in 2003 (13) and are currently
sold in many open markets in Romania.
The International Agency for Research on Cancer also classifies products
containing Aristolochia species as group 1 human carcinogens.

CONCLUSIONS
Considered an "aristocratic" plant in ancient times (hence the name), Aristolochia was used in folkloric medicine to succesfully treat various ailments, from snake
bites to cancer. It is still used as a medicinal plant in several rural places in Eastern
Europe and elsewhere. For instance, in the Balkan Peninsula, dried and powdered
leaves are mixed with pig fat and used by some people for topical application forarthritic pain or bruises; leaf decoction (usually one or two leaves boiled in one
liter of water) is also used by women for vaginal bath, however, such uses would
imply very little or insignificant exposure to AA. Topical absorption of traces of AA
might occur though, sufficient to accumulate for detection in tumours, but extensive
GC-MS urinalysis of samples from Romanian nephropathy villages by one of us (CT)has failed to find any trace of AA. Infrequent use of such A. clematitis preparations

might in part be responsible for the negative results.
In spite of being declared "bad" by the local health authorities in many
countries, in the folkloric wisdom Aristolochia has always been considered to be a"good" plant, provided several precautions were taken (i.e, only leaves and roots
collected during the flowering season and in very small amounts should be used).
Any rare intoxications with long term consequences like kidney damage or cancer
may have emerged due to improper administration and/or dosage, but this is thecase with most pharmaceutical drugs currently found in the drugstores.
REFERENCES
1. Steenkamp V, Stewart MJ, Nephrotoxicity Associated With Exposure to Plant
Toxins, With Particular Reference to Africa, Ther Drug Monit 2005;27:270–277
2. Stiborova´ M, Frei E , Arltc Volker M., Schmeiser Heinz H, Metabolic activation ofcarcinogenic aristolochic acid, a risk factor for Balkan endemic nephropathy, Mutation
Research 2008, 658, 55–67
3. Chan W, Lee KC, Liu N and Cai ZW, A sensitivity enhanced high performanceliquid chromatography fluorescence method for the detection of nephrotoxic andcarcinogenic aristolochic acid in herbal medicines. Journal of Chromatography A,
2007; 1164: 113–119.
4. Debelle FD, Vanherweghem J-L, Nortier JL, Aristolochic acid nephropathy: A
worldwide problem, Kidney International advance online publication, 16 April 2008
5. Grollman A P , Shibutani S, Moriya M, Miller F, Wu L, Moll U, Suzuki N, Fernandes A,
Rosenquist T, Medverec Z, Jakovina K, Brdar B, Slade N, Turesky R J, Goodenough
A K, Rieger R, Vukelić M, Jelaković B, Aristolochic acid and the etiology of endemic(Balkan) nephropathy, PNAS , July 17, 2007,vol.104, no. 29
6. Shibutani S, Dong H, Suzuki N, Ueda S, Miller F, Grollman A P, Selective
Toxicity of Aristolochic Acids I and II, Drug Metabolism And Disposition, 2007,
35(7):1217–1222
7. Wen Y-J, Su T, Tang J-W, Zhang C-Y, Wang X, Cai S-Q, Li X-M, Cytotoxicity of
Phenanthrenes Extracted from Aristolochia contorta in Human Proximal Tubular
Epithelial Cell Line, Nephron Exp Nephrol 2006; 103:e95-e102
8. Cosyns JP, Dehoux J-P, Guiot Y, Goebbels R-M, Robert AR, Bernard AM, vanYpersele de Strihou C, Chronic aristolochic acid toxicity in rabbits: A model of Chinese
herbs Nephropathy?, Kidney International, 2001,Vol.59 , 2164-2173
9. Stefanovic V, Toncheva D, Atanasova S, Polenakovic M, Etiology of Balkan
Endemic Nephropathy and Associated Urothelial Cancer,American Journal of
Nephrology, 2006,26:1–11
10. Orem W H, Tatu C A, Lerch H E III, Maharaj S V M, Pavlovic N, Paunescu V, Dumitrascu V, Identification and Environmental Significance of the Organic Compounds in
Water Supplies associated with a Balkan Endemic Nephropathy Region in Romania,
Journal of Environmental Health Research,2004, Volume 3, Issue 2,
11. de Jonge H, Vanrenterghem Y, Aristolochic acid: the common culprit of Chinese
herbs nephropathy and Balkan endemic nephropathy, Nephrology Dialysis Transplantation, 2008, 23: 39–41
12. Hranjec T, Kovac A, Kos J et al. Endemic nephropathy: the case for chronic
poisoning by aristolochia, Croat Med J , 2005, 46:116-125
13. Nortier JL, Martinez MC, Schmeiser HH, Arlt VM, Bieler CA, etein M, Depierreux
MF, De Pauw L, Abramowicz D, Vereerstraeten P, Vanherweghem JL: Urothelial
carcinoma associated with the use of a Chinese herb (Aristolochia fangchi), N Engl
J Med. 2000; Jun 8;342(23):1686-92
14. Ivic M. Etiology of endemic nephropathy, Lijec Vjesn, 1969; 91: 1273–1281

ARISTOLOCHIA CLEMATITIS IN MEDICINA: BENEFICII SI
EFECTE ADVERSEREZUMAT

Aristolochia clematitis, membra a familiei Aristolochiaceae este o planta medicinala folosita din timpuri imemoriale ca remediu natural in afectiunile obstetrice, in tratamentul muscaturilor de sarpe, terapia artritei, gutei, reumatismului si vindecarea ranilor. Folosirea plantei Aristolochia ca remediu natural sau farmacologic a fost doarrecent interzisa de Food and Drug Administration (FDA), deoarece contine acizi aristolohici, compusi carcinogenici si nefrotoxici, care sunt considerati a fi raspunzatori dedezvoltarea nefropatiei aristolohice (AAN) (cunoscuta anterior ca nefropatia plantelor chinizesti (CHN)) si de inducerea tumorilor de tract urinar superior. De asemenea, afost observata o corelatie interesanta intre Aristolochia clematitis si etiologia Nefropatiei Endemice Balcanice (NEB).
Scopul acestui studiu este de a furniza mai multe date despre rolul Aristolochia clematitis ca factor si cofactor in afectiunile amintite anterior si de a explica de ce aceasta
planta a fost recent introdusa pe „lista neagra” de catre comunitatea stiintifica, dupa secole in care a fost considerata un remediu important al farmacopeei naturale.
Cuvinte cheie: Nefropatie Endemica Balcanica, carcinogeneza, nefrotoxicitate

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Fiziologia - Physiology  2010.20.1 (65)BETA-ADRENERGIC STIMULATION AND PULMONARYFUNCTION MODULATION IN ASTHMA AND CHRONICOBSTRUCTIVE PULMONARY DISEASECAMELIA PESCARU1, VOICU TUDORACHE2, CRISTIAN OANCEA2
1
GlaxoSmithKline Romania
2
Victor Babes University of Medicine and Pharmacy, Timisoara, Romania

ABSTRACT
Adrenergic receptors have a wide distribution in the human body. There has been a progression in the discovery of their various subtypes, at least nine such receptors having
been described so far. The message stimulation and translation of these receptors is extremely complex, going beyond the classical description of the second-messenger
activation (cyclic adenylmonophosphate – cAMP); there is a complex chain of events which involves the participation of multiple protein isoforms/subunits (G protein,adenyl cyclase, phosphokinase, etc.) as well as their blockers. If the possible genotypic differences of β2 receptors are also taken into consideration, then it would be possible
to predict the different clinical responses to therapy1. On the other hand, the condition of the patient, whether aggravated or stable, may also influence the response to treatment. Bronchial asthma and chronic obstructive pulmonary disease (COPD) offer a wide possibility of using short- or long-term-acting β-antagonists. Their administrationin association with inhaled corticosteroids (ICS) enhances both their effectiveness and their safety.
Key words: β2 receptors, stimulation, asthma, COPD

ELEMENTS OF PHARMACOLOGY OF
Β-RECEPTORS
Pharmacological involvement in the airways is possible due to the receptors
existing in the smooth muscles of the airways, in the secretory cells, in the bronchial epithelium and in the bronchial and pulmonary vessels. The lungs containboth sympathetic (adrenergic) and parasympathetic (cholinergic) receptors; theirnames come from their respective neurotransmitter: norepinephrine (also known
as adrenalin), and acetylcholine. As cholinergic receptors can be detected both in
the neuromuscular junction and in the parasympathetic lymph nodes, the term
muscarinic agent refers only to the cholinergic receptors acting in the neuromuscularjunction. Each type of receptor may have stimulating agent, the so-called agonists,or, on the contrary, inhibiting agents, called blockers. In their turn, adrenergic
receptors have been divided into two main groups, α and β, although at least ninedistinct subtypes have been decoded based on their respective genes: α1A, α1B, α1D,
α2A/D, α2B, α2C, β1, β2, β3.
Thus, α-adrenergic stimulation generates vasoconstriction and subsequently
increases blood pressure, β-adrenergic stimulation increases heart rate and inotropism, causes bronchodilatation, stimulates mucociliary activity and induces a mild
inhibition of inflammatory mediator release. Bronchodilatation, due to β-adrenergic
stimulation, is the desired effect in the therapy of obstructive bronchial syndromes
consequently the present study focuses only on the modulation of β2-receptor
function.
Both α and β receptors have to be linked to/with G proteins. There are different
forms of α, β and γ subunits, generating a great heterogeneity of G proteins involvedin the signal-translation mechanism. Gs and Gi proteins stimulate and, respectively,
inhibit adenylate cyclase, modulating cAMP concentration. Subsequently, a rise in
the cAMP induces the activation of cAMP-dependent proteinkinase cascade. The
finding that Gs can also activate membrane C++ channels showed that G protein cancontrol several effectors. Other G proteins, such as Gq, Go, Gpic, associate receptors
with C phospholipase activation or Gk, which is connected with the K+ channel. The

activity of another enzyme, MLCK (Myosin-Light-Chain-Kinase), is essential for theactivation of actomyosin interaction. MLCK activity is mostly controlled by calmodulin intracellular Ca++. Airway relaxation modulators may alter MLCK phosphorilation
by regulating certain phosphoproteinphosphatase (1) (Fig. 1) (3,4).

Fig. 1. The relaxation of the smooth muscle is determined by the association with anagonist such as adrenalin or salbutamol to the β receptor. The association takes place in the receptor’sGs component which will stimulate adenyl cyclase, inducing the transformation of adenosine
triphosphate (ATP) into adenosine monophosphate (cAMP). An increase in the cAMP concentration will activate A protein kinase (APK) which will phosphorilate MLCK. The latter will decrease
its association with calcium/calmodulin. Besides, inositol triphosphate receptors (IP3R) from theendoplasmic reticulum (ER) are phosphorilated by the APK. This will, in turn, lead to a decrease incalcium release from ER in the cytoplasm. These two pathways will induce the depolarization andrelaxation of the smooth muscle cell.

Thus, up to the moment of bronchial relaxation, there is a cascade of events
involving multiple genotypic adenyl cyclase (AC), protein kinase (PK) and phosphodiesterase (PD) variants, as well as various blockers (scaffolds) (Fig. 2) (1). If all these
are also associated with possible genotypic differences (substitution of Arg with Gly
in the position B-16) of β2 receptors, this could predict different clinical responses

Received Fabruary 2010. Accepted March 2010. Address for correspondence: Dr. Camelia Pescaru, GlaxoSmithKline Romania, e-mail: corina.c.pescaru@gsk.com
2010.20.1(65)  Fiziologia - Physiology

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29

to treatment. On the other hand, response to treatment can also be influenced by
the condition of the patient, whether aggravated or stable.

Fig. 2. The translation of β2 adrenergic receptor stimulation is mediated by a complex
cascade of events which involves the participation of multiple G, AC, PK and PDE protein isoforms/
subunits, as well as of their scaffolds. Thus, (a) the interaction of the agonist with the β2 receptor(β2-AR) in the bronchial muscle cell membrane triggers the release of the α-subunit-stimulating
G protein (Gs) from an αβγ heterotrimetric complex. Subsequently, the released Gsα will boost theactivity of one or more AC isoforms, resulting in the intensification of cAMP formation from ATP. cAMP
will be associated with the PK (A) regulating subunit which, by target protein phosphorilation, will
induce cell bronchodilatation response. (b) K+ channels may also be targets for the PKA, opening
after phosphorilation and producing a flow of K+, which will induce a diminishing of the excitability
and the subsequent bronchial relaxation. The opening of the K+ channels may also take place throughthe direct interaction of Gs with the respective channels, independent from cAMP and PKA. (c)The β2-AR agonist couple may induce an increase in the cAMP concentration triggering bronchial
relaxation and PKG activation. (d, e) Still, the cascade initiated by β2-AR linking may follow thepathway of tyrosine kinase activation (Src), either through the Giα or the Gsα subunit, leading to theformation of Ras, Raf-1 or MKK-1 (mitogen-activated protein kinase linase) and ERK (extracellularsignal-regulated kinase) activation; or, independent from PKA, cAMP activation can, through Epac
(exchange proteins directly activated) lead to the same result (bronchodilatation) via Rap-1.

There is an extremely varied range of β-adrenergic agonist agents which
differ through their affinity to a certain subtype of receptor (α, β1, β2, etc), andthrough their pharmacodynamic properties (onset of action, peak intensity, lengthof action, etc). A general classification is also the one that delimits β-agonists toultrashort-acting agents (epinephrine, isoproterenol, isoetharine), short-acting
agents (metaproterenol, terbutaline, salbutamol, pirbuterol), and long-acting
agents (salmeterol, fenoterol).
The former, being all unspecific β2 catecholamines, had a high cardiac adverse
potential (tachiarrhythmias, increased blood pressure). Due to the strong effect of
α1 and of their vassoconstrictive effect, they are only used as ingredients in nasal
decongestants or to limit severe episodes (anaphylaxis, croup).
Short-acting β-agonists (4-6 hours) also lack 2 specificity (with the exceptionof levalbuterol, the R-isometric form of albuterol, which has β2 affinity), have been
replaced with long-acting β-agonists (~ 12 hours), which are also β2 specific.
The latter refer to salmeterol, which was introduced in 1994 and to formoterol,
introduced in 2001. Both drugs have a duration of action up to 12 hours, only
differing through the onset of their action (~ 20 minutes for salmeterol, and only
5 – 15 minutes for formoterol), and through the rapidity with which they reach
their peak effect (faster for formoterol, similar to that of salbutamol). On the other
hand, it should be taken into account the fact that salmeterol is a partial agonist,
while formoterol is a full-agonist, with greater relaxing effect, but from the point ofview of β2/ β1 selectivity = 85,000/400 = samleterol/formoterol (5).

BENEFICIAL EFFECTS OF Β-AGONISTSThe competition for the validation of the best therapeutic conduct started
between short-acting β-agonists (i.e. salbutamol) and long-acting β-agonists (i.e.
salmeterol); later on, the competition continued between long-acting β-agonists
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and combination drugs [inhaled corticosteroids (ICS) + long-acting β-agonists].
The results showed that salmeterol inhaled twice a day is more effective
than salbutamol inhaled four times a day in lessening the symptoms of moderate
bronchial asthma patients requiring maintenance therapy. There was no worseningof asthma control after using salmeterol for three months. At the same time, thepulmonary function (AUC greater for FEV1) was improved as compared to placebo
or salbutamol (6).
Both salmeterol (study conducted on 411 patients with COPD) (5) and formoterol (study conducted on 780 patients with COPD) (7) are better than ipratropium
in producing constant long-term bronchodilatation (12 hours). In a 21-week study,
the association of salmeterol with ICS resulted in a greater improvement of thepulmonary function than doubling the ICS dosage (p=0.001).
The association of a long-acting β-agonist is recommended when an average
dose of ICS is not enough to control symptoms.
The bronchodilating effect of salmeterol remains similar in time. There isno indication of tachyphylaxis (p<0.001 vs. placebo) (9). There was a direct correlation between the deterioration of the pulmonary function (assessed by FEV1or the degree of dyspnea, BDI) and the quality of life (determined by the SGRQ
questionnaire). Bronchodilators, obviously influencing FEV1 in asthma and lessin COPD, will improve the patient’s health (10). Seretide offers instant relief fromthe very beginning and a very good long-term effect (11). The quality of life will
also be improved indirectly if we take into consideration a definite decrease of
exacerbations (12,13).
Several studies have shown that combined therapy is more effective thaneach drug taken separately, having the advantage of easier administration andgreater compliance (14,15).
Still, as formoterol has a faster onset than salmeterol, is preferred both in rescue therapy and in controller therapy when used in combined therapy (budesonide+ formoterol). Both components, administered“in need”, ensure a higher protection
against severe exacerbations in patients receiving combined maintenance therapy,
thus improving low-medication asthma control (16,17).
Other beneficial effects have been noticed besides bronchodilation:
1. hyperinflation decreases as a direct consequence of bronchodilation (18);
2. mucociliary clearance improves (19,20);
3. various β-agonists increase muscle mass and strength, lessen fatigue,
showing potential for improved skeletal (and respiratory) muscle fatigue (21);
4. possible antiinflammatory action by mastocyte stabilization;
5. decreased plasma exudation in the airways by closing the inter-endothelialcell spaces in the post-capillary venules (22);
6. bacterial adherence decreases in bronchial epithelial cells (23).

ADVERSE EFFECTS
β-agonists may induce various adverse effects: cardiac (tachiarrhythmias,
palpitations, lengthening of QT), muscular (tremors, cramps), central nervous(distress, dizziness, anxiety, nausea), metabolic (hypokalemia, hyperglycemia),
respiratory (tachyphylaxis, loss of bronchoprotection, bronchospasm, hypoxemia).
Association of teophylines increases cardiovascular risk. There are two strategies
aimed at diminishing adverse effects and increasing pulmonary effectiveness: using
inhaled drugs, and including the drugs in the so-called combined medication.
The reason for using combined medication (ICS + inhaled β-agonists) is
supported by an existing mutual amplification relation (synergy) of using corticosteroids and β2-agonists:- β2-agonists: activate steroid receptors, increase the number of steroid
receptors, prevent the down regulation of these receptors, their overall effect being
Fiziologia - Physiology  2010.20.1 (65)an amplification of the steroid effect;- steroids: increase β2-receptor gene transcription, facilitate the association of β2 receptors with adenyl cyclase, their overall effect being an increased
desensitization of β2 receptors.
There has been a certain reserve towards these products due to the fact thatthese agents are systematically absorbed in the lungs and reach the heart without
being metabolized in the liver (“first pass”). Even “selective” β2-agonists may increase inotropic and chronotropic response, facilitating atrial and ventricular ectopy(24,25). As shown above, the administration of inhaled substances significantly
diminished this risk, and the fact that long-acting drugs are also β2-selective seems
to have alleviated all fears.
However, the SMART study (Salmeterol Multicenter Asthma Research Trial)
has reactivated suspicions. The trial was aimed at assessing salmeterol safety inbronchial asthma patients; it was a double-blind randomized study lasting 28
weeks that included 26,355 patients (71% Caucasians, 18 % African-Americans
and 8% Hispanics) who received either salmeterol or placebo besides their usual
medication (26).
The death rate was greater (13 out of 13,176) in the group that received
salmeterol vs. the placebo group (3 out of 13,179), i.e., 0.10% vs. 0.02%, which led
to the study being prematurely discontinued. However, statistically, the incidenceof primary events was significantly greater in the African-Americans who received
salmeterol vs. the placebo group (RR=4.1)
Patients who reported using ICS at the beginning of the trial had an occurrence
rate of key primary and secondary events similar for both salmeterol and placebogroups (RR=1.2). Patients who did not report using ICS at the beginning of thestudy, the asthma-related death rate or the development of life-threatening eventswas significantly higher in the salmeterol group vs. the placebo group (RR=1.6).
Whether considering the asthma-related death rate or the development of lifethreatening events, differences were even greater in the African-American subgroup
who did not use ICS (RR=5.61).
A careful analysis of the SMART data did not yield any clear explanation ofthese results. Still, possible explanations point rather to a genetic predisposition, to
ICS neutralization, or to patient-related factors such as late diagnosis or inconsistent
medication, than to a pharmacological effect. Consequently, the GINA guidelines
maintain that “long-acting β2-agonists are not a substitute for inhaled or oral
glucocorticosteroids, and should only be used in combination with an appropriate
dose of inhaled glucocorticosteroid as determined by a physician (27).”
The SMART results are similar to those of another trial, Salmeterol Nationwide
Surveillance (SNS) that was conducted for 16 weeks in 1990, and included 25,180
asthma patients from England. SNA showed that the incidence of asthma- andrespiratory-related deaths, although statistically insignificant, was greater insalmeterol patients (12/16787) vs. salbutamol patients (2/8393) combined with
their usual asthma treatment (28).
In 2004, Salpeter (29) published a meta-analysis conducted on 19 studies. His conclusion was that in long-term studies, the use of β2-agonists was
associated with the RR increase of cardiovascular adverse reactions vs. placebo(99=2.45, 95% CI, 1.59, 4.05, p=0.00001). The most common adverse reaction was sinusal tachycardia, which was significantly increased by β2-agonists
vs. placebo (RR=3.06, 95% CI, 1.7, 5.5), while the combined incidence of otheradverse effects (ventricular tachycardia, atrial fibrilation, syncope, cardiac failure,
myocardial infarction, heart arrest, sudden death)was not statistically significant(RR=1.61, 95% CI, 0.76, 3.42).
But the problem with this meta-analysis is both that it lacks homogeneity(it included both asthma and COPD) and that it lacks clarity: we do not know thenumber of persons that also received ICS, nor the number of patients that received
2010.20.1(65)  Fiziologia - Physiology

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short-acting β-agonists, long-acting β-agonists or combined therapy (29).
Researching MEDLINE, EMBASE, CINAHL and Cochrane, Gustavo Rodrigo
identified 27 studies including 8,400 patients, dealing with the safe use of long-acting
β-agonists in stable COPD. The analysis of the studies that reported deaths did not show
any significant differences between the patients who used long-acting β-agonists andthose who received placebo (RR=0.90; 95% CI, 0.76 to 1.06; p=0.20).
The study represents a turning point in the traditional view according to which
the use of long-acting β-agonists would represent a risk for patients with COPD
associated with cardiovascular disease (30).
Still, precaution should be taken in using short-acting β-inhalers (as MDI) in
smoker cardiovascular COPD patients, where the risk of an acute cardiac ischemic
event (infarction or angina) is dose-dependent: OR=1.38 for those who used 1-2
canisters/month, OR=1.58 for those who used 3-4 canisters/month, and OR=1.93
for those using 6 or more canisters/month. This situation is the result of the factthat dyspnea or thoracic discomfort, often induced by myocardial ischemia, ismisinterpreted as being of pulmonary nature and, consequently, β-agonists that willkeep being used leading to a heart attack as a result of overdose. Administration of
β-blockers significantly reduces and even eliminates cardiac risk (31).

CONCLUSIONS

Between 1994 – 1998, salmeterol was approved for the treatment of bronchial asthma and of COPD, currently being used in over 100 countries, either as aunique drug or in combination with fluticasone (Seretide, Viane, Advair).
In 2006, the FDA reported a worldwide exposure to salmeterol, estimating
45.2 million patients/year, as follows (32):- 24.3 million patients/year for salmeterol;- 20.9 million patients/year for salmeterol associated with fluticasone
propionate in a single device.
Following the publication of the results of the most representative metaanalyses regarding the widespread use of this medication, we can conclude thatthe use of long-acting β2-agonists both in asthma and in COPD is safe, and theyshould be associated with ICS which enhance both their effectiveness and theirsafety. Special precautions are needed in patients with severe cardiac disorders or
thyroid disorders (33)
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3. Witek TJ, Schahter EN., Pharmacology and therapeutics in Respiratory Care., edit.
Saunders Company., 1994
4. Trevor T.Hansel and Barnes, An atlas of COPD. 2004.
5. Donald A. Mahler, James F. Donohue, Robert A. Barbee, Michael D. Goldman,
Nicholas J. Gross, Michael E. Wisniewski, Steven W. Yancey, Bradford A. Zakes,
Kathleen A. Rickard, Wayne H. Anderson. Efficacy of Salmeterol Xinafoate in theTreatment of COPD Chestl 1999 115:957-965
6. D'Alonzo GE, Nathan RA, Henochowicz S, Morris RJ, Ratner P, Rennard SI. Salmeterol xinafoate as maintenance therapy compared with albuterol in patients withasthma. JAMA. 1994 May 11;271(18):1412-6.
7. R. Dahl et al. Inhaled Formoterol Dry Powder Versus Ipratropium Bromide in
Chronic Obstructive Pulmonary Disease. AJRCCM 2001; 164: 778 - 784.
8. A.P Greening, P.W Ind, M Northfield, G. Shaw. Added salmeterol versus higher-dose
corticosteroid in asthma patients with symptoms on existing inhaled corticosteroid. The
Lancet, vol. 344 no. 8917 pp 219-224
9. Kemp JP, DeGraff AC Jr, Pearlman DS, Wang Y, Arledge TE, Welch MB, O'Riordan
TG, Herrle MR, Stahl EG. A 1-year study of salmeterol powder on pulmonary function
and hyperresponsiveness to methacholine. J Allergy Clin Immunol 1999;104:1189-

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10. R Shoup, G Dalsky, S Warner, M Davies, M Connors, M Khan, F Khan, R ZuWallack.
Body composition and health-related quality of life in patients with obstructive
airways disease. ERJ 1997; 10: 1576 - 1580.
11. Calverley PMA, Anderson JA, Celli B, Ferguson GT, Jenkins C, Jones PW, Yates
JC, Vestbo J. Salmeterol and Fluticasone Propionate and Survival in Chronic Obstructive Pulmonary Disease. N Engl J Med 356:775, 2007
12. P.M. Calverley, W. Boonsawat, Z. Cseke, N. Zhong, S. Peterson, and H. Olsson.
Maintenance therapy with budesonide and formoterol in chronicobstructive
pulmonary disease. ERJ 2003; 22: 912 - 919.
13. W. Szafranski, A. Cukier, A. Ramirez, G. Menga, R. Sansores, S. Nahabedian,
S. Peterson, and H. Olsson. Efficacy and safety of budesonide/formoterol in the
management of chronic obstructive pulmonary disease. ERJ 2003; 21: 74 - 81.
14. Lalloo UG, Malolepszy J, Kozma D, et al. Budesonide and formoterol in a single
inhaler improves asthma control compared with increasing the dose of corticosteroid
in adults with mild-to-moderate asthma. Chest 2003;123(5):1480-7.
15. Kips JC, O'Connor BJ, Inman MD, et al A long-term study of the antiinflammatory
effect of low-dose budesonide plus formoterol versus high-dose budesonide in asthma.
Am J Respir Crit Care Med 2000;161(3 Pt1):996-1001.
16. Kuna P, Peters MJ, Manjra AI, Jorup C, Naya IP, Martínez-Jimenez NE, Buhl R.
Effect of budesonide/formoterol maintenance and reliever therapy on asthma exacerbations. Int J Clin Pract. 2007 May;61(5):725-36.
17. Klaus F Rabe, Tito Atienza, Pál Magyar , Per Larsson, Carin Jorup, Umesh G
Lalloo. Effect of budesonide in combination with formoterol for reliever therapy inasthma exacerbations: a randomised controlled, double-blind study. The Lancet, vol.
368 no. 9537 pp 744-753
18. O’Donell DE Lam M, Webb KA. Measurement of symptoms, lung hyperinflationand endurance during exercise in chronic obstructive pulmonary disease., Am J Respir
Crit Care Med., 1998; 158: 1557-6
19. Devalia JL, Sapsford RJ , Rusznak C et al., The effect of salmeterol and salbutamolon cilliary beat frequency of cultured human bronchial epithelial cells, in vitro. Pulm
Pharmacol., 1992; 5: 257-63
20. Melloni B, Germouty J. The influence of a new beta agonist: formoterol on mucociliary function. Rev Mal Respir., 1992; 9: 503-7

21. Giembycz MA. Phosphodiesterase 4 inhibitors and the treatment of asthma: whereare we now and where do we go from here?. Drugs. 2000, 59(2):193-212.
22. Proud D., Reynolds CJ, Lichtenstein LM et al. Intranasal salmeterol inhibits allergeninduced vascular permeability but not mast cell activation or cellular infiltration. Clin
Exp Allergy 1998; 28: 868-75
23. Dowling RB Johnson M Cole PJ et al. effect of salmeterol on Hemophillus influenzae
infection of respiratory mucosa in vitro. Eur Respir J 1998; 11: 86-90
24. Cazzola M, Imperatore F, Salzillo A, et al. Cardiac effects of formoterol andsalmeterol in patients suffering from COPD with preexisting cardiac arrhythmias andhypoxemia. Chest 1998; 114:411–415
25. Newhouse M, Chapman K, McCallum A, et al. Cardiovascular safety of high doses
of inhaled fenoterol and albuterol in acute severe asthma. Chest 1996; 110:595–603
26 Nelson HS, Weiss ST, Bleecker ER, Yancey SW, Dorinsky PM; SMART Study
Group. The Salmeterol Multicenter Asthma Research Trial: a comparison of usual
pharmacotherapy for asthma or usual pharmacotherapy plus salmeterol. Chest. 2006
Jan;129(1):15-26. Erratum in: Chest. 2006 May;129(5):1393.
27. Global Strategy for Asthma Management and Prevention, 2006., pg 31
28. W Castle, R Fuller, J Hall, J Palmer. Serevent nationwide surveillance study:
comparison of salmeterol with salbutamol in asthmatic patients who require
regular bronchodilator treatment. BMJ 1993; 306: 1034 - 1037;
29. http://www.ginasthma.com
30. Shelley R. Salpeter, Thomas M. Ormiston, Edwin E. Salpeter. Cardiovascular Effects of β-Agonists in Patients With Asthma and COPD: A Meta-Analysis. Chest 2004
125:2309-2321; doi:10.1378/chest.125.6.2309
31. Rodrigo GJ, Castro-Rodriguez JA, Plaza V. Safety and efficacy of combined longacting beta-agonists and inhaled corticosteroids vs long-acting beta-agonists monotherapy for stable COPD: a systematic review. Chest. 2009 Oct;136(4):1029-38.
32. David HJ, Curtis R, Every NR, et al. Association Between Inhaled β-Agonists andthe Risk of Unstable Angina and Myocardial Infarction, Chest 2002;121;846-851
33. (http://www.fda.gov/ohrms/dockets/ac/05/slides/2005-4148S1_03_01-GSK-Main_files /frame.htm)
34. Hansel T, Barnes P. An atlas of COPD. 2004, ed. Parthenon Publishing ed.
Parthenon GroupSTIMULAREA BETA-ADRENERGICA SI MODULAREA FUNCTIEIPULMONARE IN ASTM SI IN BRONHOPNEUMOPATIA CRONICA
OBSTRUCTIVAREZUMAT

Receptorii adrenergici au o largă distribuție în corpul uman, stimularea lor însoțindu-se de o pleiadă variată de efecte. Decodificarea genelor de transcripție a demonstrat
existența a nu mai puțin de 9 subtipuri. Distribuția receptorilor Beta (β1, β2, β3 ) arată o predominanță β1 la nivelul sistemului cardiovascular și β2 la nivelul aparatului
respirator. Stimularea receptorilor β2 s-a dovedit că pe lângă efectul primordial de brohodilatație este susceptibilă, într-o măsură mult mai redusă, de îmbunătățirea
clearanceului mucociliar, de o activitate antiinflamatorie, de reducerea exudării plasmatice în căile aeriene, de reducerea aderenței bacteriene la celulele epiteliale bronșice,și chiar are potențialul de a crește masa musculară scheletică, puterea acesteia și ameliora fatigabilitatea, etc.
Mecanismul prin care este posibilă inducerea acestor efecte depășește clasicul releu al ”celui de-al doilea mesager”, - rectae, creșterea adenosinmonofosfatului ciclic (AMPc)
pe calea activării adenyl ciclazei, - antrenând multiple izoforme/subunități de proteine (proteina G, adenilciclaze, phosphokinase, etc) care interacționează în cascadă.
Fenotipurile enzimatice codificate genetic, sub influiența unui mediu inflamator local aflat în stare de activitate sau nu, a interferențelor cu medicamentele concomitentadministrate, a statusului de fumător sau nu, a vârstei etc, vor modula suplimentar răspunsul la stimularea beta terapeutică. Astmul bronșic, cât și bronhopneumopatia cronică
obstructivă (BPOC), oferă un câmp larg de aplicare și testare a diferitelor tipuri de agenți β2 agoniști (de scurtă, lungă și ultralungă durată de acțiune), cât și a combinațiilor
care le sporesc eficiența și siguranța (asocierea în același preparat cu un corticosteroid).
Cuvinte cheie: receptori β2, stimulare, astm, BPOC

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Fiziologia - Physiology  2010.20.1 (65)

MATHEMATICAL MODELS OF BREAST CANCER RISKCALCULATION AND ITS APPLICATIONSDANA CARMEN ZAHA1, LIANA ANTAL1, IOAN MAGYAR1, ELENA LAZAR2
1
Faculty of Medicine and Pharmacy, Oradea University, Oradea
2“Victor Babes “University of Medicine and Pharmacy, Timisoara

ABSTRACT
A complete assessment of a woman’s risk for breast cancer is important. The five year breast cancer risk can vary from 0.4% for a woman age 40 years with no risk to 6%
for a woman age 49 years with several risk factors. The 2006 American Cancer Society guidelines recommend early mammograms starting at the age of 40 and continuing
for as long as a woman is in good health. The large scale introduction of mammography screening has had as consequences the increasing of breast cancer rate, but also
the mortality reduction through the diagnostic in early stages. Current evidence shows variation among women in terms of benefits and harms associated with screening
mammography. A new method of calculating breast cancer risk can help to predict a woman's risk of developing an invasive form of the cancer. The technique can provide
useful information to women who may be contemplating the risks and benefits of breast cancer preventative strategies. The Gail Model is one of the models that havebeen developed to estimate a woman’s risk of developing breast cancer. The model incorporates a series of questions related to breast cancer risk factors and answers arecalculated into a Gail risk score. The model uses a woman's own personal medical history (number of previous breast biopsies and the presence of atypical hyperplasia in
any previous breast biopsy specimen), her own reproductive history (age at the start of menstruation and age at the first live birth of a child) and the history of breast cancer
among her first-degree relatives (mother, sisters, daughters) to estimate her risk of developing invasive breast cancer over specific periods of time. The Breast Cancer Risk
Assessment Tool is an interactive tool designed by scientists at the National Cancer Institute (NCI) and the National Surgical Adjuvant Breast and Bowel Project (NSABP) toestimate a woman's risk of developing invasive breast cancer (NSABP model 2).Because of the complexity of interpretation of some of the input information, the information
should be entered by a health professional with some experience in oncology. The combination of these mathematical models with other early diagnostic methods such as
mammography or preventive chemotherapy or other can lead to survival rates improving in breast cancer.
Keywords: breast cancer risk, assessment tool, survival rates

INTRODUCTION
Invasive breast cancer is the most common carcinoma in women; it accounts
for 22 % of all female cancers. The areas of high risk are the populations of North
America, Europe and Australia; the risk is low in the less developed regions of subSaharian Africa and Southern and Eastern Asia, including Japan. The prognosis of
the disease is very good if detected at an early stage. Breast cancer incidence, aswith most epithelial tumours, increases rapidly with age.
The aetiology of breast cancer is multifactorial and involves diet, reproductive
factors (young age at menarche, older age at first full-term birth, nulliparity, older
age at menopause, oral contraceptives) and related hormonal imbalances.
For almost half a century, the events of reproductive life have been considered
to be risk factors for breast cancer in women. Breast cancer occurs more frequently
among women who have an early menarche, remain nulliparous or, if parous, havefew children with a late age at first delivery. Infertility appears to be a risk factor
as may be lack of breast- feeding. Also, late age at menopause increases the risk.
Recent data indicates that the age at any delivery, not just the first is associatedwith breast cancer risk, with deliveries occurring before the age of 30 having aprotective effect.
Another important risk factor is exogenous hormones. Two major types ofhormonal compounds have been evaluated in relation to breast cancer: oral contraceptives and menopausal replacement therapy. The evidence suggests a small
increase in the relative risk associated with the use of combined oral contraceptives,
especially among current and recent users, which is not related to duration of theuse and type or dose of preparation and may be partly linked to detection bias.
Data on injectable pure progestogen contraceptives shows relative risks from 1 to
3 wich are not statistically significant.

Epidemiological studies on postmenopausal estrogen therapy show a small
increase in risk with longer duration of use in current and recent users. Informationon the effect of postmenopausal estrogen- progesterone therapy was provided inonly a minority of studies, but indicates that the increased relative risk in long- termusers is not significantly different from that for long-term use of estrogens alone.
Yet it should be noted that, among hormone replacement therapy users, there is anover representation of tumours that, with regard to tumour stage, type and grade
are associated with a more favorable prognosis.
There is overwhelming evidence from epidemiological studies that sex steroids(androgens, estrogens, progestogens) have an important role in the developementof breast tumours. Breast cancer incidence rates rise more steeply with age before
menopause than after, when ovarian synthesis of estrogens and progesterone ceases
and ovarian androgen production gradually diminishes.
The estrogen excess hypothesis is central, stipulating that breast cancer
risk depends directly on breast tissue exposure to estrogens. In vitro studies show
increases breast cell proliferation and inhibition of apoptosis. Animal studies show
increased rates of tumour development when estrogens are administered. The risk
is higher among postmenopausal women who have elevated plasma levels of
testosterone and androstenedione, reduced levels of sex hormone-binding globulin(SHBG), and increased levels of oestrone, oestradiol and bioavailable oestradiol
not bound to SHBG.
A second major theory , the estrogen plus progesterone hypothesis, postulates
that , compared to exposure to estrogens alone ( as in postmenopausal women notusing exogenous hormones ) , risk of breast cancer is futher increased in women
who have elevated plasma and tissue levels of estrogens in combination withprogestogens. This theory is supported by observations that proliferation of mammary epithelial cells is increasing during the luteal phase of the menstrual cycle,

Received December 2009. Accepted February 2010. Address for correspondence: Dr. Zaha Dana Carmen, 1 decembrie Square No. 10 Oradea, e-mail: danazaha@rdslink.ro,phone: 0723 604496
2010.20.1(65)  Fiziologia - Physiology

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33

compared to the follicular phase. Among pre-menopausal women, several studies
have not shown any clear association between breast cancer risk and circulatinglevels of androgens, estrogens or progesterone.
In addition their research showed a higher risk among long term oral
contraceptives users; especially those had started to take the pill before age 18.
Women who have a first- degree family of breast cancer and oral contraceptives
exposure may want to be particularly vigilant regarding appropriate breast cancer
screening practices.
A metabolic consequence of excess body weight and lack of physical activity
is development of insulin resistance. Elevated insulin levels may lead to increased
ovarian and / or adrenal synthesis of sex steroids, particularly of androgens,and decrease the hepatic synthesis and circulating levels of SHBG. Especially inpostmenopausal women, elevated plasma androgens lead to increased estrogenformation in adipose tissue and hence to increased levels of oestrone and oestradiol.
The hypothesis that chronic hyper-insulinemia might explain the observed associations of breast cancer risk with low plasma SHBG and elevated androgens andestrogens, among postmenopausal women has, however, received only limited
support . Insulin-growth factor I (IGF-I) and IGF- binding proteins (IGFBP) appear
to be significant risk predictors.
Recent studies have shown a fairly consistent though small effect of alcohol
consumption on breast cancer risk. In a summary analysis of epidemiologic
studies, breast cancer risk increased between 40 and 70 percent with about two
drinks daily (9).
A part of these risk factors have been considered for the conception of sometools or mathematical models for the breast cancer risk evaluation. This paper aims
for the presentation of two tools such as the ones above and their applicability. The
Gail model is named after Mitchell H. Gail who published several famous papers
describing the scientific basis for the risk calculation. National Surgical Adjuvant
Breast and Bowel Project is an important organization that has conducted many
important research studies. The NSABP used the original Gail model and modified
it for a research study. That is why it is called NSABP model 2 or Gail model 2, butthat would be even more confusing.
The Gail model
The Gail Model incorporates a series of questions 1 through 6 related tobreast cancer risk factors and answers are calculated into a Gail risk score, a summary relative risk (3). The model uses a woman's own personal medical history(number of previous breast biopsies and the presence of atypical hyperplasia in any
previous breast biopsy specimen), her own reproductive history (age at the start ofmenstruation and age at the first live birth of a child), and the history of breast cancer
among her first-degree relatives (mother, sisters, daughters) to estimate her risk ofdeveloping invasive breast cancer over specific periods of time. A medical historyof ductal carcinoma in situ (DCIS) or lobular carcinoma in situ (LCIS) increases therisk of developing invasive breast cancer.
The Gail model has been tested in large populations of white women and has
been shown to provide accurate estimates of breast cancer risk (2, 13, 11). In otherwords, the model has been "validated" for white women. It has also been tested
in data from the Women’s Health Initiative for African American women and themodel performs well, but may underestimate risk in African American women withprevious biopsies. The model still needs to be validated for Hispanic women, Asian
women, and other subgroups, and results should be interpreted by a health careprovider for women with special risk factors, such as women treated for Hodgkin’s
disease with radiation to the chest and carriers of gene mutations that increasebreast cancer risk. Researchers are conducting additional studies, including studies
with minority populations, to gather more data and to test and improve the model.
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While race/ethnicity is included in the calculation, it does not influence breast cancer
risk as much as other factors. The model for African American women was derived
from the Women’s Contraceptive and Reproductive Experiences (CARE) Study and
NCI’s SEER Program. For Hispanic women, part of the model is derived from white
women who participated in the Breast Cancer Detection Demonstration Project
and from SEER data. The risk estimates for Hispanic women are therefore subject togreater uncertainty than those for white women. Calculations for American Indian,
Alaskan Native, Asian, and Pacific Islander women are based entirely on data forwhite women and may not be accurate. Researchers are conducting additional
studies, including studies with minority populations, to gather more data and toincrease the accuracy of the tool for women in these populations.
Although the tool has been used with success in clinics for women withstrong family histories of breast cancer, more specific methods of estimating risk
are appropriate for women known to have breast cancer-producing mutations in
the BRCA1 or BRCA2 genes (10). Other factors may also affect risk and are notaccounted for by the tool. These factors include previous radiation therapy to thechest for the treatment of Hodgkin lymphoma or recent migration from a region
with low breast cancer rates, such as rural China. A woman who does not havemammograms will have somewhat lower chances of a diagnosis of breast cancer.
The risk of developing breast cancer increases with age. The great majority of breast
cancer cases occur in women older than age 50. Most cancers develop slowly overtime. For this reason, breast cancer is more common among older women. Incidence rates in women before the age of 45 are higher among blacks; after the ageof 45, they are higher for whites. Women of higher socioeconomic status, married
women, women living in urban versus rural areas and women in northern states
have the highest rates.
The questions are:
1.Does the woman has a medical history of any breast cancer or of ductal carcinoma
in situ (DCIS) or lobular carcinoma in situ (LCIS)?
2. What is the woman's age?
3. What was the woman's age at time of her first menstrual period?
4. What was the woman's age at her first live birth of a child?
Risk depends on many factors, including age at first live birth and family historyof breast cancer. The relationship of these two factors is shown in the following tableof relative risks. (Table 1) For women with 0 or 1 affected relative, risks increase
with age at first live birth. For women with 2 or more first degree relatives, risks
decrease with age at first live birth.
5 .How many of the woman's first-degree relatives - mother, sisters, daughters have had breast cancer?
6. Has the woman ever had a breast biopsy?
6a: How many previous breast biopsies (positive or negative) has the woman
had?
6b: Has the woman had at least one breast biopsy with atypical hyperplasia?
7. If known, please indicate the woman's race/ethnicity.
A woman's risk is considered low if her five-year risk of developing breast
cancer is less than 1.66 percent; it is considered high if she scores above 1.66 percent.
All women who are over 60 have a score of at least 1.66 and are considered highrisk, based on the Gail Model. Women younger than 60 have to have the score of
a 60-year-old woman [1.66] to be considered high risk. The sensitivity of the Gailmodel (the number of women who had breast cancer who were actually deemedhigh risk by the test) was 14, 7% and the specificity (the number of women who
did not have breast cancer and who test found to be low risk) was 93, 9%.
NSABP model 2
The Breast Cancer Risk Assessment Tool is an interactive tool designed by
Fiziologia - Physiology  2010.20.1 (65)

scientists at the National Cancer Institute (NCI) and the National Surgical Adjuvant
Breast and Bowel Project (NSABP) to estimate a woman's risk of developing
invasive breast cancer. The tool should not be used to calculate breast cancer risk
for women who have already had a diagnosis of breast cancer, lobular carcinoma
in situ (LCIS) or ductal carcinoma in situ (DCIS). The Breast Cancer Risk Assessment
Tool will estimate a woman's risk of developing invasive breast cancer during thenext 5-year period and up to age 90 (lifetime risk) based on the woman's age andthe risk factor information provided. For comparison, the tool will then calculate
5-year and lifetime risk estimates for a woman of the same age who is at averagerisk for developing breast cancer. A positive response to any of the questions (exceptage) equals an elevated risk of breast cancer (2,4).
The internal workings of my NSABP model 2 calculators also start by calculating the summary relative risk, using the basic Gail model method. However, model
2 calculator uses slightly different values for relative risks. Model 2 does not include
the risk of developing Ductal Carcinoma In-Situ (DCIS).
The components of this model are:1. Age at menarche in years
2. Number of Biopsies
3. Number of first degree relatives with breast cancer
4. Biopsy with atypical hyperplasia?
5. Mammographic Density
6. Taking Tamoxifen?
7. Alcohol use
8. LCIS on biopsy?
9. Used Oral Contraceptives?
The answer of to each of the questions above is represented by a score andsummary relative risk is calculated multiplying the nine scores between them. Aftercalculating summary relative risk, then the absolute risk is determined using polynomial
equations. For the NSABP model 2 calculator, there are separate curves for 5-year andlifetime absolute risk, for ages 20, 30, 40, 50, 60 and 70. The calculated by the toolrisk are estimates of absolute breast cancer risk. Absolute breast cancer risk is the
chance or probability of developing invasive breast cancer in a defined age interval.
One way to evaluate the accuracy of the estimated risk is to determine whether it
correctly predicts average risk in a group of women with the same risk factors and age.
Although a woman's risk may be accurately estimated, these predictions do not allow
one to say precisely which woman will develop breast cancer. In fact, the distributionof risk estimates for women who develop breast cancer overlaps the estimates of risk
for women who do not.
Table 1 Relative Risk of Developing Breast Cancer (Gail MH et al., 1999)

Age at first# of affected relatives

live birth0

1

2 or more

20 or younger

1

2.6

6.8

20-24

1.2

2.7

5.8

25-29 or no child

1.5

2.8

4.9

30 or older

1.9

2.8

4.2

CONCLUSIONSIn women after 40 years of age, clinicians should periodically perform
individualized assessment of risk for breast cancer to help guide decisions about
screening mammography. The most important benefit of screening mammography
every 1 or 2 years in women past 40 years of age is a potential decrease in breast
cancer mortality, despite the increasing disease incidence. Use of mammography
has been associated with increased diagnosis of DCIS. The natural history of DCIS
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is unknown, as the percentage of these tumors that will progress to more serious
disease. Because the evidence shows variation in risk for breast cancer and benefits
and harms of screening mammography based on an individual woman risk profile,a personalized screening strategy based on a discussion of the benefits and potential
harms o screening and understanding of a woman’s preferences will help identify
those who will most benefit from screening mammography. Women who scorehigh on the test are encouraged to have increased surveillance and to consider
breast cancer risk-reduction options.
Recent guidelines suggest that chemoprevention with tamoxifen may beappropriate for women who have a 5-year risk of breast cancer greater than 1.66%
calculated using the Gail model (11).
Given the close balance between the risk and benefits of tamoxifen for most
women considering chemoprophylaxis, discovering new strategies to improve theidentification of women at very high risk for developing breast cancer is clinicallyimportant. Adding information from biological measurements to the risk model
may improve prediction of the near-term risk of breast cancer. NAF cytology has
the potential to improve prediction models of breast cancer incidence, particularly
for high-risk women. Nipple aspiration is a minimally invasive procedure originallydeveloped as a form of Papanicolau test for breast cancer. Prospective cohort studies
have shown that cytology information from cells obtained from nipple aspiration
predicts breast cancer incidence independent of traditional risk factors (12, 15).
Adding NAF cytology results to the predictor variables used to calculate the Gailrisk for women modestly improved the discriminatory accuracy of the model (fromc-statistic of 0.62 to 0.64). Clinically, the test information may be most useful for
women at highest absolute risk by the Gail model because modest differences inrelative risk are amplified (6).
Premalignant cell damage in this lining may produce biochemical signalsthat deliver inflammatory proteins to the site. The presence of C-reactive protein(CRP) in nipple aspirate fluid (NAF) may reflect an inflammatory state indicativeof a premalignant breast microenvironment. CRP is differentially present in NAF
and varies by Gail model risk factors. CRP in NAF holds promise as a noninvasive
biomarker that detects a precarcinogenic breast ductal microenvironment and may
contribute to the diagnosis of breast cancer early in the course of the disease when
prognosis is most favorable (7).
Researchers are conducting additional studies to gather more data and to
determine whether including information on other risk factors can strengthen themathematical models.
REFERENCES
1. Chemoprevention of breast cancer: recommendations and rationale. Ann Intern
Med 2002; 137:56–8.
2. Costantino JP, Gail MH, Pee D, et al. Validation studies for models projecting the riskof invasive and total breast cancer incidence. J Natl Cancer Inst 1999; 91:1541–8.
3. Gail MH, Brinton LA, Byar DP, Corle DK, Green SB, Shairer C, Mulvihill JJ: Projecting individualized probabilities of developing breast cancer for white females who arebeing examined annually. J Natl Cancer Inst 81(24):1879-86, 1989.
4. Gail MH, Costantino JP, Bryant J, Croyle R, Freedman L, Helzlsouer K, Vogel V:
Weighing the risks and benefits of tamoxifen treatment for preventing breast cancer.
J Natl Cancer Inst 91(21):1829-46, 1999.
5. Gail MH, Costantino JP, Pee D, Bondy M, Newman L, Selvan M, Anderson GL,
Malone KE, Marchbanks PA, McCaskill-Stevens W, Norman SA, Simon MS, Spirtas R,
Ursin G, and Bernstein L. Projecting Individualized Absolute Invasive Breast Cancer
Risk in African American Women. JNCI. Vol. 99, No. 23.
6. Jeffrey A. Tice, Rei Miike2, Kelly Adduci et al, Nipple Aspirate Fluid Cytology and
the Gail Model for Breast Cancer Risk Assessment in a Screening Population, Cancer
Epidemiol. Biomarkers Prev. March 1, 2009 18:732-738
7. Diana Lithgow, Adeline Nyamathi, David Elashoff, Otoniel Martinez-Maza,
Chandice Covington , C-reactive Protein in Nipple Aspirate Fluid Associated With

35

Gail Model Factors Biological Research For Nursing, Vol. 9, No. 2, 108-116 (2007)
DOI: 10.1177/1099800407306426
8.C.Suzanne Lea, Nancy P.Gordon, Lee Ann Prebil,Rochelle Ereman,Connie
S.Uratsu,Mark Powell, Differences in reproductive risk factors for breast cancer
in middle-aged women in Marin County and sociodemographically similar area ofNorthern California, BioMed Central,2009,9: 6
9. David Heber, George L.Blackburn,Vay Liang W.Go,John Milner, Nutitional Oncology, second edition,2006
10. Milne et al.,Oral contraceptive use and risk of early- onset breast cancer in carriers
and noncarriers of BRCA 1 and BRCA 2 mutations, Cancer Epidemiol.Biomarkers
Prev.,2005;14 : 350- 356
11. Rockhill B, Spiegelman D, Byrne C, Hunter DJ, Colditz GA. Validation of the Gailet al. model of breast cancer risk prediction and implications for chemoprevention. J

Natl Cancer Inst 2001;93:358–66.
12. Sartorius OW, Smith HS, Morris P, Benedict D, Friesen L. Cytologic evaluation of breast fluid in the detection of breast disease. J Natl Cancer Inst 1997;
59:1073–80.
13. Spiegelman D, Colditz GA, Hunter D, Hertzmark E. Validation of the Gailet al. model for predicting individual breast cancer risk. J Natl Cancer Inst
1994;86:600–7.
14. World Health Organization, International Classification for Disease Oncology,
3 rd Edition, 2000
15. Wrensch MR, Petrakis NL, Miike R, et al. Breast cancer risk in women withabnormal cytology in nipple aspirates of breast fluid. J Natl Cancer Inst 2001;
93:1791–80.

MODELE MATEMATICE DE EVALUARE A RISCULUI DE CANCER DESÂN ŞI APLICAŢIILE LORREZUMAT

Evaluarea completă a riscului de cancer de sân este importantă în practica medicală. Riscul de cancer de sân la cinci ani poate fi cuprins între 0,4% pentru o femeie de 40
de ani fără factori de risc şi 6% pentru femeia de 49 ani care asociază mai mulţi factori de risc. Ghidul Asociaţiei Americane de Cancer din 2006 recomandă mamografii
timpurii începând cu vârsta de 40 ani, continuând atât timp cât femeia prezintă o stare bună de sănătate. Introducerea pe scară largă a screening-ului mamografic a avut
drept consecinţe creşterea ratelor de cancer de sân, dar şi reducerea mortalităţii prin diagnosticul în stadii incipiente. Date recente arată variaţii în ceea ce priveşte beneficiile
şi prejudiciile asociate screeningului mamografic. O nouă metodă de calcul a riscului de cancer de sân poate aprecia riscul de a dezvolta o formă invazivă de cancer de sânla femei. Tehnica poate furniza informaţii utile femeilor în ceea ce priveşte riscul şi beneficiile strategiilor preventive ale cancerului de sân. Modelul Gail este unul dintre aceste
modele care a fost dezvoltat pentru estimarea riscului de a dezvolta cancer de sân. Modelul cuprinde o serie de întrebări în legătură cu factorii de risc ai cancerului de sân,iar răspunsurile se calculează într-un scor de risc Gail. Modelul utilizează istoricul medical personal al femeii (numărul biopsiilor de sân anterioare şi prezenţa hiperplaziei
atipice în orice biopsie anterioară), istoricul reproductiv (menarha, vârsta primei naşteri) şi istoricul de cancer de sân la rudele de grad I (mama, sora, fiica) pentru a estima
riscul său de a dezvolta într-o anumită perioadă de timp o formă invazivă de cancer de sân. Breast Cancer Risk Assessment Tool este un instrument interactive proiectat despecialişti ai National Cancer Institute (NCI) and the National Surgical Adjuvant Breast and Bowel Project (NSABP) pentru a estima la femei riscul de a dezvolta un cancer
invaziv (NSABP model 2). Deoarece complexitatea interpretării a unora dintre informaţiile introduse se recomandă ca introducerea lor sa fie realizată de către personal cu ooarecare experienţă în oncologie. Prin combinarea acestor modele matematice cu alte metode de diagnostic timpuriu cum este mamografia sau chimioterapia preventivă
sau altele se poate duce la îmbunătăţirea ratelor de supravieţuire în cancerul de sân.
Cuvinte cheie: risc cancer mamar, instrumente de analiza, rata de supravietuire

36

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Fiziologia - Physiology  2010.20.1 (65)STROMAL CELLS - TUMOR MICROENVIRONMENT
INTERACTIONS – Part I
FLORINA BOJIN1, CALIN TATU2,3, OANA GAVRILIUC1, ALEXANDRA GRUIA3,GABRIELA TANASIE1,3, CARMEN TATU1,3, MIRABELA CRISTEA3, FELICIA CIUCULESCU1,ALEXANDRU TOCUT1, DANIELA CRISNIC1,3, ADRIAN CARABINEANU4, DACIANANISTOR1,3, CARMEN BUNU1, VIRGIL PAUNESCU1
1
Physiology-Immunology Department, “Victor Babes” University of Medicine and Pharmacy Timisoara
2
Biology Department “Victor Babes” University of Medicine and Pharmacy, Timisoara
3
Regional Center for Transplant Immunology, Emergency Clinical County Hospital Timisoara
4
Oncologic Surgery Department, Municipal Emergency Hospital Timisoara

ABSTRACTThe complex cellular tumor micro(environment) comprises immunocompetent and inflammatory cells, endothelial cells and fibroblasts. All these cell types may influence
the multi-step process of carcinogenesis and malignant phenotype. Fibroblasts are known to take part in immune reaction during tissue damage and injury by modulating
local cellular and cytokine milieu, adjusting the kinetics and components of the inflammatory infiltrate, and by modulating the functional status of the immunocompetentcells. Purpose of the study was to investigate stromal cells role in immune suppression, which can profoundly alter tumor cells progression. Mesenchymal stem cells (MSCs)
and tumor-associated fibroblasts (TAFs) were used in a comparative study for providing morphological and immunophenotypical characterization of stromal compartment
interactions with tumorigenic cells.
Key words: mesenchymal stem cells, tumor-associated fibroblasts, tumor cells, microenvironment

INTRODUCTION
Mesenchymal stem cells (MSCs), most commonly isolated from bonemarrow, are defined as cells that display fibroblastic morphology in cell culture,
exhibit a robust self-renewal capacity, and retain the ability to undergo trilineage
differentiation into adipocytes, chondrocytes, and osteoblasts. Recent evidence
suggests that bone marrow MSC can be mobilized into the periphery to serve
as regenerative stem cells at sites of injury and inflammation (1,2). On the other
hand, in vivo biology of MSC is poorly understood, several studies demonstratingthat MSCs can be selectively recruited into tumors. Following engraftment withintumor stroma, MSC proliferate and acquire an activated phenotype similar tumorassociated fibroblasts (TAF). Tumor-homing properties of MSC have lead to theirutility as therapeutic cell-based antitumor protein delivery vehicles (3-6). However,with a greater appreciation for the influential role that the tumor microenvironment
can serve during tumor initiation, promotion, and progression, MSC may enhancetumor progression following acquisition of TAF-like characteristics. A more comprehensive delineation of the biological role of MSC within tumor stroma will improve
our understanding of tumor-stroma interactions and facilitate future developmentof MSC-based clinical therapies.
Carcinomas are solid tumors that arise from malignant epithelial cells andrepresent the most common type of cancer in humans. However, it is becoming
increasingly clear that malignant cells are supported by non-epithelial tumor stromathat shape a given tumor microenvironment. Tumor stroma can be generally divided
into four main components: 1) tumor vasculature, 2) inflammatory immune cells,3) extracellular matrix (ECM)/soluble factors, and 4) tumor-associated fibroblasts(TAF). Bidirectional paracrine communication between connective tissue fibroblasts
and epithelial cells are vital for normal tissue homeostasis, and a comparable but
progressive intimacy continues throughout tumor development.

Tumors are thought to develop their stroma from different sources, so thatstromal cells are characterized by Wels et al. as “migratory neighbors and distantinvaders” (7). In literature, there are four origins mentioned with this regard: 1)
recruitment of resident tissue cells, 2) epithelial to mesenchymal transition, 3)
fibroblasts recruitment, and 4) recruitment of bone marrow-derived cells fromthe circulation (8-11).
Tumors arise from cells that have sustained genetic mutations resulting inderegulation of several of their normal growth-controlling mechanisms. Much of
the research concerning the origins of cancer has focused on the genetic mutation
within tumor cells, treating tumorigenesis as a cell-autonomous process governedby the genes carried by the tumor cells. However, it is increasingly apparent thatthe stromal microenvironment in which the tumor cells develop profoundly influences many steps of tumor progression. In various experimental tumor models,the microenvironment affects the efficiency of tumor formation, the rate of tumor
growth, the extent of invasiveness, and the ability of tumor cells to metastasize.
In carcinomas, the influences of the microenvironment are mediated in large partby paracrine signaling between epithelial tumor cells and neighboring stromal
fibroblasts. Paracrine signaling between other cell types within the carcinomas,
such as endothelial cells and inflammatory cells may play equally important roles
in tumor formation. Although mesenchymal cell fibroblasts can enhance tumor
growth and metastasis, there is also evidence that healthy tumor microenvironment
can act in a dominant manner to inhibit tumor growth.
Direct contact between bulk tumor cells and bone marrow-derived MSCs or
TAFs in coculture models could reveal the true nature of stromal-tumor cell interaction, and could clarify the perspective of using MSCs as a strategy for deliveringanti-tumor agents directly into tumors (Figure 1).

Received January 2010. Accepted February 2010. Address for correspondence: Florina Bojin MD, Physiology-Immunology Department, “Victor Babes” University of Medicine andPharmacy Timisoara, 2A Eftimie Murgu Square, RO-300041, Phone/Fax: +40256490507, e-mail: florinabojin@umft.ro2010.20.1(65)  Fiziologia - Physiology

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37

n other cell types within the carcinomas, such as endothelial cells and inflammatory cells
important roles in tumor formation. Although mesenchymal cell fibroblasts can enhanced metastasis, there is also evidence that healthy tumor microenvironment can act in ar to inhibit tumor growth.
ntact between bulk tumor cells and bone marrow-derived MSCs or TAFs in coculture
eal the true nature of stromal-tumor cell interaction, and could clarify the perspective ofstrategy for delivering anti-tumor agents directly into tumors (Figure 1).
Dubelcco’s Modified Eagle’s Medium (DMEM, Sigma), supplemented with F12,
B27, epidermal growth factor (EGF) and fibroblast growth factor (FGF) in variable
Bulk Tumor Cells
Enzymatic proportions.
digestion: Breast cancer surgical pieces from 20 patients (Ductal infiltrative
All usingtissue Collagenasesamples weretypeobtained
after signing
the informed
consent
carcinoma) were processed
IV-S (Sigma)
after cutting
in smallpieces, placed atminutes.under
Cellularsuspension
wasaccording
centrifugedminutes
at 300g,
pellet is washed 3
37C°, 5% CO2 for 30
elaborated
an approved
protocol,to the7 World
Medical
Association
times using PBS (Sigma) and filtered through 70 µm strainers (BD), then cultured in suspension cell
Declarationof Helsinki.
culture flasks. Tumor
cells were
cultured in 6-well suspension culture flask in presence of Dubelcco’s

Modified Eagle’s Medium (DMEM, Sigma), supplemented with F12, B27, epidermal growth factor(EGF) and fibroblast growth factor (FGF) in variable proportions.
Additional Procedures on Tumor-Derived Cells
All tissue samples were obtained after signing the informed consent elaborated under an approved
1 weekof suspension
cell culture,
tumorofcells
formed spheres,protocol, according to theAfter
World
Medical
Association
Declaration
Helsinki.

identified using vital staining ethidium bromide/acridine orange (Figure 2). The

Additional Procedures on Tumor-Derived Cellsof the cells
removedpositive
microbeads
using
After 1 weekremainingof suspension
cell were
culture,tumorbycells
formedspheres,selection
identified
using vital staining
Dead Cell orange
Removal(Figure
Kit (MACS)-which
identifies even
apoptotic
with an by positive
ethidium bromide/acridine2). The remainingof early
the cells
werecells
removedmicrobeads selection
using
Cell Removal
KitLineage(MACS)-which
identifies
even early apoptotic cells
intactcellDeadmembrane,followed by
Cell Depletion
Kit (MACS)-includes
with an intact cell membrane, followed by Lineage Cell Depletion Kit (MACS)-includes CD2, CD3,
CD2,CD16,
CD3, CD11b,
CD14,
CD15,CD123,
CD16, CD19,
CD56, CD123, CD235a, so that theCD11b,
CD14,
CD15,
CD19,
CD56,
arding formation of tumors and possible role of MSCs/TAFs fromtumor
stromain tumor
progression
andCD235a, so that the morphological aspect of thecells was changed. morphological aspect of the cells was changed.

Fig.
1. Concepts regarding
formation ofpotential
tumors and possible
role ofwhile
MSCs/TAFsMSCs/TAFs
from tumor stroma role is not completely elucidated.
r stem cells may
generatetumorigenic
cells,
in tumor progression and development. Cancer stem cells may generate tumorigenic potential cells,
while MSCs/TAFs role is not completely elucidated.

ND METHODS

MATERIAL AND METHODS

nd Culture
m Cells (MSCs)Cell Isolation and Culture
Mesenchymal Stem Cells (MSCs)
uman mesenchymal
stem cells (MSCs) were obtained from bone marrow of 6 healthy
Normal human mesenchymal stem cells (MSCs) were obtained from boneents undergoinghip
replacement
surgery.
Approximately
marrow of 6 healthy
Orthopedics patients
undergoing
hip replacement surgery.10 ml of bone marrow weree plates, andthe fibroblastic-like,plastic
fraction,
was isolated following
Approximately
10 ml of bone marrow wereplaced inadherent
culture plates, andthes and used infibroblastic-like,our experiments.
The
MSCs
were
further
culturedand expanded in alphaplastic adherent fraction, was isolated following multiple pasFig. 2.
Vitalgrownstaining
tumor cellscellgrown
in suspension
cellaggregates
cultures. CellularaggregatesFig. 2. Vital staining of tumor
cells
in of
suspension
cultures.
Cellularstainedin greenstained
contain viable cells,
and usedGibco
in our experiments.
MSCs were further cultured
andwhich
expanded
ial medium sages(MEM;
BRL,TheInvitrogen,
Carlsbad,CA,USA),
supplemented
with
were
further
processed
andculturedas cells,
singlecellsweresuspension.
in green
containviablewhich
further processed and cultured as single cells suspension.
in alpha-minimum essential
medium (MEM;
Gibco BRL, Invitrogen,
Carlsbad,erum (FCS; PromoCell,
Heidelberg,
Germany)
and 2%
Penicillin/Streptomycin mixture
Cocultures
CA, USA), supplemented
10% fetal calf serum
Heidelberg,
MSCs
and TAFsCocultures
at passage
P4 and P5waswere cultured in 6-wells culture plates for 2 hours
C in 5%CO
Medium00 IU/ml; PromoCell),bywithincubation
at (FCS;
37oPromoCell,
2 atmosphere.
(adherence
tumor
cells
were
addedP4
inand
different
on topof MSCs/TAFs:
1:10,
Germany) and 2% Penicillin/Streptomycin mixture (Pen/Strep, 10,000
IU/ml; purpose), andMSCs
and
TAFs
at
passage
P5 werecellular
culturedratios
in 6-wells
cultureplateshree days and when the confluence
was 80-90% the
cells
were
passed100,000
usingcells0.25%
1:20,
and 1:50.
Approximately
were cultured per well, and tumor cells were added in
PromoCell), by incubation at 37oC in 5% CO2 atmosphere. Medium was
replaced
for
2
hours
(adherence
purpose),
and
tumor
cells
were
added
in
different
cellularadequate proportions.
Cells weregrown intypes of media – tumor cells culture media andolution (Sigma)
followed by confluencecentrifugation(10 minutes,300g) andreplated
intwoT75
every three days and when thewas 80-90% the cells were MSCs/TAFs
passed us- specificratios
culturemedia.
on topof MSCs/TAFs: 1:10, 1:20, and 1:50. Approximately 100,000 cells
2a density of ing
10,000cells/cm
to(Sigma)ensureoptimalproliferation.
with passage two,
0.25% Trypsin-EDTA
solution
followedby centrifugation(10 minutes, Starting
were cultured per well, and tumor cells were added in adequate proportions. Cells
MTT-Based
Toxicology
Assay2
were used 300g)
for andfurtherphenotypicalanalysesanddifferentiation
assays,MSCs
replated in T75 culture flasks at a density of 10,000 cells/cm to ensure
werecellsgrownconditioned
in twowhile
typesmedia
of media– tumorculture media
and MSCs/TAFs
Tumor-derived
wasused cells
to identify
the influence
on MSCs/TAFs
ages 2-5 wereusedin theStarting
subsequent
optimalproliferation.
with passageexperiments.
two, part of the cells wereused for capacity.
proliferation
An MTT
specific
culturebased
media.in vitro toxicology assay kit (Tox-1, Sigma) was used to determine

proliferation
further phenotypical analyses and differentiation assays, while MSCs expanded
to rate of MSCs and TAFs at 24 and 48 hours. Trypan Blue cell counting and viability
assessment were simultaneously performed at the same time intervals for better correlation of the results.
2-5 were used in the subsequent experiments.
Toxicology
Assay
Cells were seededMTT-Basedat 2,000 cells/wellin 96-well plates
in quadruplicate and the average value ofd fibroblastspassages(TAFs)
specific medium extinction
was subtracted
from the media
samples
extinction
readtheatinfluence570/655onnm using a
Tumor-derived
cells
conditioned
wasused to identify
umor-associated fibroblasts (TAFs) were isolated using
both PR
the2100
explant
andcollagenase
benchmarkmicroplate
readerfrom
Bio-Rad(Hercules,
CA,
USA).
Conditioned
media wasTumor-associated fibroblasts (TAFs)
MSCs/TAFs
proliferation
capacity.
An MTT based50%-M2,
in vitro toxicology
assay kit +(Tox-1,addedmethods.
in progressively
dilutedconcentrations(100%-M1,
and 25%-M3)
10% Fetal Calf
Clostridium histolyticum(Sigma,St.
Louis,MO,
USA)
Breastcancersurgicalboth (FCS,
the PromoCell),so thatinfluencerate
ofofFCS
culturemedium.
Sigma) was
usedtotoeliminate
determinethe
proliferation
MSCslack
andinTAFs
at 24 and48 hours.
2 Human tumor-associated fibroblasts (TAFs) were isolated usingSerumwereobtained
femalehistolyticum
patients,
withthe histopathologicaldiagnosis
mately 5 cmexplantand collagenase
typeform
IV-S from8Clostridium(Sigma,St. Louis,
Trypan Blue cell counting and viability assessment were simultaneously performed
and Chemokines
Profile times withuctal mammary
Tissue-isolated
cells 5Cytokines
were
MO, USA)carcinoma.
methods. Breast cancer
surgical pieces of approximately
cm2 were washedat theseveral
same time intervals
for better correlation of the results.
ed saline (PBS,
andpassed diagnosis
through0.70/0.40 μmCells
strainerand in 96-well plates in quadruplicate andobtainedSigma)
form 8 femalesolution,
patients, with thehistopathologicalof infiltrative
were seededfiltersat 2,000 cells/wellductal mammaryin
carcinoma.
Tissue-isolated
cells were
washedplates.
several times withsingle-cell suspension
adherent
plastic
culturethe average value of specific medium extinction was subtracted from the samples
phosphate buffered saline (PBS, Sigma) solution, and passed through 0.70/0.40
extinction read at 570/655 nm using a benchmark PR 2100 microplate reader fromμm strainer filters and were replated as single-cell suspension in adherent plastic
Bio-Rad (Hercules, CA, USA). Conditioned media was added in progressively diluted
culture plates.
concentrations (100%-M1, 50%-M2, and 25%-M3) + 10% Fetal Calf Serum (FCS,
PromoCell), so that to eliminate the influence of FCS lack in culture medium.
Bulk Tumor Cells
Enzymatic digestion: Breast cancer surgical pieces from 20 patients (Ductal
Cytokines and Chemokines Profile
infiltrative carcinoma) were processed using Collagenase type IV-S (Sigma) after
ELISA analysis of supernatant was performed 2 weeks from the beginning
cutting in small pieces, placed at 37C°, 5% CO2 for 30 minutes. Cellular suspensionof cocultures, or the next day from plating MSCs and TAFs using Quantikine Imwas centrifuged 7 minutes at 300g, pellet is washed 3 times using PBS (Sigma)
munoassay kits (R&D Systems, Minneapolis, MN, USA) specific for IL-4, IL-10, IL-13,
and filtered through 70 µm strainers (BD), then cultured in suspension cell culture
TGF-β1, TNF-α, IFN-γ, and VEGF. Supernatants of all MSCs, TAFs and cocultures were
flasks. Tumor cells were cultured in 6-well suspension culture flask in presence ofcollected 24 hours after medium replacement, filtered through 0.22 µm strainers,

38

cmyk

Fiziologia - Physiology  2010.20.1 (65)Fig. 3. Morphological aspect of tumor-isolated cells before (a) and after (b) removal of dead cells and committed
cells. Cells were grown in suspension cultures and are heterogeneous, with rounded-shape morphology, large
variations in nuclei size and cellular diameter.

Fig. 3. Morphological aspect of tumor-isolated cells before (a) and after (b) removal of dead cells and committed
When co-cultured together with MSCs or TAFs, direct contact between cells lead to isolated
cells. Cells were grown in suspension cultures and are heterogeneous, with rounded-shape morphology, large
cellular clusters with round radial shape. Morphological changes in co-cultures aspect were induced after
variations in nuclei size and cellular diameter.
one week of co-culture, while in week 2 adherent cells started to detach from the culture plate (Figure 4).
As long as we did not inactivate the underlying cells (MSCs or TAFs), the results could indicate either an
Whenco-cultured
togetherwithcells
MSCs
or TAFs,
directcontact
betweencells
increased
proliferation
rate of theseand unfitted
culturesurface,or aggressive
actionfromleadtumorto isolated
Fig. 3. Morphologicaltumor-isolated
cells before (a) andafter (b)removalof deadcells
and induced
committed
cellularclusters
withaspectroundofradial
shape.
Morphologicalchangesinco-culturesaspectwere
after
cells against
adhesionmolecules
or inhibition/stimulatingprocesses
targetingspecificsurfacemarkersofcells. Cellsthese
werecells.
grown in suspension cultures and are heterogeneous, with rounded-shape morphology, largeone
weekinofnuclei
co-culture,indiameter.
week 2 adherent cells started to detach from the culture plate (Figure 4).
variations
size andwhilecellularand stored at -70o C until analyzed in 96-well plates on the reader.

or aggressive action from tumor cells against adhesion molecules or inhibition/
stimulating processes targeting specific surface markers of these cells.

As long as we did not inactivate the underlying cells (MSCs or TAFs), the results could indicate either an
Immunocytochemical Analysis
increased
proliferation
ratetogetherof thesewith
cells
and unfitted
or aggressive
actiontumor
When
co-cultured
MSCs
or TAFs,culture
direct surface,contact between
cells leadto fromisolated
Cells prepared for these analyses were grown in 4-well glass chamber slides, cells
againstadhesionmolecules
inhibition/stimulatingprocesses
targetingaspectspecificsurfacemarkers
cellularclusters
with roundradial or
shape.
Morphological changes
in co-cultures
were
inducedafter ofone weekthesecells.of co-culture, while in week 2 adherent cells started to detach from the culture plate (Figure 4).
and 3-5 days from plating medium was removed, cells were washed, fixed with 4% As long as we did not inactivate the underlying cells (MSCs or TAFs), the results could indicate either anparaformaldehyde and permeabilized with 0.1% Triton X-100 and then investigated increased proliferation rate of these cells and unfitted culture surface, or aggressive action from tumor
cells against adhesion molecules or inhibition/stimulating processes targeting specific surface markers offor expression of the proteins of interest, using for labeling the following antibodies: these cells.
monoclonal mouse anti-swine Vimentin (clone V9), and monoclonal mouse antihuman Cytokeratin (clone MNF116), respectively. Cocultures of both cellular types,abgrown in 2 different media (tumor cells media and media specific to each cellular
Fig.
4. Morphologicalaspect of aspect
coculturesbulk tumor andcells).bulk
Twotumor
types ofcells).
media wereFig.
4. Morphologicalof(MSCs/TAFs
cocultures and(MSCs/TAFs
Two used
typesforofcocultures:
media
layer cells specific media and tumor cells specific media. Cellular behavior was similar using both type of media andtype) were also analyzed considering the same monoclonal antibodies.
were used
for cocultures:
cells
specific
media
and tumor
cellsandspecificmedia.
Cellularbehavior
underlying
cellularsubstrate afterlayer
1 week(a) and2 weeks(b), forming
aggregatesstarting todetach fromthe culture
plate.
was similarusing both type of media and underlying cellular substrate after 1 week (a) and 2 weeks
Immunocytochemical staining revealed increased Vimentin expression in co-cultured TAFs andTumor cells were stained for specific tumor markers: anti-human estrogen
MSCs, mainly when
they wereaggregates
cultured inandtumor
specific
media (Figure
Expression(b), forming
starting
to detachfrom the5).culture
plate.of Cytokeratin in
receptor (ER) and andti-human p53, while cocultured cells were tested for maintumor cells showed they can maintain an epithelial-like phenotype even in culture conditions (Figure 6).
abtenance of tumor specific markers in coculture after 2 weeks.
Immunocytochemical staining revealed increased Vimentin expression in co4. Morphological aspect of cocultures (MSCs/TAFs and bulk tumor cells). Two types of media were used for cocultures:
All primary antibodies were provided by DakoCytomation (Glostrup, Den- Fig.
layer cells cultured
specific media
cells
specificwhen
Cellularwasinsimilarboth media
type of media
amedia.theyb andTAFsandandtumor
MSCs,
mainlywerebehavior
culturedtumorusing
specificmark) and tested for human specificity and cross-reactivity. Staining protocol underlying cellular substrate after 1 week (a) and 2 weeks (b), forming aggregates and starting to detach from the culture plate.
(Figure 5).
Expression of(MSCs/TAFs
Cytokeratin inbulk
tumor
cells
showedthey can maintainan
4. Morphologicalaspect of cocultures
tumorVimentin
cells). Two expression
types of mediainwere
used for cocultures:
Immunocytochemical
staining revealedandincreased
co-cultured
TAFs andcontinued with secondary biotinylated antibody binding, substrate addition, and Fig.
layer cells specific media and tumor cells specific media. Cellular behavior was similar using both type of media andepithelial-likephenotypeevenweeks
intumorcultureconditions
6).
MSCs,
mainlythey
wereculturedmedia (Figure(Figure5).toExpressionof culture
Cytokeratin
underlying
cellularwhen
substrate
after
1 week(a) and 2in(b), specificforming aggregatesand starting
detach from theplate. inhematoxylin counterstaining of the nuclei (LSAB2 System-HRP, Dako) following tumor cells showed they can maintain an epithelial-likeabphenotype even in culture conditions(Figure 6).
a
Fig.
5.
OverexpressionofVimentinin
MSCs
cocultures:a.
tumor-specificmedium,b.
MSCs
specific
medium.
Different
Immunocytochemical
staining revealed increased Vimentin expression in co-cultured TAFs andthe manufacturer
procedures.
Microscopy
performed
on a Nikon
ELISA analysis ofsupernatant was
performed 2 analysis
weeks fromwas
the beginningof cocultures,or themorphological aspects are underlined, MSCs specific medium being more beneficial for proper development of cocultures
next day from plating MSCs and TAFs using Quantikine Immunoassay kits (R&D Systems, Minneapolis, MSCs, mainly when they were cultured in tumor specific media (Figure 5). Expression of Cytokeratin in
Eclipse
E800
microscope.
MN,
USA)
specificfor IL-4, IL-10, IL-13, TGF-β1, TNF-α, IFN-γ, and VEGF. Supernatants of all MSCs, tumor cells showed they can maintain an epithelial-like phenotype even in culture conditions (Figure 6).
TAFs and cocultures were collected 24 hours after medium replacement, filtered through 0.22 µm
strainers, and stored at -70o C until analyzed in 96-well plates on the reader.

Statistic analysis
Statistic analysis was performed using Excell Microsoft Office 2003 (Microsoft
Corporation) software. The central tendencies of the variables were expressed as amean (M), and the dispersion ones as standard deviation (sd). In order to performthe statistic comparisons, „t”-Student test and the variance analysis (ANOVA)
were used for continuous variables. Differences were considered significant for
p < 0.05.

Immunocytochemical Analysis
Cells prepared for these analyses were grown in 4-well glass chamber slides, and 3-5 days fromplating medium was removed, cells were washed, fixed with 4% paraformaldehyde and permeabilizedwith 0.1% Triton X-100 and then investigated for expression of the proteins of interest, using for labelingthe following antibodies: monoclonal mouse anti-swine Vimentin (clone V9), and monoclonal mousebaab
anti-human Cytokeratin (clone MNF116), respectively. Cocultures of both cellular types, grown in 2adifferent media (tumor cells media and media specific to each cellular type) were also analyzed Fig. 5. Overexpression of Vimentin in MSCs cocultures: a. tumor-specific medium, b. MSCs specifica medium.
Different
considering the same monoclonal antibodies.
morphological aspects are underlined, MSCs specific medium being more beneficial for proper development of cocultures
Fig. 5. Overexpression of Vimentin inaMSCs cocultures: a. tumor-specific medium, b. MSCsb
Tumor cells were stained for specific tumor markers: anti-human estrogen receptor (ER) andandti-human p53, while cocultured cells were tested for maintenance of tumor specific markers in Fig. 5. Overexpression
amoreDifferent
specific medium.
Differentin morphologicalaspects
are underlined,
MSCs specificmediumof Vimentin
MSCs cocultures:a. tumor-specificmedium,b. MSCs
specificbeing
medium.
coculture after 2 weeks.
morphological aspects are underlined, MSCs specific medium being more beneficial for proper development of cocultures
beneficial for proper development of cocultures
All primary antibodies were provided by DakoCytomation (Glostrup, Denmark) and tested forhuman specificity and cross-reactivity. Staining protocol continued with secondary biotinylated antibody
binding, substrate addition, and hematoxylin counterstaining of the nuclei (LSAB2 System-HRP, Dako)following the manufacturer procedures. Microscopy analysis was performed on a Nikon Eclipse E800
microscope.

RESULTS

Statistic analysis
Statistic analysis was performed using Excell Microsoft Office 2003 (Microsoft Corporation)
software. The central tendencies of the variables were expressed as a mean (M), and the dispersion ones
as standard deviation (sd). In order to perform the statistic comparisons, „t”-Student test and the varianceanalysis (ANOVA) were used for continuous variables. Differences were considered significant for p <0.05.

Morphological changes of co-cultured cellsabaIsolated bulk tumor cells were cultivated in suspension 6-wells culture flasksab(Gibco)intumor
medium
and
grown
for
1
week.
Mediumwasreplaced
every
3
days
a
RESULTS
Fig. 6. Cytokeratin expression in TAFs cocultures: a. tumor-specific medium, b. TAFs specificwith freshly prepared medium. Removal of dead cells and depletion of committed
Morphological changes of co-cultured cells
medium. Morphological aspects suggest that FCS containing medium could prolong survival rates of
Isolatedtumor cells
were cultivated insuspension
6-wells
culture flasks(Gibco) ofin cells
tumor
fraction
lead bulk
to changed
morphologicalaspectas wellas decreased
numbermedium and grown for 1 week. Medium was replaced every 3 days with freshly preparedFig.
medium.
by increasing theof substratemedium,cells.
6. Cytokeratin expression incocultures
TAFs cocultures:a. adherence
tumor-specificb. TAFs specific medium. Mor
Removalof deadcells and depletionof committed
fraction lead to changed
morphologicalas well(Figure 3),
suggestingthat manyof the tumor-isolated
cells diein cultureaspectmedium,as decreased number of cells (Figure 3), suggesting that many of the tumor-isolated cells die aspects
in culture suggest that FCS containing medium could prolong survival rates of cocultures by increasing the ad
substrate cells.
medium,are alreadycommitted towards
towards otherspecific
lineages.
or they oraretheyalreadycommitted
otherspecific
lineages.
Interestingly, tumors expressing estrogen receptor (ER) (50% of our tumor
samples werepositivefor ER) maintained
theirreceptor
expression(ER)in cocultures,
Interestingly,
tumorsexpressing
estrogen(50% inofboth
ourtypestumor samples werefor ER) maintained
their expression
cocultures,
incould
bothbetypes
experiments,
of experiments,
showing thatincoculturesmodelsa validofin vitro
model for showing that cmodels could tumor
be a valid
in vitromodel for (Figure
tumor7).progression
andprogression
and development
Presence of p53
wasdevelopment
not detected (Figure 7). Pre
p53 was not detected by immunocytochemistry in primary tumors, nor in cocultures.
by immunocytochemistry in primary tumors, nor in cocultures.
abaFig. 3. Morphological aspect of tumor-isolated cells before (a) and after (b) removal of dead cells
and committed cells. Cells were grown in suspension cultures and are heterogeneous, with roundedshape morphology, large variations in nuclei size and cellular diameter.

When co-cultured together with MSCs or TAFs, direct contact between cells
lead to isolated cellular clusters with round radial shape. Morphological changes
in co-cultures aspect were induced after one week of co-culture, while in week 2
adherent cells started to detach from the culture plate (Figure 4). As long as we
did not inactivate the underlying cells (MSCs or TAFs), the results could indicate
either an increased proliferation rate of these cells and unfitted culture surface,
2010.20.1(65)  Fiziologia - Physiology

cmyk

a

b

a
Fig. Fig.
7. Estrogen
receptor
expressionin cocultures:
a. MSCsb.cocultures,
7. Estrogen
receptor(ER)(ER) expressionin cocultures:
a. MSCs cocultures,
TAFs coculturesb. TAFs cocultures

Microenvironment-induced changes in cocultured cells
39
Conditioned medium decreased the MSCs proliferation rate, at all cellulardensities, independent manner, and the results of the MTT assay were evaluated on a BioRad Plate
subtracting the background absorbance at 590 nm from the specrophotometrically measured ab
at 570 nm (Figure 8). TAFs proliferation rate decreased in a dose-dependent manner, but for
50% and 100% conditioned media, the cellular expansion reached a plateau phase.

IL-10pg/ml pg/ml

16
14
16
12
14
10
12
8
10
6
8
4
6
2
4
0
2

TAFs

MSCs

TAFs coculture

MSCs coculture

0

a

b

MSCs
coculture
Fig. 9. Increased IL-10 secretion in (p<0.001) TAFs cocultures comparedwithboth TAFs and MSCs
TAFs

MSCs

TAFs coculture

pg/ml pg/ml

Microenvironment-induced changes
in coculIL-4
9. Increaseda Fig.
Fig. 7. Estrogen receptor (ER) expression in cocultures: a. MSCs cocultures,b. TAFs
coculturesIL-10 secretion in (p<0.001) TAFs cocultures compared with both TAFs and MSC
tured cells
0.18
IL-4
0.16
Conditioned medium decreased the MSCs proliferation rate, at all cellular
0.18
0.14
roenvironment-inducedin cocultured
densities, inchanges
a dose-dependent
manner, cells
and the results of the MTT assay were
0.16
0.12
Conditioned evaluatedmediumondecreasedtheMSCs
proliferation
rate,
at
all
cellulardensities,
ina dose0.14
0.1
a BioRad Plate Reader, subtracting the background absorbance at 590
endent manner, and the results of the MTT assay were evaluated on a BioRad Plate 0.12
Reader,0.08
nm from the specrophotometrically measured absorbance at 570 nm (Figure 8). TAFs
0.1
0.06
racting the background absorbance at 590 nm from the specrophotometrically measured absorbance
0.08
0.04
proliferation
rate decreased inrate
a dose-dependent
manner,but for values of 50%
and but for values
70 nm (Figure 8).
TAFs proliferation
decreased ina dose-dependent
manner,
of
0.06
0.02
100% conditioned
media,
the cellularexpansion
reachedaa plateau
plateau phase.
% and 100% conditioned
media, thecellular
expansion
reachedphase.
0.04
0
0.02

MTT MSCs 24h
0,2
0,16
0,14

TAFs coculture

MSCs coculture

TGF beta1

4500

0,1

4000
5000
3500
4500

0,08

pg/ml

0,06
0,04
0,02
0
20% IM

30% IM

50% IM

100% IM

Imduced medium proportions

MTT TAFs 24 h
0,12
0,1

590 nm extinction

MSCs

5000

0,12

0,08

pg/ml

590 nm Extinction

0,18

TAFs

0

Fig.10. IL-4 secretion is decreased in bothTAFs
types of cocultures.
Probably
the effectMSCs
ofcoculture
direct inhibition of imm
MSCs
TAFs coculture
Fig.10. IL-4 secretion is decreased in both types of cocultures. Probably the effect of direct inhibifrom the TAFs
Fig.10. IL-4 secretion is decreased in both types ofProbablytion cocultures.
of immune response
from the TAFsthe effect of direct inhibition of imm
TGF beta1
from the TAFs

3000
4000
2500
3500
2000
3000
1500
2500
1000
2000
500
1500
0
1000

500

TAFs

MSCs

TAFs coculture

MSCs coculture0
Fig. 11. TGF-β1 secretion in coculturesmedia
is significantly
decreasedcomparedMSCs
withboth normal stromal ce
TAFs
MSCs
TAFs coculture
coculture(p<0.001), consistent with its role in cellular apoptosis, proliferation inhibition and cell deathFig. 11. TGF-β1 secretion in cocultures media is significantly decreased compared with both normal stromal c(p<0.001), consistent with its roleFig.
in cellular
proliferation
inhibition
andcell death
11. TGF-β1apoptosis,
secretion in cocultures
media is significantly
decreasedcomparedwith both

Our results indicate that cytokines profile is definitely modified within the cocult
normal stromal cells and TAFs (p<0.001), consistent with its role in cellular apoptosis, proliferationcompared with the MSCs or TAFs alone. IL-4inhibition
and TGF-β1
is decreased, while IL-1
death secretion
Our results indicate that cytokines profile andiscelldefinitely
modified within the cocul
increased values in both coculture types, thus supporting the evidence that stromal cell com
0,02
compared with the MSCs or TAFs alone. IL-4 and TGF-β1 secretion is decreased, while IL-10
required for maintenance and proliferation
of
cells
9,
10, 11).
Our results
indicatethus
thattumor
cytokines
profile(Figuresisthedefinitely
modifiedwithin stromal
the
20% IM
30% IM
50%
IM
100 IM
increasedvaluesin both coculture
types,
supporting
evidencethatcell com
Induced medium proportions
cocultures,
while comparedofwith
the MSCsor TAFs
alone. IL-4 andTGF-β111).
secretionrequiredformaintenanceandproliferation
tumor
cells
(Figures
9,
10,
DISCUSSIONS
is decreased, while IL-10 presented increased values in both coculture types, thus
Fig. 8. MTT assay of MSCs and TAFs cultured in tumor conditioned media.Soluble
In both cases thefactors secreted by TAFs (and in lesser extent by MSCs) such as IL-10 are
supporting the evidence that stromal cell compartment is required for maintenanceproliferation and expansion of cells decreases progressively, reachinga plateau (lag phase) for 50%
DISCUSSIONS
synthesis of pro-inflammatory
cytokines
like
IL-2, IL-3, TNF-α and GM-CS
and 100% conditioned mediuminhibiting
and proliferationof tumorcells (Figures
9, 10,IFN-γ,
11).
Solublefactorssecreted
byexpansion
TAFs
in lesser
extent by the
MSCs)
such
as IL-10areassay of MSCs and TAFs cultured in tumor conditioned media.
In bothcases
the proliferation
ofhelperby cells
suchmacrophages
andandthetype
1 T(andcells infiltratingtumor.
IL-10
also dispinhibiting
synthesisofpro-inflammatory
cytokines
like
IFN-γ,
IL-2,
IL-3,
TNF-α
and
GM-CS
s progressively, reaching a plateau (lag phase) for 50% and 100%
conditioned
mediumabilities
tonormalized
suppress
presentation capacity of antigen presenting cells.
Cell culture supernatants were collected 24 hoursby
fromplating,
to the antigen
cells
such
macrophages
andthe type 1 T helper cells infiltrating the tumor. IL-10 also dis
DISCUSSIONS
cell number, filtered through 0.22 μm strainer, and stored
at -80oCtountil
analyzed. the antigen
abilities
suppress
presentation
culture supernatants were collected 24 hours from plating, normalized to cell number, filteredcapacity of antigen presenting cells.
ELISA method revealed important differences in IL-10, IL-4 and TGF-β1 secretion,
Soluble importantfactors secreted by TAFs (and in lesser extent by MSCs) such as IL-1022 μm strainer, and stored at -80oC until analyzed. ELISA method revealed
when comparing MSCs and TAFs, as well as their complementary cocultures.
arecapableofsynthesis of pro-inflammatory cytokines like IFN-γ, IL-2,
in IL-10, IL-4 and TGF-β1 secretion, when comparing MSCs and TAFs, as wellinhibiting
as their
Secretion of cytokines with direct suppressive effect, such as IL-10 was significantly
IL-3,
TNF-α
and
GM-CSF
produced by cells such macrophages and the type 1 T
tary cocultures. Secretion of cytokines with direct suppressive effect, such as IL-10 wasincreased, thus suggesting the role of stromal cell in supporting and promoting
helpercells
infiltratingthey increased, thus suggesting the role of stromal cell in supporting and promoting tumor celltumor. IL-10 also displays potent abilities to suppress
tumor cell invasiveness and progression. TGF-β1 was determined as active form
the antigen presentation
capacity of antigen presenting cells.
s and progression. TGF-β1 was determined as active form in cocultures supernatant,the entire
in cocultures supernatant, the entire amount of latent form being activated by
TGF-β1ismultifunctional
latent form being activated by the ELISA kit used for this assay, thus being impossible to peptide that controls proliferation, differentiation,
the ELISA kit used for this assay, thus being impossible to determine the active
and other functions in many tissues. TGF-β1 inhibits proliferation and can induce
he active amountab initio.
amount ab initio.
apoptosis in various cell types, but on the other hand, most human tumors produce
this cytokine whose autocrine and paracrine actions promote cancer cell invasiveness
IL-10
and metastasis, epithelial to mesenchymal transition and regulates intratumoral
16
angiogenesis [20]. Enhanced TGF-β1 production by TAFs may be a critical factor
14
12
in tumor homeostasis not so much for the cancer cells directly, but for the TAFs
10
themselves, via autocrine mechanisms, allowing them to proliferate and preserve8
their activated fibroblast behavior and supportive activity for the tumor. Moreover,6
TGF-β1 secreted by TAFs could suppress activation, proliferation and differentiation of42
the cytotoxic T lymphocytes, NK cells and macrophages recruited within the tumor,
0
impairing immune surveillance and antitumoral immunity. The TGF-β-pathway is
TAFs
MSCs
TAFs coculture
MSCs coculture
one MSCs.
of the major pathways altered in tumors, including breast cancer (12). Bindig. 9. Increased IL-10 secretion in (p<0.001) TAFs cocultures compared with both TAFs andingof
TGF-β to the TGF-β receptor II (TβRII) activates the signal-transducing TGF-β
IL-4
Fig. 9. Increased IL-10 secretion in (p<0.001)
TAFs cocultures compared with both
receptor
I (TβRI) that phosphorylates latent cytoplasmic transcriptional activators,
TAFs and MSCs.
0.18
Smad2 and Smad3. They then, together with Smad4, translocate into the nucleus
0.16
0,06

pg/ml

0,04

0.14
0.12

Fiziologia - Physiology  2010.20.1 (65)pg/ml

40 0.1
0.08
0.06

cmyk
0.04
0.02

0
TAFs

MSCs

TAFs coculture

MSCs coculture

ecretion is decreased in both types of cocultures. Probably the effect of direct inhibition of immune responsewhere they regulate TGF-β-dependent gene transcription (13, 14, 15). In breast
cancer, TGF-β-signaling was shown to reduce growth of the primary tumor but
also to promote metastasis, indicating that the apparent effect of TGF-β depends
on its cellular context (16).
Both stromal cells types that we considered for our in vitro design had aprofoundly altered morphological aspect, cells loosing the spindle shape, suffering
destruction of cell membranes. Over-expression of Vimentin in co-cultured stromalcells may be a result of cellular injury induced by the tumor cells through their directcontact or different factors secreted into the microenvironment.
Relatively dose-dependent and time-dependent decrease of MSC/TAF
proliferation after exposure to conditioned tumor medium may suggest that initialstroma-tumor microenvironment interaction may lead to drastic changes in MSCsor TAFs behavior. Long-term culture in conditioned media induces habituation ofstromal cells, and even normal proliferation behavior.
We may also comment on tumor cells ability to maintain expression of tumor
markers estrogen receptors - ER (breast cancer) in cocultured cells. Although initiallythe tumors presented low expression of these markers (50%), we were able to detect
their presence in coculture conditions. There is evidence that estrogen, by regulating
expression and secretion of angiogenic factors such as VEGF, can also stimulate angiogenesis (17,18), so that presence and maintenance of ER in coculture is required
for formation of vessels and supportive role in tumor progression.
MSCs and TAFs can decrease in vitro proliferation of tumor cells, but theirpotential to inhibit tumor progression and development may be limited under the
influence of different tumor aggressive factors secreted into the microenvironment,
as well as by stroma-tumor cells cross talk.
Acknowledgements
This work was supported by PNCDI-II – Parteneriate, project number 41019/2007 and by CNCSIS –UEFISCSU, project number PNII – IDEI 1748/2008
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3. Studeny M, Marini FC, Dembinski JL, Zompetta C, Cabreira-Hansen M, et al.

Mesenchymal stem cells: potential precursors fro tumor stroma and target-delivery
vehicles for anticancer agents. J Natl Cancer Inst 2004; 96: 1593-1603.
4. Studeny M, Marini FC, Champlin RE, Zompetta C, Fidler IJ, et al. Bone marrowderived mesenchymal stem cells as vehicles fro interferon-beta delivery into tumors.
Cancer Res 2002; 62: 3603-3608.
5. Nakamizo A, Marini F, Amano T, Khan A, Studeny M, et al. Human bone marrowderived mesenchymal stem cells in the treatment of gliomas. Cancer Res 2005;
65: 3307-3318.
6. Sonabend AM, Ulasov IV, Tyler MA, Rivera AA, Mathis JM, et al. Mesenchymal
stem cells effectively deliver an oncolytic adenovirus to intracranial glioma. StemCells 2008; 15: 124-129.
7. Wels J, Kaplan RN, Rafii S, Lyden D. Migratory neighbors and distant invaders:Tumor-associated niche cells. Genes Dev 2008; 22: 559-574.
8. Udagawa T, Puder M, Wood M, Schaefer BC, D’Amato RJ. Analysis of tumorassociated stromal cells using SCID GFP transgenic mice: contribution of local andbone marrow-derived host cells. FASEB J 2006; 20: 95-102.
9. Koyama H, Kobayashi N, Harada M, Takeoka M, Kawai Y, et al. Significance oftumor-associated stroma in promotion of intratumoral lymphangiogenesis: pivotal roleof hyaluronan-rich tumor microenvironment. Am J Pathol 2008; 172: 179-193.
10. Dong J, Grunstein J, Tejada M, Peale F, Frantz G, et al. VEGF-null cells requirePDGFR alpha signaling-mediated stromal fibroblasts recruitment for tumorigenesis.
EMBO J 2004; 23: 2800-2810.
11. Jodele S, Chantrain CF, Blavier L, Lutzko C, Crooks GM, et al. The contributionof bone marrow-derived cells to the tumor vasculature in neuroblastoma is matrix
metalloproteinase-9 dependent. Cancer Res 2005; 65: 3200-3208.
12. Roberts AB, Wakefield LM. The two faces of transforming growth factor beta in
carcinogenesis. Proc Natl Acad Sci U S A. 2003; 100(15):8621-3.
13. Heldin CH, Miyazono K, ten Dijke P. TGF-beta signalling from cell membrane
to nucleus through SMAD proteins. Nature 1997; 390(6659): 465-71.
14. Massagué J, Blain SW, Lo RS. TGFbeta signaling in growth control, cancer,
and heritable disorders. Cell 2000;103(2): 295-309.
15. Wakefield LM, Roberts AB. TGF-beta signaling: positive and negative effects
on tumorigenesis. Curr Opin Genet Dev. 2002;12(1):22-9.
16. Muraoka RS, Koh Y, Roebuck LR, Sanders ME, Brantley-Sieders D, Gorska
AE, Moses HL, Arteaga CL. Increased malignancy of Neu-induced mammary
tumors overexpressing active transforming growth factor beta1. Mol Cell Biol.
2003; 23(23): 8691-703.
17. Babischkin JS, Bonagura TW, Udoff LC, Vergara CO, Johnson HW, Atlas RO,
Pepe GJ, Albrecht ED. Estrogen stimulates the human endometrium to express afactor(s) that promotes vascular smooth muscle cell migration as an early step inmicrovessel remodeling. Endocrine, 2009 Feb; 35(1): 81-8.
18. Albrecht ED, Babischkin JS, Lidor Y, Anderson LD, Udoff LC, Pepe GJ. Effectof estrogen on angiogenesis in co-cultures of human endometrial cells and microvascular endothelial cells. Hum Reprod. 2003;18(10):2039-47.

INTERACTIUNEA DINTRE MICROMEDIUL TUMORAL SI
COMPARTIMENTUL STROMAL – Partea I
REZUMAT
Micromediul tumoral cuprinde celule imunocompetente si inflmatorii, celule endoteliale si fibroblasti. Toate aceste tipuri celulare prezinta o influenta
graduala si etapizata asupra procesului carcinogenetic si aparitiei fenotipului malign. Este cunoscut faptul ca fibroblastii participa la reactiile imune in
timpul distrugerii sau injuriei tisulare prin modularea mediului local celular si secretiei de citokine, ajustand cinetica si componentele infiltratului inflamator,
precum si prin modularea statusului functional al celulelor imunocompetente. Scopul acestui studiu a fost investigarea rolului celulelor stromale in supresia
raspunsului imun, ceea ce ar putea avea o influenta remarcabila asupra progresiei celulelor tumorale. Celulele stem mezenchimale (MSCs) si fibroblastii
tumorali (TAFs) au fost folositi intr-un studiu comparativ pentru caracterizarea morfologica si imunofenotipica a interactiunilor dintre compartimentul
stromal si celulele cu potential tumorigenic.
Cuvinte cheie: celule stem mezenchimale, fibroblasti tumorali, celule tumorale, micromediu

2010.20.1(65)  Fiziologia - Physiology

cmyk

41

FROM INACTIVE CARRIER TO REACTIVATION OF
HEPATITIS B VIRUS
DIANA NICOLIŢĂ1, IOAN SPOREA1, ROXANA ŞIRLI1, ISABEL DAN1, CORINA VERNIC2
1
Department of Gastroenterology and Hepatology
2
Department of Medical Informatics and Biostatistics“Victor Babes” University of Medicine and Pharmacy, Timisoara

ABSTRACT
Background and aim: The natural evolution of the hepatitis B virus infection depends on the host immune system status. The aim of this paper is to evaluate
time dynamics of the HBV-DNA viral load in HBsAg carriers in order to see how many of them remain in an inactive state and how many progress towards
chronic hepatitis. Material and method: We included in our study 34 patients diagnosed initially as hepatitis B virus carriers for which we analyzed repeated
HBV-DNA viral loads starting from 2 to 6 quantitative determinations, as well as levels for alanine aminotransferase (ALT). According to recent guides, wedefined the upper limit of the serum HBV DNA viral load to be bellow 2.000 IU/ml (<10.000 copies/ml) in order to consider the patient an HBsAg carrier.
Eco-guided liver biopsy was performed to those patients who developed chronic hepatitis and histologic activity index was taken into consideration.
Results: From the total number of 34 patients, 17 were women (50%) and 17 men (50%). The mean for age was 49.02 ±11.6 years (minimum 23 years,
maximum 69 years). The average for the patient’s follow up period was 35.47±3 months with a minimum of 14 months and a maximum of 118 months.
Nine patients (26.47%) developed significant viral replication during this period of time: 4 women (44.4%) and 5 men (55.5%) after a medium periodof 38.4 months. During the follow up period, the data showed that there was a weak positive association between the viral load value and the one of theserum ALT, though not statistically significant (Pearson correlation r=0.425, p=0.115).
Conclusions: Approximately 1 out of 4 hepatitis B virus carriers developed significant viral replication after an average follow up period of 3 years. The hepatitis
B virus carriers should be evaluated periodically in order to discover a possible significant viral replication that could beneficiate from proper treatment.
Key words: hepatitis B virus carrier, reactivation, HBV-DNA viral load

INTRODUCTION

MATERIAL AND METHOD

Hepatitis B virus (HBV) is a worldwide spread viral pathogen with multiple genotypes andmolecular variants, leading to a great variety of stages for chronic HBV infection. Understanding the dynamic of chronic HBV infection is essential in the future management of HBVcarriers. Four phases of HBV infection are described: immune tolerance, immune clearance,
inactive carrier state and last but not least reactivation (28). If the disease is not controlled
and the virus keeps replicating, complications such as cirrhosis and hepatocellular carcinoma
can become inevitable.
It is known that in the Asia-Pacific region approximately 10% of people infected with HBV
are actually in the carrier state. Approximately 25-40% of them will eventually die of liverdisease (cirrhosis complicated or not with hepatocellular carcinoma with a death rate of
50% for male carriers and 15% for female carriers). That is why patients with chronic HBV
infection must be continuously monitored and treated at the right moment, in order not towaste precious time (11,17).
In highly endemic regions, such as Asia and Africa, 8-15% of the population has positive
HBsAg mostly due to perinatal transmission and horizontal infection in childhood, while in
low endemic areas, such as Western countries, the infection occurs mostly in adolescentsand adults, due to high risk sexual behaviors, drug injections or insufficiently sterilizedmaterial (18).
Continuous discoveries are being made in order to have a better understanding of the natural
evolution of HBV infection. It is well known that the host immune system status is essential forthe further development of the disease. Not all patients go through all stages, some of themremain carriers all their life, and others develop chronic hepatitis with further progression tocirrhosis or hepatocellular carcinoma (3).
The aim of this paper is to evaluate time dynamics of the HBV-DNA viral load in HBsAg carriers
in order to see how many of them remain in an inactive state and how many progresses
towards chronic hepatitis.

We performed a retrospective study in which we included 34 patients
initially diagnosed as inactive HBsAg carriers, according to the following criteria:
positive HBsAg for more than 6 months; negative HBeAg; positive HBe antibodies;persistently normal AST, ALT levels; serum HBV DNA <2.000 IU/ml (<10.000
copies/ml). Further on, we established two groups referring one that remained
inactive carrier (25 patients) and another one that developed significant viral
replication (9 patients).
Both HBeAg and HBe antibodies, as well as HBsAg were determined by using
an Axsym Analizor, by means of MEIA Abbott method.
Each patient had repeated HBV-DNA viral load measurements (2 up to 6
quantitative determinations), during a maximum of 118 months follow-up period.
The viral load was quantified by means of real-time PCR (m2000sp/m2000rt Abbott) with a detection limit of 51 copies HBV DNA/ml (1.71 log copies HBV DNA/ml). PCR assays allow the detection of very low serum HBV DNA levels and they
are more reliable than hybridization techniques (24), so this particular technique
is more frequently used nowadays.
According to recent guidelines, an arbitrary serum HBV DNA level of 2.000
IU/ml has been proposed to differentiate HBeAg-negative chronic hepatitis B fromthe inactive carrier state (14).
Alanine aminotransferase (ALT) is the most commonly used enzyme forthe evaluation of liver disease status. In our group of patients the upper normal
limit for ALT was 41 U/l, while for AST it was 38 U/l. All patients had serum ALT
determined every 6 months and some of them underwent liver biopsy in cases ofrepeated increased ALT activity.

Received December 2009. Accepted February 2010. Address for correspondence: Diana Nicoliţă, Department of Gastroenterology and Hepatology, “Victor Babes” University of
Medicine and Pharmacy Timisoara, RO-300041, Ofcea Street No. 19, e-mail: diana1310tm@yahoo.com
42

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Fiziologia - Physiology  2010.20.1 (65)

RESULTS
Out of the 34 patients initially diagnosed as inactive HBsAg carriers, thedistribution according to gender was equal: 17 women (50%) and 17 men (50%).
The mean age was 49.02±11.6 years (minimum 23 years, maximum 69 years).
Referring to the follow-up period, our goal was to include in the study thoseparticular patients who were monitored during an extensive period of time, in order
to be able to detect a possible reactivation of the disease. For fulfilling that purpose,the mean follow-up period was 35.47±3 months, with a minimum of 14 months
and a maximum of 118 months. Nine patients (26.4%) developed significant viral
replication, 4 women and 5 men, after a mean follow-up period of 38.4 months.
The rest of 25 patients remained inactive carriers during the entire follow-up period.
There was no significant statistical difference between the mean age of the group
who remained inactive carriers compared to the one who became replicative(p=0.64). Further on, the results will show the fluctuation of liver enzymes for all
patients involved in this study.
All 9 patients presented ALT flares during their follow-up period. Moreover, whenhigh viral replication occurred, concomitant increase of serum alanine aminotransferase(ALT) level was observed in 66.6% of cases (6 out of 9 patients) (Fig. 1).
250
225

Patient 1

200

Patient 2

ALT (U/l)175

Patient 3

150

Patient 4

125

Patient 5

100

Patient 6

75

Patient 7
Patient 8

50

Patient 9

25
0
1

2

3

4

5

6

repeated ALT values

Fig. 1. ALT variation in patients with significant viral replication

The situation was slightly different for the 25 patients who remained inactive
2010.20.1(65)  Fiziologia - Physiology

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carriers according to repeated serum HBV DNA levels smaller than 2.000 IU/ml(<10.000 copies/ml). More exactly, 7 out of 25 patients (28%) had elevated ALT
values at least once during their follow-up period (Fig.2). There were no significant
statistical differences concerning the frequency of increased ALT levels between thetwo groups (p=0.0995).
300
250
200

ALT 1
ALT 2
ALT 3

150

ALT 4
ALT 5

100
50

25

23

nt

pa

tie

21

nt

pa

tie

19

nt
tie

17

nt
tie

pa

15

nt
tie

pa

pa

13

nt

nt

tie

tie

pa

9

11
nt

tie

pa

7

nt
tie

pa

pa

5

nt
tie

3

in
t
te

pa

nt

pa

tie

tie
pa

pa

nt

1

0

Fig. 2. Alanine aminotransferase variation in HBsAg carriers

We could not find a correlation between the initial viral load value and theone of the serum ALT (Pearson correlation r=0.003, p=0.99), but during the follow
up period, the data showed that there was a weak positive association between theviral load value and the one of the serum ALT, though not statistically significant(Pearson correlation r=0.425, p=0.115).
Among patients initially diagnosed as hepatitis B virus carriers, 9 (26.4%)of them developed significant viral replication together with the increase of ALT
values, though without a direct correlation between them. (Fig. 3). Liver biopsy was
mandatory in order to be able to correctly quantify the damage of the liver.
Patient 1
Patient 2

viral load (copies/ml)

All serum samples were processed in the same laboratory.
In some of the patients, liver biopsy was performed echoassisted using
Menghini type modified needles, 1.4 and 1.6 mm in diameter. Only liver biopsy
fragments with a length of at least 2 cm, including at least 8 portal tracts wereconsidered adequate for pathological interpretation. The liver biopsies were assessed
according to the Knodell score by a senior pathologist. The Knodell score is composedof four individually assigned numbers that summarize a single score reflecting theextent of liver inflammation and fibrosis. The first component (periportal and/or
bridging necrosis) was scored from 0 up to 10. The next two components (intralobular degeneration and portal inflammation) were scored 0-4. The combinationof these three markers led towards the following description:
0 = no inflammation
1-4 = minimal inflammation
5-8 = mild inflammation
9-12 = moderate inflammation
13-18 = marked inflammation.
The fourth component indicates the amount of fibrosis: 0=no fibrosis up to 4=
cirrhosis. Each patient had signed an informed consent form before the liver biopsy.
Statistical analysis
Data were expressed as mean ± standard deviation, frequency; Pearson
correlation coefficient was calculated and afterwards t-test was used to determine
the significance of correlation between level of viral load and level of serum ALT. We
used unpaired t-test for comparison the means between the two groups, as well as
chi square test to compare percentages. A p value <0.05 was considered statisticallysignificant. The statistical analysis was performed using specialized programs suchas SPSS 10, Open Epi 2.3 and Microsoft Excel 2007.

Patient 3

100000000

Patient 4

1000000

Patient 5

10000

Patient 6
Patient 7

100

Patient 8

1
1

2

3

4

5

6

7

Patient 9

repeated HBV-DNA values

Fig. 3. Viral load variation in patients who developed significant viral replication

In our study, liver biopsy was performed in 77.7% (7 out of 9) patients inwhich reactivation occurred. No cases of cirrhosis were found. The mean value forfibrosis was 1.4±1.1 for those who developed significant viral replication, while
the mean value for activity (HAI) was 8.8±3.2. (Table I)
Table I. Histologic activity index in patients who underwent liver biopsy
Knodell
Necro-inflammatory
Fibrosis
Score
activity
Patient 1
12
3
Patient 2
9
1
Patient 3
2
0
Patient 4
11
1
Patient 5
10
1
Patient 6
9
3
Patient 7
91

DISCUSSIONS
Hepatitis B virus carriers have three possible ways of evolving: they can either
develop protective HBs antibodies together with HBsAg clearance, or remain in an
inactive state, while some of them may develop chronic hepatitis together with its
complications: cirrhosis and hepatocellular carcinoma.
The bright side of being an inactive carrier is that some of them eventually
become HBsAg negative and develop protective HBs antibodies. The approximate
HBsAg clearance is estimated to be 1%-2% per year in Western countries, where HBV
infection is acquired during adulthood, and 0.05%-0.8% per year in endemic areas,
where HBV infection is mostly acquired prenatally or in early childhood (21,8).
43

For a better understanding of the natural evolution of the disease, it would
be appropriate to describe briefly its four phases.
Immune tolerance stage
HBV infection acquired perinatally or in early childhood has a tendency to
evolve towards an initial tolerance stage characterized by: positive HBeAg, highDNA levels, but with normal ALT and minimal or no inflammation on liver biopsy(12). This is rarely the case of adult-acquired disease which usually evolves towards
chronic hepatitis. Positive HBeAg is proven to function as an immunoregulatory
protein that promotes chronicity (20). This may be a logical explanation for thehigh chronic HBV infection rate (90%) observed in babies infected by their HBeAg
positive mothers. Published data proved that the immune tolerance stage can last
1 to 4 decades. During this phase, the rate of spontaneous or treatment induced
HBeAg seroconversion is less than 5% (5).
Immune clearance stage
This phase is characterized by: positive HBeAg, high serum HBV DNA and ALTlevels, accompanied by active inflammation and fibrosis in the liver. These flares ofhepatitis may precede the disappearance of HBeAg and the appearance of HBeAg
antibodies. Seroconversion is benefic for the patients’ health status, as several
studies had shown, being associated with biochemical and histological remissionof inflammatory activity in most patients (19,7,25). Usually regression of fibrosis
occurs several months or years after the HBeAg seroconversion. Both the durationof immune clearance phase and the frequency and severity of flares are responsible
for the progression towards cirrhosis and hepatocellular carcinoma (16).
Inactive HBsAg carrier stateThis stage is characterized by positive HBsAg for more than 6 months, negative
HBeAg, positive HBe antibodies, serum HBV DNA < 2.000 IU/ml (<10.000 copies/ml), persistent normal AST, ALT levels, as well as by the absence of significant hepatitis on liver biopsy (22). Those patients are not candidates for treatment initiation,
but they should be periodically monitored in order to discover a possible reactivationin an early phase, so that they should be treated properly and effectively.
Reactivation stage
Approximately one third of the inactive HBsAg carriers without serum reversion of
HBeAg may develop an HBeAg-negative chronic hepatitis (27). It can occur either spontaneously or due to immune suppression in inactive carriers. Long term prognosis is poor.
Long term longitudinal studies on inactive HBsAg carriers have reported that
15% up to 24% of patients developed in time HBeAg negative chronic hepatitis;
20%-30% had moderate to severe inflammation, while up to 20% had advanced
fibrosis or cirrhosis (15,9).
Fattovich G and al. performed a study on a cohort of 70 Caucasian patientsobserved during a median follow-up period of 25 years. The study showed thatsixty-one (87%) patients underwent spontaneous HBeAg seroconversion. During a
median period of 22.8 years after HBeAg seroclearance, 40 (66%) patients became
inactive carriers, whereas the remaining 21 (34%) showed alanine aminotransferase
elevation: one (1%) had HBeAg reversion, nine (15%) detectable serum HBV DNA
but were negative for HBeAg, eight (13%) concurrent virus infection and three(5%) concurrent non-alcoholic fatty liver disease. Liver-related death occurred in
11 (15.7%) patients, caused by hepatocellular carcinoma in five and liver failurein six (9). Most patients with HBeAg seroconversion became inactive carriers withvery good prognosis.
The REVEAL (Risk Evaluation of Viraemia Elevation and Associated Liver
Disease)-HBV study group followed a cohort of 3653 HBsAg-positive patients in
Taiwan (4). All of them acquired HBV infection perinatally. A baseline high HBV-DNAlevel > 10 000 copies/mL was associated with a significant increased risk of HCC (4)and with progression towards cirrhosis (13). Persistently high levels of viral replication, for up to four decades were correlated to highest risk for HCC/cirrhosis (1).
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The viral evolution in inactive HBsAg carriers is usually benign. A long term
follow-up (up to 18 years) of these carriers showed a very low risk of developing cirrhosis or hepatocellular carcinoma (2). Published data showed that the major factor
for the development of HBV related hepatocarcinoma is the immune system (10).
These patients must be periodically monitored by means of noninvasive methods
such as abdominal ultrasound and transient elastography and also, by repeated viralload and AST and ALT measurements, since it is well known that multiple episodesof reactivation can lead to progressive liver destruction. It is extremely important forphysicians to screen for HBV infection and to monitor liver disease progression in HBVcarriers by using both serological and virological markers, so that effective treatment
can be initiated early before the development of advanced liver disease (14).
According to literature, ALT levels can continue to increase without flare or haveintermittent flares on a continuous elevation (22). It is crucial to be able to identify
the patients that are no longer healthy carriers and who require proper treatment
in order to have a chance of healing. Tai DI, Lin SM, Sheen IS et al. (26) studied theprognostic value of ALT in patients with chronic hepatitis B virus infection. A totalof 4376 asymptomatic hepatitis B e antigen (HBeAg) negative, surface antigen(HBsAg) carriers with baseline ALT less than 2 times the upper limit of normal (ULN)
were monitored with periodical ALT measurement during a period of three years.
Baseline ALT level was normal in 3673 subjects and increased to abnormal levelin 1720 (46.8%) cases. The incidence of liver cirrhosis, hepatocellular carcinoma
and mortality was correlated with increasing maximal ALT level during follow-up,
especially in those with maximal ALT of at least 2 times ULN, as compared to those
who maintained normal ALT values. The conclusion was that persistently normal ALT
values was associated with excellent long-term prognosis, while increased ALT levelsof at least 2 times ULN was associated with increasing morbidity and mortality. ALTof at least 2 times ULN is therefore an appropriate threshold for anti-HBV therapy,
whereas those with ALT 1 to 2 times ULN require liver biopsy for decision. Our study
had also shown that increased ALT values were correlated with reactivation of thedisease as demonstrated by the increase of HBV-DNA viral load.
A Greek study performed by George Zacharakis and al. revealed that 4 out
of 195 (2%) patients initially classified as inactive carriers developed reactivationand had HBV-DNA viral load > 2000 IU/ml (29). The follow-up period was ofmaximum 12 years.
In the study performed by Chu and al. (6), a total of 1,965 inactive HBsAg carriers were evaluated (mean age 35.6 years; males: 1,076). During an 11.5 years meanfollow-up, 314 carriers (13%) developed HBV hepatitis reactivation (ALT flares morethan twice the upper limit of normal and positive HBV DNA by means of hybridization
assays). The risk of HBV reactivation significantly correlated with advanced age whenentering the study (p<0.0001) and male gender (p<0.0001). A total of 57 patientsdeveloped cirrhosis, the cumulative incidence being 15% after 25 years. The risk of
cirrhosis significantly correlated with advanced age at entry (p=0.004) and HBV reactivation (p<0.0001). From the 1,651 carriers without HBV reactivation, 10 developed
cirrhosis, and advanced age at entry was the only significant factor (p=0.03). From the314 patients with HBV reactivation, cirrhosis developed in 47, the cumulative incidence
being 8, 16, 27, and 46% at 5, 10, 15, and 20 years after reactivation. Male gender(p=0.037) and advanced age at reactivation (p=0.006) were the two independentrisk factors. The conclusion of this study is that the so-called inactive carrier state cannot
be generally viewed as an innocent, long-lasting condition with good prognosis, sothat regular follow-up is necessary.
Another Greek study conducted by Papatheodoridis GV and al. (23) evaluatedthe severity of liver histology and the presence of histological indication for treatment
in patients with HBeAg-negative chronic HBV infection focusing on those with low
viremia and/or normal alanine aminotransferase. They included 399 patients withincreased ALT and detectable serum HBV DNA (chronic hepatitis B patients) and 35
Fiziologia - Physiology  2010.20.1 (65)cases with persistently normal ALT and HBV DNA <2.000 IU/mL (inactive carriers).
Histological indication for treatment was present in 17% (6/35) of inactive carriers
due to moderate (stage 2) fibrosis without active necroinflammation. Minimal
histological lesions were observed in the majority of HBeAg-negative patientswith persistently normal ALT and low HBV DNA viral load, who may not require
immediate liver biopsy and treatment but only close follow-up.
Our study showed that the mean value for fibrosis was 1.4±1.1 for those who
developed significant viral replication, while the mean value for activity (HAI) was
8.8±3.2 showing a mild/moderate inflammation of the liver. Moderate fibrosis wasfound in 28.5% of patients. The beginning of an antiviral treatment is mandatory.
Our study had also its limitations such as: the small number of patients, aswell as the absence of histology in “healthy” carriers.

CONCLUSIONSIn our study, approximately 1 out of 4 inactive HBsAg carriers (26.4%)
developed significant viral replication after an average follow up period of 3 years(mean 38.4 months).
Inactive HBsAg carriers should be evaluated periodically by repetitive viralload assessment every 3-6 months, so that disease reactivation is diagnosed assoon as possible and a proper treatment is started.
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1. Bárcena Marugán R, García Garzón S. DNA-guided hepatitis B treatment, viral load
is essential, but not sufficient. World J Gastroenterol. 2009 Jan 28;15(4):423-30.
2. Brunetto MR, Giarin MM, Oliveri F et al. Wild type and e antigen-minus hepatitis B viruses and course of chronic hepatitis. Proc Natl Acad Sci USA 1991; 88:4186-4190.
3. Carneiro de Moura M, Marinho R. Natural history and clinical manifestations ofchronic hepatitis B virus. Enferm Infecc Microbiol Clin. 2008 May;26 Suppl 7:11-8.
4. Chen CJ, Yang HI, Su J et al. Risk of hepatocellular carcinoma across a biologicalgradient of serum hepatitis B virus DNA level. JAMA. 2006;295:65–7.
5. Chen M, Sallberg M, Hughes J et al. Immune tolerance split between hepatitis B
virus precore and core proteins. J Virol 2005; 79:3016-3027.
6. Chu CM, Liaw YF: Incidence and Risk Factors of Progression to Cirrhosis in Inactive
Carriers of Hepatitis B Virus. Am J Gastroenterol. 2009 Jul;104(7):1693-9.
7. Elgouhari HM, Abu-Rajab Tamimi TI, Carey WD. Hepatitis B virus infection:
understanding its epidemiology, course and diagnosis. Cleve Clin J Med 2008;
75:881-889.
8. Fattovich G, Bortolotti F, Donato F. Natural history of chronic hepatitis B:
special emphasis on disease progression and prognostic factors. J Hepatol.
2008;48:335–352.
9. Fattovich G, Olivari N, Pasino M, et al. Long-term outcome of chronic hepatitis B
in Caucasian patients: mortality after 25 years. Gut 2008; 57:84-90.

10. Ganem D, Prince AM. Hepatitis B virus infection-natural history and clinicalconsequences. N Engl J Med 2004; 350:1118-1129.
11. He Yl, Zhao YR, Zhang SL, et al. Host susceptibility to persistent hepatitis B virus
infection. World Journal of Gastroenterology 2006; 12:4788-4793.
12. Huang CF, Lin SS, Ho YC, et al. The immune response induced by hepatitis B
virus principal antigens. Cell Mol Immunol 2006; 3:97-106.
13. Iloeje UH, Yang HI, Jen CL et al. Risk and predictors of mortality associated withchronic hepatitis B infection. Clin Gastroenterol Hepatol. 2007;5:921–931.
14. Kao JH. Diagnosis of hepatitis B virus infection through serological and virologicalmarkers. Expert Rev Gastroenterol Hepatol. 2008 Aug;2(4):553-62.
15. Lin CL, Liao LY, Liu CJ et al. Hepatitis B viral factors in HBeAg-negative carriers
with persistently normal serum alanine aminotransferase levels. Hepatology 2007;
45:1193-1198.
16. Lu YW, Tan TL, Zhang J, et al. Cellular apoptosis induced by replication ofhepatitis B virus: possible link between viral genotype and clinical outcome. Virol
J 2007; 4:117.
17. Lupberger J, Hildt E. Hepatitis B virus-induced oncogenesis. World Journal of
Gastroenterology 2007; 13:74-81.
18. Mahtab MA, Rahman S, Khan M, et al. Hepatitis B virus genotypes: an overview.
Hepatobiliary Pancreat Dis Int 2008; 7:457-464.
19. Mc Mahon BJ. Epidemiology and natural history of hepatitis B. Semin Liver Dis
2005; 25 Suppl 1:3-8.
20. Milich D, Liang TJ. Exploring the biological basis of hepatitis B e antigen inhepatitis B virus infection. Hepatology 2003; 38:1075-1086.
21. Ni YH, Chang MH, Chen PJ et al. Viremia profiles in children with chronic hepatitis B virus infection and spontaneous e antigen seroconversion. Gastroenterology
2007; 132:2340-2345.
22. Papatheodoridis GV, Manolakopoulos S, Dusheiko G, et al. Therapeutic strategies in the management of patients with chronic hepatitis B virus infection. Lancet
Infect Dis 2008; 8:167-178.
23. Papatheodoridis GV, Manesis EK, Manolakopoulos S et al. Is there ameaningful serum hepatitis B virus DNA cutoff level for therapeutic decisions inhepatitis B e antigen-negative chronic hepatitis B virus infection? Hepatology. 2008
Nov;48(5):1451-9.
24. Pawlotsky JM. Molecular diagnosis of viral hepatitis. Gastroenterology 2002;
122:1554-1568.
25. Sharma SK, Saini N, Chwla Y. Hepatitis b virus: inactive carriers. Virol J 2005;
2:82-85.
26. Tai DI, Lin SM, Sheen IS, et al. Long-term outcome of hepatitis B e antigennegative hepatitis B surface antigen carriers in relation to changes of alanine
aminotransferase levels over time. Hepatology. 2009 Jun;49(6):1859-67.
27. Victoria Fda S, Oliveira CM, Victoria MB, et al. Characterization of HBeAgnegative chronic hepatitis B in western Brazilian Amazonia. Braz J Infect Dis 2008;
12:27-37.
28. Ying-Hui Shi, Chang-He Shi. Molecular characteristics and stages ofchronic hepatitis B virus infection. World Journal of Gastroenterology 2009 July 7;
15(25):3099-3105.
29. Zacharakis G, Koskinas J, Kotsiou S et al. The role of serial measurement ofserum HBV DNA levels in patients with chronic HBeAg(−) hepatitis B infection:
Association with liver disease progression. A prospective cohort study. J Hepatol
2008 Dec; 49(6):884-91.

DE LA STAREA DE PURTĂTOR LA REACTIVAREA VIRUSULUI HEPATIC B
REZUMAT

Obiective: evoluţia naturală a infecţiei cu virus hepatic B depinde de starea sistemului imun al gazdei. Scopul acestei lucrări este de a evalua dinamica în timp a viremiei
HBV-DNA la purtătorii de AgHBs pentru a vedea câţi dintre ei rămân în stare inactivă şi câţi progresează către hepatită cronică.
Material şi metodă: Am inclus în studiu 34 pacienţi diagnosticaţi iniţial ca purtători de virus hepatic B la care am urmărit incărcătura virală pornind de la 2 până la 6 determinări
cantitative, precum şi nivelul alanin+aminotransferazei (ALT). Conform ghidurilor actuale, am definit starea de purtător ca fiind aceea în care limita superioară a viremie
HBV DNA a fost mai mică decât 2,000 IU∕ml (‹10,000 copii∕ml). A fost efectuată puncţie biopsie hepatică ecoghidată pentru a urmări indexul de activitate histologică laacei pacienţi care au dezvoltat hepatită cronică.
Rezultate: Din totalul de 34 pacienţi, 17 au fost femei (50%) şi 17 bărbaţi (50%). Vârsta medie a fost 49.02 ±11.6 ani (minim 23 ani, maxim 69 ani). Perioada medie deurmărire a fost 35,47±3 luni cu un minim de 14 luni şi un maxim de 118 luni. Nouă pacienţi (26,47%) au prezentat replicare virală semnificativă: 4 femei (44,4%) şi 5bărbaţi (55,5%) după o perioadă medie de 38,4 luni. Pe timpul perioadei de urmărire s-a constatat un grad rezonabil de corelatie între valoarea viremiei si valoarea ALT
serice, deşi nesemnificativă statistic (r=0.425, p=0.115).
Concluzii: Aproximativ 1 din 4 purtători inactivi de virus hepatic B au dezvoltat replicare virală semnificativă după o perioadă medie de urmărire de 3 ani. Purtătorii de virus
hepatic B trebuie monitorizaţi periodic pentru a putea surprinde o posibilă activare a replicării virale ce ar putea beneficia de pe urma unui tratament antiviral adecvat.
Cuvinte cheie: purtător inactiv de virus hepatic B, reactivare, viremie HBV-DNA
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45

ELECTRODIAGNOSTIC OF THE NEUROMUSCULAR
JUNCTION IN MYASTHENIA GRAVISBOGDAN ISTRATE1,2, DANIEL DUMITRU BANCIU1, CRISANDA VÎLCIU2, ANA CÂMPEANU2,MARIA-LUISA FLONTA1
1
Department of Neurobiology and Molecular Physiology,University of Bucharest, Romania
2
Department of Neurology,Clinical Institute Fundeni,University of Medicine and Pharmacy, Bucharest, Romania

ABSTRACTThe electro diagnostic examination in patients with neuromuscular junction disorders requires a good comprehension on the physiology and pathphysiology of neuromuscular transmission. S.F.E.M.G. and R.N.S. are an essential part of the electro diagnostic study of the patients suspected by Myasthenia Gravis, being in particular useful inpatients with the negative or uncertain pharmacological test (Tensilon) or minimal neurological findings, besides patients with seronegative Myasthenia Gravis. Also, theelectro diagnostic of Myasthenia Gravis should be performed according to the patients’ symptoms, medical and family history. Electromiography is a precious instrument
in a complete and certain diagnostic of Myasthenia.
Key words: myasthenia gravis, electromyography, diagnostic tests

INTRODUCTION
Myasthenia Gravis is an autoimmune disease which results in muscle
weakness, more pronounced during periods of physical activity and decreasing
after periods of rest. The pathogenesis of Myasthenia is triggered by a defect in thetransmission of nerve impulses to muscle. The immune phenomena are caused by
the neurochemical mechanism in which acetylcholine auto antibodies attacks the receptor areas of the muscle, especially at the level of the neuromuscular junction.
Electromiography is an electrical recording of muscle activity that aids in the
diagnosis of certain neuromuscular disease. As a neurophysiologic diagnostic thistechnique of investigation offers the most sensitive complete diagnostic test, but
this fact doesn’t exclude that is absolutely specific. Two major tests for Myasthenia
Gravis diagnostic are used from electromyography techniques panel: Single Fiber
Electromiography and Repetitive Nerve Stimulation.
Repetitive nerve stimulation shows a decremented response that is characteristic for myasthenia, the decrement being most pronounced in proximal muscles.
Single fiber electromyography, is much more sensitive.
Repetitive nerve stimulation
It is used to diagnose Myasthenia Gravis and Lambert Eaton Myasthenic
Syndrome. The recordings of the electrical stimulation are performed with surface
electrodes. The intensity of stimulation must be supramaximal. The selected muscle
will be chose in respect with the patient symptoms. In Myasthenia Gravis, axial andproximal body muscles are predominantly affected by a bulbar involvement. For
E.M.G. we can select a muscle with moderate dysfunction degree. It is not easy
to find a muscle like this, especially in myasthenia with ocular involvement. It is
desirable to test a facial or a proximal muscle in these patients or in the patientswith moderate symptoms. Among proximal muscle, most used is the deltoid. In
this case, the electrical stimulation must be done in the supraclavicular fossa. In
ocular myasthenia, the facial muscle is easy to stimulate too.
The recordings are acquired after orbucularis oculi stimulation being performed by placing the active electrode at the level of inferior eyelid, and the referenceelectrode placed on the wing of the nose. For nasalis muscle the active electrode isput on the wing of nose too. The facial nerve is stimulated under the ear. The standard
protocol of stimulation says that the normal muscle may present a decrement of 8%

percentage. A decrement up to 10% percentage is considered to be abnormal andpathologic. In order to confirm the fact that a resulting decrement after a repetitive
stimulation is caused by the neuromuscular junction pathology, we can repeat theelectromyographyc test after anti-cholinesterase administration (edrophonium or
neostigmine) which leads to the remission of decrement.
Single fiber electromyography is a special technique discovered by Ekstedt
and Stalberg, which offer information concerning the structure and function of the
motor unit. In present S.F.E.M.G. is used in the diagnostic of the neuromusculartransmission defects. The “jitter” measurement is considered the most accurate
electrophysiological method with higher sensibility.
Physiology of the Repetitive Nerve Stimulation
When repetitive stimuli are applied, at the level of neuromuscular junction,
two different processes take place.
1. The pre-synaptic depression: the progressive decreasing of the acetylcholine
clusters from synaptic endings. These are manifested trough a progressive reductionof the synaptic potentials, by a decreasing number of acetylcholine quanta that isreleased along with each nervous impulse, as measure of the acetylcholine depredation of clusters is instantly available. An increased synthesis and mobilization
partially compensates this phenomenon.
2. Facilitated release of acetylcholine may be progressive as a consequence ofthe calcium ions that are present at the level of the nervous ending. This phenomenon is electrophysiologically translated by increasing amplitude of the compoundmuscle action potential area, after supported physically effort.
Tested muscles
In Myasthenia Gravis, predominantly affected is the bulbar musculature of themembers (axial and proximal). Proximal muscles-frequently the deltoid muscle istested. The electrical stimulation is performed into supraclavicular fossa. The active
electrode is placed on the muscle and the reference electrode on the acromion.
The voluntary activity will be attained by the arm abduction against resistanceat an angle of 45-90°.The stimulation causes discomfort and activation of othermuscles is practically inevitable. The trapezium muscle – is easy to be stimulate,the produced pain being much smaller. The spinal accessory nerve is stimulated
at the half of sternocleidomastoidian muscle, on his posterior brink. The brachial

Received February 2010. Accepted March 2010. Address for correspondence: Dr. Bogdan Istrate, Department of Neurobiology and Molecular Physiology, University of Bucharest, Romania,
e-mail: bogdan_ellis@yahoo.com

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Fiziologia - Physiology  2010.20.1 (65)

biceps is stimulated with the pacient placed in clinostats, with the arm in abduction
at 90°. The musculocutaneous nerve is stimulated in axial. The anconeus muscle isrelatively easy to stimulate, less painful and with rough and ready sensitivity. In thesesituations, the patient is asked to stand in ortostatism, with forearm in pronationand sustained on examination table. The radial nerve is stimulated at the level oflateral intermuscular septum, at 1-2 cm upper to the lateral epicondilus. The active recording electrode is placed on the muscle, at three width of digital finger byolecran, in the top of an isosceles triangle, his basis being the line between olecranand medial epicondilus. The reference electrode is placed on the dorsal face of theforearm. Muscle activation is made by the elbow extension against resistance. Easy
to be stimulated are facial muscles too. The electrical recordings for orbicularis oculi
muscle is made by placing the active electrode at the level of inferior eyelid andwith the reference electrode placed on the ala of nose.
For nasalis muscle the active electrode will be placed at the level of the ala ofnose, on the right side. The facial nerve is stimulated under the ear. Easier to stimulate
is the A.D.M. muscle, with distal stimulation of hand joint of ulnar nerve.
Single fiber electromyography
Technically speaking, S.F.E.M.G. consists in the extracellular recording of theindividuals action potentials (with a needle-specific electrode) of the muscle fiber. In
normal subjects sarcolema activated threshold will determine the mean variation of
the neuromuscular transmission. The normal jitter is variable at the level of different
synapses of the same muscle, even the same motor unit. Also, the medium valuesof jitter are variable on several muscles.
In Myasthenia Gravis the retrenchment of the post synaptic responsivity,
decrease the amplitude of end plate potentials, which leads to a prolonged timein order to reach the discharge threshold. The jitter can be determined either ofmuscle voluntary activation or electrical stimulation of the tested muscle. At
voluntary activation of muscle the activations potentials are recorded from two
muscle fibers of the same motor unit. The first fiber is used as “trigger”, that serve as
reference in order to measure prior in time, the interval variability up to the second
muscle fiber activity. The needle electrode is placed in the muscle that is in a mild
syncopation, near to the motor endplate. The position in which two single fiber
potential is recorded will be searched. The first potential will represent the “trigger”
of the signal acquisition. Then, the variability of inter-potential interval (jitter) will
be measured, after 50-100 successive discharged impulses.
For the majority of muscles, the normal range of jitter is situated between
5-55µs. The first sign of the neuromuscular transmission defect/impairment is increasing of jitter. When the jitter is up to 70µs, a neuromuscular block will appear.
In order to illustrate the things above said, in following images we can see
the aspect of normal R.N.S (Fig.1) and S.F.E.M.G. (Fig.2) (with jitter) and alsocharacteristic for Myasthenia.

Fig.2. Normal and myasthenic aspect of jitter during S.F.E.M.G. examination

MATERIAL AND METHODSWe chose to present three clinical cases of which one is normal, without signsof neuromuscular impairment (control) in order to have criteria for comparison. The
patients were directed to electromyography exam as a complementary test to confirm
the diagnostic, besides the blood test for anti-acetylcholine receptor antibodies.
The electromyography test was performed with a Neropack 9100 portable electromiograph.The R.N.S. was performed wit surface electrodes and S.F.E.M.G. with needle
electrodes.The characteristic electromyographic routs images of the clinical cases taken as
examples are illustrated and interpreted in the section results of the article.
Case 1. A.L; sex F, 48-years-old. The electromyogram was performed bystimulating the right facial nerve and nasalis acquisition. (Table I, II, III; Fig. 3, 4)
Results: At the repetitive nerve stimulation exam (3Hz) of the right facial
nerve and nasalis acquisition, there are no significant recordings changes of thecompound muscle action potential. The electromiographic route above illustrated
is normal in the examined area.
Table I. Repetitive Nerve Stimulation (case 1)

Table II. Needle EMG (case 1)

Fig.1. The normal aspect compared with the characteristic aspectof decrement in Myasthenia during R.N.S

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Table III. Repetitive Nerve Stimulation (case 1)

Fig.5. Repetitive nerve stimulation of CNS with a significant voluntary post activation (case 2)
Table IV. RNS Report. Nerve: Cubital. Muscle: ADM (case 2)

Fig.3. The EMG route after RNS (3Hz) (case 1)

Case 3. S.G; sex F, 54-years-old,which besides the myasthenia wassuspected by amyotrophic lateral screlosis,reason for why the Single Fiber Electromiograpgy was performed, in right EDC muscle.Repetitive nerve stimulation wasmade too. (Fig.6, 7; Table V, VI)
Results: The electromyographic route in this context, show at the exam ofrepetitive nerve stimulation (3Hz) of right cubital nerve and ADM data acquisition,a decrement of 16%, with significant improvement in conditions of effort, followedby post activation exhaustion. A severe decrement was reported (45%). The single
fiber electromyography, performed on EDC muscle, reveal an increased jitter withfrequently blockage. The electromyographic route pleads for Myasthenia Gravis.
Table V. RNS Report. Nerve: Cubital. Muscle: ADM (case 3)

Fig.4. EMG aspect after RNS (3Hz)(case 1)
Case 2. F.K; sex F, 12-years –old. The electromyographic test in this casewas performed by slowly repetitive electrostimulation of the right cubital nerveand ADM acquisition of currents (Fig.5, Table IV).
Results: At the slowly repetitive nerve stimulation (3Hz), of the rightcubital nerve and ADM acquisition, an” U-shape” decrement is present, after asignificant voluntary post activation. The electromyographic route is compatible
and characteristic for Myasthenia Gravis.
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Fiziologia - Physiology  2010.20.1 (65)Table VI. SFEMG of right EDC (case 3)

CONCLUSIONS
When myasthenia is already confirmed, with an MDS lower score (and themyasthenic deficit is also small), then the diagnosis must be made with certainty.
Obviously, the positive response to the pharmacological test(with miostin) makes
clear the evidence of myasthenia, but through electromyographic exam is importantto see what kind of neuromuscular block is present: presynaptic or postsynaptic
Electromiography is useful for confirming the diagnosis of myasthenia, whenis performed in the time of a myasthenic burst, in the territory of the neighboring
muscle with an important deficit and within 12 hours after anticholinesterase
administration.
An EMG performed outside the mysthenic burst or under the anticholinesterase effect, could offer a normal route and a false guidance in order to put thediagnosis. Even if the electromyographic route is normal doesn’t mean that thediagnosis is excluded.
REFERENCES

Fig.6. RNS after 20 seconds of required
effort (case 3)

Fig.7. SFEMG on EDC muscle (case 3)

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1. Amato AA, James RA. Neuromuscular Disorders. Mc.Graw Hill Medical, 2008.
2. Brust JCM, List TA, Catalano LW, Lovelace R. Ocular myasthenia gravis,
mimicking progressive external ophthalmoplegia. Rare case of myasthenia
associated with peripheral neuropathy and spastic paraparesis. Neurology(Minneap.) 1975, 24, 755-760.
3. Cogan DG, Yee RD, Gittinger J. Rapid eye movements in myasthenia
gravis. I. Clinical observations. Arch. Ophthalmol., 1976; 94: 1083-1085.
4. Kaminsky JH. Myasthenia Gravis and related disorders. Second edition.
Humana Press, 2009.
5. Keane JR, Hoyt WF. Myasthenic (vertical) nystagmus: Verification byedrophonium tonography. J. Amer med. Asso. 1970; 212:1209-1210.
6. Kelly JJ, Daube JR, Lennon VA, Howard FM, Younge BR. The laboratory
diagnosis of mild myasthenia gravis. Ann. Neurol, 1982;12: 238-242.
7. Konishi T, Nishitani H, Matsubara F, Ohta M. Myasthenia gravis: Relation
between jitter in single-fiber EMG and antibody to acetylcholine receptor.
Neurology 1981; 31:386-392.
8. Lindstrom JM, Seybold ME, Lennon VA, Whittingham S, Duane DD.
Antibody to acetylcholine receptor in myasthenia gravis. Neurology (Minneap.) 1976; 26:1054-1059.
9. Oh SJ, Ealami N, Nishihira T, Sarala PK, Kuba T, Elmore RS, Sunwoo
IN, Ro YI. Electrophysiological and clinical correlation in myasthenia gravis.
Ann. Neurol 1982, 12, 348-354.
10. Osher RH. Myasthenic "oculomotor" palsies. Ann. Ophthalmol. 1979;11:
31-34.
11. Osher RH, Griggs RC. Orbicularis fatigue: The `peek' sign of myasthenia
gravis. Arch. Ophthalmol.1979;97: 677-679.
12. Retzlaff JA, Kearns TP, Howard FM, Cronin ML. Lancaster red-green
test in evaluation of edrophonium effect in myasthenia gravis. Amer. J.
Ophthalmol. 1969; 67: 13-21.
13. Sanders DB, Howard JF, Johns TR. Single-fiber electromyography inmyasthenia gravis. Neurology (Minneap.) 1979;29: 68-76.
14. Simpson JA. Myasthenia gravis and myasthenic syndromes. In:
Disorders o f Voluntary Muscle, 4th Edition, 1981, edited by S.J. Walton,
Churchill, Livingstone-Edinburgh, pp. 587-589.
Abbreviations and defining terms
E.M.G. electromyography
M.D.S. mysthenic deficit score
R.N.S. repetitive nerve stimulation
S.F.E.M.G. single fiber electromyography
JITTER mean variation in interpotential interval between single-fiber action
potentials of the same motor unit

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A.D.M. abductor digiti minimi (muscle)
E.D.C. extensor digitorum communis(muscle)
S.P.E. (sciaticus popliteus external (nerve)
T.A tibial anterior (muscle)

Abbreviations and defining terms
E.M.G. electromyography
M.D.S. mysthenic deficit score
R.N.S. repetitive nerve stimulation

S.F.E.M.G. single fiber electromyography
JITTER mean variation in interpotential interval between single-fiber action
potentials of the same motor unit
A.D.M. abductor digiti minimi (muscle)
E.D.C. extensor digitorum communis(muscle)
S.P.E. (sciaticus popliteus external (nerve)
T.A tibial anterior (muscle)

ELECTRODIAGNOSTICUL JONCTIUNII NEUROMUSCULAREIN MIASTENIA GRAVISREZUMAT
Examinarea electro-diagnostica a pacientilor care prezinta afectarea jonctiunii neuromusculare necesita o buna intelegere a fiziologiei sifiziopatologiei transmiterii neuro-musculare.
S.F.E.M.G. si R.N.S. sunt o parte esentiala a studiilor electro-diagnostice in cazul pacientilor suspectati de miastenia gravis, fiind in mod
particular utile in cazul acelor pacienti cu teste farmacologice negative sau incerte (Tensilon), in cazul pacientilor cu afectare neurologicalminimala si pentru pacientii seronegativi pentru miastenia gravis. De asemenea, electro-diagnosticul miasteniei gravis ar trebui efectuat inconcordanta cu simptomatologia pacientilor, precum si cu istoricul medical si antecedentele heredocolaterale ale acestora. Electromiografia
este un instrument pretios pentru stabilirea si certificarea diagnosticului de miastenie.
Cuvinte cheie: miastenia gravis, electromiografie, teste diagnostice

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LIPOPEROXIDES –MARKERS OF OXIDATIVE STRESS IN THE
NEWBORN WITH PERINATAL ASPHYXIAGABRIELA ZAHARIE1, MELINDA MATYAS1, ADINA MUREŞAN2, MONICA POPA1
Department of Neonatology, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca
2
Department of Physiology, “Iuliu Hatieganu” University of Medicine and Pharmacy Cluj-Napoca

1

ABSTRACT

Background: Oxidative stress was included in the pathogenesis of asphyxia and ischemic hypoxic encephalopathy in the last years because of certain
particularities of the central nervous system, which increase the vulnerability in the oxygen reactive species.
Objective: The study aimed to identify lipoperoxides in the newborns with perinatal asphyxia.
Materials and Methods: The study group included term newborns with asphyxia at birth, in the period January 2005- June 2006, in the Department of
Neonatology of the 1st Gynecology Clinic, Cluj-Napoca, Romania. Lipoperoxides were measured in dynamics on the first and third days of life. The results
were processed with the STATISTICA program.
Results: The study group included 11 term newborns of 38.91±0.70 weeks of gestation and 3391±380.90g body weight respectively. The term newborns
presented: mild asphyxia 45.45%, moderate asphyxia 19.19% and severe asphyxia 36.36%. The oxygen concentration that was necessary on the first day oflife was of 30.18±10.06% as compared to that of the third day, which was of 24.82±4.98% (p=0.0258). The level of MDA lipoperoxides was higher on thethird day (4.72±1.97nmol/ml) than that on the first day (4.46±2.09nmol/ml).
Conclusions: Lipoperoxides represent a marker for oxidative stress in the newborn with perinatal asphyxia, which is a major source of morbidity, mortality
and neurological consequences in the surviving cases.
Key words: asphyxia, lipoperoxides, newborn

INTRODUCTION
Perinatal hypoxic stress is a frequent cause of morbidity, mortality andof neurological sequelae in surviving cases suffering from neonatal asphyxia(3,6,12,15).
The hypoxic - ischemic brain lesion begins with the insult and continues
during the rehabilitation period after reperfusion (6,17,18).
Oxidative stress has been lately considered as part of the pathogenesis ofasphyxia and of perinatal AIE because of certain CNS particularities, which increase
its vulnerability to ORS as follows: cell membranes rich in polyunsaturated fatty
acids and shortage of antioxidants in the brain, especially in catalase and superoxide
dismutase (SOD). Brain regions, which are rich in iron, during low antioxidative
defense, highlight the discharge of iron with small molecular mass, leading tohydroxyl radical and lipid peroxidation (10,11).
Oxygen reactive species act upon biomolecules causing their degradation.
The ORS effect upon these biomolecules can be identified by indirect methods(1,2,4). Focus was laid on the study of the ORS effect upon fats in asphyxia as the
lipoperoxides are the markers most widely used to identify oxidative stress in the
newborn during the first week of life.
All these theoretical considerations justify the study of lipoperoxides inneonatal asphyxia. The objective of this study was to identify lipoperoxidation in
the term newborn with various types of asphyxia.

malonilaldekide (MDA). The MDA is the most important aldekide, which resultsfrom lipid peroxidation.
Two measurements from the venous blood were made in all newborns of thestudy group, on the first and third day respectively. In the newborns of the control
group only one measurement was made on the first day of life.
The dosing method used in this study was Satoh’s method, whose testing isbased on the reaction of an MDA molecule with two molecules of tiobarbituric acid,
which results in a pink production with 530 nm absorption. The absorption intensity
is directly proportional to the MDA quantity in the sample. The results are expressedin nmols MDA/ml of serum. The normal level in adults is of 1.29±0.37 nmol/ml.
Along with the dosing of lipoperoxides, the Astrup parameters were alsomeasured on the first and third days of life, considering that in the neonatal period
the precipitating conditions for ORS occurrence are: acidosis, hypoxia, hyperoxia andinfections. Asphyxia and AIE constitute frequent pathological conditions associatedwith hypoxia and hyperoxia following oxygen therapy.
The Pearson and Spearman correlations, simple linear regressions, comparisons of initial and final measurements (on the first day and the third day respectively)
were used for data processing. The Student Test was used in pairs of the study groupfor the data following a normal distribution and the Mann-Withney Test was used
for the data not following a normal distribution.

RESULTS
MATERIALS AND METHODS
The study group included 11 term newborns with asphyxia of different degrees
of severity. The control group included 20 healthy term newborns. In order to study
oxidative stress in asphyxia at birth, lipid peroxidation was studied by identifying

The study group included eleven term newborns. Asphyxia severity wasassessed according to the Apgar score at 5 minutes. Thus, three types of asphyxia
were found:
1. Mild – Apgar score 7
2. Moderate –Apgar score between 6 and 4

Received January 2010. Accepted February 2010. Address for correspondence: Dr. Gabriela Zaharie, Department of Neonatology, “Iuliu Hatieganu” University of Medicine andPharmacy, Cluj-Napoca, Romania
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3. Severe – Apgar score between 3 and 1
The study group included eleven term newborns, four female newborns[36.36%, IC 95% (9.92-71.90)] and seven male newborns [54.565, IC 95%(28.10-80.99)](Figure 1).

group.
The parameters were analyzed in pairs of the study group by using the Student
Test. The results are shown in Table III.
Table III. Parameters analysis within the study group

36%

64%

F
M

Fig.1. Gender incidence in term newborns with asphyxia

The mean gestational age of term newborns from the study group was of
38.91weeks with IC 95% (38.44-39.38), and the mean weight was of 3397 g withIC 95% (3141-3653).
The variation of the Apgar score in the study group of term newborns isshown in Table I.
Table I. Variation of Apgar score at 1 minute and 5 minutes in the term newborns

Table data analysis revealed a significant difference between the FiO2 valueon the first and third days (p<0.05). The correlation for the quantitative variablesin the studied term newborns is presented in Table IV.
Table IV. Pearson correlation for the quantitative variables in the studied term newborns
GA = gestational age; W = weight; MDA = lipoperoxides; FiO2 = concentration of oxygenthat newborns were exposed to; pCO2 = partial pressure of carbon dioxide; pO2 =partial pressure ofoxygen; SaO2 =oxygen saturation.

The incidence of the various types of asphyxia in the group of term newborns
is shown in Table II.
Table II. Asphyxia distribution in the term newborns

Cerebral hemorrhage was present in 2 cases and AIE in 4 cases of the study
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The Spearman correlation test for quantitative and qualitative variables is
Fiziologia - Physiology  2010.20.1 (65)presented in Table V.

Table VIII. Comparison of MDA in term newborns with asphyxia and in adults

Table V. Spearman correlation for quantitative and qualitative variables

In order to study lipoperoxidation the MDA value registered on the first andthird days in the studied term newborns was compared with the MDA value in thecontrol group that consisted of healthy term newborns. The results are illustrated
in Tables VI and VII.

DISCUSSIONS

Table VI. Comparison between MDA value on the first day in term newborns with asphyxia
versus controls

MDA D1 = MDA value on the first day; Rank Sum = sum of ranks; U=; N prem = sample sizeof prematures; sample size of controls
Table VII. Comparison between MDA values on the third day in term newborns
with asphyxia versus controls

MDA D3 = MDA value on the third day; Rank Sum = sum of ranks; U=; N prem = sample sizeof prematures; sample size of controls

The comparison between the MDA value in the studied group and normal
values in adults is presented in Table VIII.

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The MDA in term newborns with asphyxia was determined in order to study
lipid peroxidation. Table II data analysis revealed that mild asphyxia prevailed(45.45%), followed by severe asphyxia (36.36%) and moderate asphyxia (18.18%).
All newborns with severe asphyxia also presented AIE. In intranatal asphyxia ORS
are released in the central nervous system and will contribute to the genesis ofencephalopathy lesions (52,110,157,158). Oxygen reactive species are producedin the reoxygenation stage following perinatal asphyxia. The brain is at major riskof developing oxidative lesions since membrane lipids are rich in polyunsaturated
fatty acids and the quantities of some antioxidative defense enzymes (catalase andsuperoxide dismutase) may be reduced. Some brain areas are rich in iron. Iron from
cells is released under the action of cell lesions caused by asphyxia. Non-transferin
bound iron generates hydroxyl radicals, which, in their turn, will trigger a chain
peroxidation reaction that increases MDA levels in blood and urine (5,7,8). Cerebral
hemorrhage, which occurred in two cases, was caused by obstetrical trauma.
In the study group oxygen therapy was applied at a median FiO2 of 30.18%
on the first day (95% CI (23.42-36.94)) as compared with a median FiO2 of 24.82%
on the third day (95% CI (21.48-28.16)). The oxygen supply required was low. On
the third day the median value of the FiO2 applied was close to the concentrationof oxygen in air. The difference between the FiO2 value on the first day and on thethird day was statistically significant (p = 0.0298). Respiration improved rapidly
after asphyxia thus reducing oxygen administration. The clinical picture was replacedby neurological manifestations once the vital functions became stable and therespiratory symptomatology improved (3,13,14,16).
The median value of the pH in the studied group was the same on the first
and third days, with a median of 7.34 (95% CI (7.30-7.39)). The values of pCO2,
pO2 and SaO2 did not register statistically significant differences (p > 0.05) on
the first and third days.
The median MDA value in the studied newborns was higher on the third day(4.72nmol/ml; 95% CI (3.39-6.04)) as compared with the first day (4.46nmol/ml; 95% CI (3.05-5.87)). However, the difference was not statistically significant(p > 0.05). The more intense peroxidation process registered on the third day may
be interpreted as a consequence of perinatal hypoxic stress, which is an importantsource of morbidity, mortality and neurological consequences in surviving newborns(109,117,120).
The comparison between the MDA in the control group and the MDA value
in the studied term newborns with asphyxia was not statistically significant (p >53

0.05) (Tables VI,VII). The comparison between the median MDA value in the studied
newborns and the median MDA value in adults revealed significant lower values inadults (p < 0.001). Therefore the peroxidation process is more intense in newborns.
This is generated by poor antioxidative defense mechanisms and circumstances thatfavor the production of oxidative stress: perinatal hypoxic stress, birth, the transition
from intrauterine to extrauterine life and neonatal asphyxia.

CONCLUSIONS

1. The studied newborns presented all types of asphyxia: mild, moderate andsevere. The mild form prevailed.
2. The studied newborns had other associated pathologies: cerebral hemorrhage (18%) and AIE (36%).
3. The control group consisted of 20 term newborns without associatedpathology, with gestational age over 38 weeks and a mean weight of 3500 g.
4. Exposure to oxygen was significantly lower on the third day as comparedto the first day (p = 0.0298) due to the rapid restoration of the vital functions andthe replacement of respiratory distress with neurological distress (9,11,16).
5. There were no statistically significant differences between the values
registered on the first and third day of life in the studied term newborns as far as
pH (7.34; p > 0.05), pO2, pCO2 and SaO2 values were concerned.
6. Lipid peroxidation reflected by the median MDA value was higher on thethird day as compared with the first day. However, it was not statistically significant(p > 0.05) since the peroxidation process was more intense on the third day of life
given the postasphyxia context.
7. The median MDA value in the newborns with asphyxia was significantly
higher than normal values in adults.
REFERENCES
1. Muresan A. Speciile reactive ale oxigenului in patologia clinica,Editura
Dacia, ISBN 35-0703-2973- 1997
2. Sies H. Oxidative stress. Oxidants and antioxidants. Experim Physiol.
1997; 82: 291-295.
3. Taeusch Ballard: Avery s diseases of the newborn, 8th edition 1998,
1123-1177.
4. Young IS, Trimble ER. Measurement of maloniladehyde in plasma byhigh performance liquid chromatography with flourimetric detection. Ann
Clin Bioch 1991; 29:708-712.

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5. Halliday HL, Dumpit FM, Brady JP. Effects of inspired oxygen on echocardiographic assesment of pulmonary vascular resistance and myocardial
contractility in bronchopulmonary dysplasia. Pediatrics 1980; 65:536-40.
6. Mishra OP, Delivoria-Papadopoulos M. Cellular mechanism of hypoxic
injury in the developing brain. Brain Res Bull 1998; 48: 233-8.
7. Varsila F, Hallman M, Anderson S. Free radical-induced lipid peroxidation
during early neonatal period. Acta Paediatr 1994; 83: 692-5.
8. Carney J, Starke-Reed P, Oliver C, Landum RW, Cheng MS, Wu JF, etall. Reversal of age-related increase in brain protein oxidation, descreasein enzyme activity, and loss in temporal and spatial memory by chronic administration of the spin-trapping compound N-tert-butyl-alpha-phenilnitron.
Proc Natl Acad Sci USA 1991; 88: 3633-6.
9. Domergues MA, Gallego J, Evrard P, Gressens P. Iron supplementation
aggravates periventricular cystic white matter lesions in newborn mice. Eur
J Pediatr Neurol 1998;2: 313-318.
10. Palmer C, Menzies SL, Roberts RL, et al. Changes in iron histochemistry
after hypoxic-ischemic brain injury in the neonatal rat. J Neurosci Res
1999; 56:60-71.
11. Sarco DP, Becker J, Palmer C, et al. The neuroprotective effect ofdeferoxamine in the hypoxic-ischemic immature mouse brain. Neurosci
Lett 2000; 282:113-116.
12. Rosenberg AA, Murdaugh E, White CW. The role of oxygen free radicals
in postasphyxia cerebral hypoperfusion in newborn lambs. Pediatr.Res.
1989; 26:215-219.
13. Mori H, Oikawa M, Tamagami T, Kumaki H, Nakaune R, Amano J,
Akinaga Y, Fukui K, Abe K, Urano S. Oxidized proteins in astrocytes generated in a hyperbaric atmosphere induce neuronal apoptosis. J Alzheimers
Dis 2007: 11 (2):165-174.
14. Sridhar MG, Setia S, John M, Bhat V, Nandheesa H, Sathiyapriya V.
Oxidative stress varies with the mode of delivery in intrauterine growth
retardation: association with Apgar score. Clin Biochem 2007; 40(9-10):
688-691.
15. Inder TE, Volpe JJ. Mechanisms of perinatal brain injury. Semin Neonatol 2000;5: 3-16.
16. Palmer C, Menzies SL, Roberts RL, Pavlick G, Connor JR. Changes iniron histochemistry after hypoxic-ischemic brain injury in the neonatal rat.
J Neurosci Res 1999; 56: 60-71.
17. Ciccoli L, Rossi V, Signorini C, Leoncini S, Buonocore G, Paffetti P,
et al. Iron release in newborn and adult erythrocytes exposed to hypoxiareoxygenation. Biol Neonate 2001; 80: 102.

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LIPOPEROXIZII – MARKERI AI STRESULUI OXIDATIV LANOU-NASCUTII CU ASFIXIE PERINATALAREZUMAT

Introducere: In ultimii ani, stresul oxidative a fost inclus in patogeneza asfixiei si a encefalopatiei ischemice hipoxice datorita anumitor particularitati ale
sistemului nervos central, care duc la cresterea vulnerabilitatii la speciile reactive ale oxigenului. Studiul urmareste identificarea lipoperoxizilor la nou-nascutii
cu asfixie perinatala.
Materiale si metode: Grupul de studio a inclus nou-nascuti la termen cu asfixie la nastere, din perioada Ianuarie 2005-Iunie 2006, din Departamentul deNeonatologie al Clinicii de Ginecologie Nr. 1, Cluj-Napoca, Romania. Lipoperoxizii au fost masurati in dinamica in prima si a treia zi de viata. Rezultatele aufost prcesate folosind programul computerizat STATISTICA.
Rezultate: Grupul de studio a inclus 11 nou-nascuti la termen la 38.91±0.70 saptamani de gestatie, avand greutate la nastere de 3391±380.90g. Nou-nascutii
la termen au prezentat: 45.45% asfixie usoara, 19.19% asfixie moderata si 36.36% asfixie severa. Concentratia oxigenului necesar in prima zi de viata a fost
30.18±10.06%, comparative cu ziua a treia, cand oxigenul necesar a fost in concentratie de 24.82±4.98% (p=0.0258). Nivelul lipoperoxizilor MDA a fost2010.20.1(65)  Fiziologia - Physiology

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mai crescut in ziua a treia (4.72±1.97nmol/ml) comparativ cu
prima zi (4.46±2.09nmol/ml).
Concluzii: Lipoperoxizii reprezinta un marker de stres oxidative la nou-nascutii cu asfixie perinatala, ceea ce face sa fie ocauza majora de morbiditate, mortalitate si induce consecinte
neurologice in cazurile care supravietuiesc.
Cuvinte cheie: asfixie, lipoperoxizi, nou-nascut

The XXIVth National Conference of Romanian Society of Physiological
Sciences
Prioritair Directions in Experimental and Clinical Physiology
Honorary President
Ioan Hăulică (Iaşi)SRSF President
Adriana Mureşan (Cluj-Napoca)

Conference President
Cismaş-Pruteanu Petrişor

Honorary Presidium
Valeriu Neştianu (Craiova)
Marius Sabău (Tg. Mureş)Francisc Schneider (Arad)
Scientific committee

Carmen Bunu (Timişoara)
Anca Bădărău (Bucureşti)Petrişor Cismaş (Oradea)
Dan Dobreanu (Tg. Mureş)Smaranda Goţia (Timişoara)
Maria Luiza Flonta (Bucureşti)Maria Grama (Sibiu)
Simona Gusti (Craiova)
Maria Iancau (Craiova)
Ileana Ion (Constanţa)Rodica Mateescu (Timişoara)
Georgeta Mihalaş (Timişoara)
Daniela Motoc (Arad)
Adriana Mureşan (Cluj Napoca)
Virgil Păunescu (Timişoara)
Gheorghe Petrescu (Iaşi)Manuela Pumnea (Sibiu)
Aurel Saulea (Chişinău)Simona Slătineanu (Iaşi)Simona Tache (Cluj-Napoca)
Maria Vrabete (Craiova)
Leon Zăgrean (Bucureşti)Organizing committee
Adriana Ardelean
Florina Bojin
Ovidiu Burtă
Olivia Burta
Cornel Cheregi
Claudia Ciuciu
Ramona DanCătălin Florescu
Melania Forogău
Ioan Magyar
Daniela Matei
Rodica Negrean
Alina PopRăzvan Oros
Carmen Tatu
Dana Carmen Zahaşi studenţii:Mădălin Bulzan
Loredana Corha
Carmen Stoica
Ioana Zaha

Main topics:
1. Experimental Physiology
4. Oxidative Stress
2. Clinical Physiology
5. Vascular Endothelium
3. Exercise Physiology
6. Neurosciences

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cmyk

Fiziologia - Physiology  2010.20.1 (65)